Experimental Biology and Medicine

Gonadectomy of adult male rats resulted in hypertrophy of the adrenal glands. Total adrenal steroid (blue tetrazoli-um positive) production in vitro was similarly augmented. Corticosterone secretion both in vitro and in vivo was unaffected. The ratio of corticosterone to total steroid, therefore, gradually declined following removal of the testes. This effect is explained by a concomitant increase in adrenal 5a-reductase activity after orchiectomy, resulting in greater conversion of corticosterone to 5a-reduced metabolites.

The authors are grateful to Mrs. Nancy Swygert and Mrs. Andrea Newsome for expert technical assistance.

The mechanism by which dietary trypsin inhibitor induces excessive pancreatic secretion was investigated in rats with bile-pancreatic duct fistulas. It was found that removal of bile-pancreatic juice from the intestine resulted in a large increase in pancreatic enzyme secretion. Infusion of trypsin or chymotrypsin as well as bile-pancreatic juice suppressed the secretion of pancreatic enzymes. When trypsin was present in the intestine, a large pancreatic enzyme response was obtained by infusion of trypsin inhibitors. A similar increase in pancreatic enzyme output was evoked when trypsin infusion was stopped. The results indicated that pancreatic enzyme secretion in the rat is subject to feedback inhibition from intestinal trypsin and chymotrypsin, and that trypsin inhibitors stimulate pancreatic enzyme secretion indirectly, by binding or neutralizing trypsin and thereby removing its feedback inhibition.

The authors express their thanks to Miss Chris Giotas for her technical assistance.

Interruption of suckling in rats for a single period of up to 12 hr in the immediate postpartum period or on day 10 of lactation had no significant effect on subsequent litter weights or mammary nucleic acid content. Interruption for 24 hr in both early and midlactation reduced litter weights and mammary gland RNA content but had no significant effect on weight of anterior pituitary, adrenal, or mammary gland DNA content. The results suggest that the effect of interrupted nursing is to reduce mammary gland metabolic activity without altering cell number and furthermore, that the effect probably occurs locally within the mammary gland.

A distinct band of lymphocytes was observed at the interface of subcapsular renal implants of C3H mammary tumors and kidneys of syngeneic C3H mice with sine-comitant and concomitant immunity and the allogeneic DBA/2J strain. This reaction was either absent or significantly less in nonim-munized C3H mice and the C3D2F₁/J hybrid. This information is interpreted as evidence against the immunologic nature of so-called allogeneic inhibition or hybrid effect as it relates to tumor growth.

Studies are reported on the ability of the hatchling of the lizard Calotes versicolor to engage in immunological reactions. The experiments demonstrate that even within the first 24 hr after hatching the lizard is able to mount an immune reaction against sheep red blood cells, in a manner not readily distinguished from adult animals.

The effects of norepinephrine and epinephrine (0.1 and 1.0 μg kg- 1 ) upon portal, arterial, and central venous pressures were measured during drug injections into the portal vein, vena cava, and hepatic artery of anesthetized dogs. The small dose of norepinephrine and epinephrine evoked maximal increases in portal pressure when injected into the portal vein. Except for the responses to portal vein injections, which remained unchanged, these effects were attenuated by splenectomy. The large dose of epinephrine brought about portal pressure increases by all three routes of injection; the maximal change occurred following vena caval injection. These effects were attenuated by splenectomy and by alpha adrenergic blockade. Beta adrenergic blockade potentiated specific portal pressure responses to epinephrine. Norepinephrine elicited the same directional responses as epinephrine but they were lesser in magnitude. The data suggest that portal pressure responses to injected catecholamines are secondary to splenic contraction as well as direct effects on portal venules. The emptying mechanism of the canine spleen appears to be mediated by alpha adrenergic mechanisms.

Technical assistance of Randall W. Barton is gratefully acknowledged.

The cow red cell membrane was demonstrated to possess a Mg-dependent adenosine triphosphatase (Mg-ATPase) and a Na-K-dependent ATPase (Na-K-ATPase), the activity of which was completely abolished by the presence of ouabain. The pH optimum, the optimum Mg^{2 +}:ATP concentration ratio, the K_m ATP for the enzyme, and the ouabain concentration required for inhibition of activity were similar to those of the membrane ATPase of human erythrocytes. The cow enzyme was also sensitive to the medium Na⁺:K⁺ concentration ratio. The cow red cell membrane, like that of other species, was confirmed to possess an enzyme system with the characteristics of a transport ATPase.

In an effort to elucidate the mechanism of the hypocholesterolemic effect of glutamic acid previously demonstrated in the Mongolian gerbil, several amino acid formula diets have been studied. The diets containing amino acids which could be readily converted to α-ketoglutarate were hypocholesterolemic, while the diets containing amino acids not readily converted to α-ketoglutarate were not. The diet containing α-ketoglutarate was the most effective.

No significant correlation could be made between the serum cholesterol and the hepatic squalene or cholesterol content. However, the animals with the lowest serum cholesterol levels had correspondingly low hepatic cholesterol levels.

The authors thank Miss Carole Williams for her technical assistance and Miss Shannon Smith for the preparation of the manuscript.

In vitro studies of lymphocyte reactivity to mitogens, soluble antigens and homologous cells were performed by diluting whole blood with culture medium and using 24-hr labeling with ³H-thymidine. Sensitivity, reproducibility and correlations with delayed skin test responses of the blood donors were superior to those obtained with conventional techniques using lymphocyte concentrates. At appropriate reactive cell densities, supplementation of the culture medium with agents such as fetal calf serum has not been necessary. This simplified technique offers considerable savings in effort, cost and volumes of blood required.

Several recent studies have shown that N ⁶, O ²-dibutyryl-3',5'-cyclic adenosine monophosphate (dibutyryl cyclic AMP) can transform cells of various tumor lines so that they take on a more normal structural appearance. The initial observations were made by Hsie and Puck (1) using Chinese hamster ovarian tumor cells and by Johnson, Friedman, and Pastan (2) using rat sarcoma and neoplastic mouse fibroblast lines.

Similar observations have been made with tumors of nervous tissue origin. For example, morphologic changes in glioblastoma cells under the influence of dibutyryl cyclic AMP have been reported by Perkins and MacIn-tyre (3), and neuroblastoma cells have been shown to differentiate, as judged by neurite extension, following exposure to dibutyryl cyclic AMP (4, 5).

An effect of dibutyryl cyclic AMP on the morphology of nonneoplastic mammalian nervous tissue has not yet been reported. The studies presented below reveal that treatment of fetal mammalian sensory ganglia with dibutyryl cyclic AMP produces increased neurite outgrowth and that this outgrowth mimics that observed in cultures exposed to nerve growth factor (NGF). After the work reported here was completed, we learned that Roisen et al. (6) had made similar observations with chick embryo ganglia.

Methods. Gasserian ganglia from rat or mouse fetuses, 17-19 days in utero, were placed on collagen-coated coverslips in Max-imow double coverslip assemblies, as described earlier (7). A drop of F-10 feeding medium (Gibco) containing 25% fetal calf serum and 750 mg of glucose/100 ml was added, and the cultures were incubated at 35° for 24 hr. Dibutyryl cyclic AMP (Sigma) was prepared as a 50 mM stock solution in F-10, frozen in aliquots, and thawed and diluted for use as needed. All growth media containing dibutyryl cyclic AMP contained fetal calf serum and glucose at the concentrations given above.

In PHA-induced blastogenesis of mouse spleen lymphocytes cultured in a medium supplemented with serum, the specific response obtained with any combination of these three variables was governed by their relative concentrations, indicating that the three factors interacted with each other.

The PHA-binding function of serum exerted a buffering effect which was expressed as a suppression of the activity of very low concentrations of PHA and as a tolerance of levels of PHA which otherwise would have been inhibitory. The PHA-binding caapcity of lymphocytes resulted in a similar effect.

A medium containing no supplement supported a moderate degree of blastogenesis. Supplementation with either serum albumin, gelatin or methylcellulose provided a chemically defined medium which supported blastogenesis nearly as well as with whole serum. However, cells in such media were very sensitive to the toxic effects of PHA in concentrations above a sharply defined maximum.

The technical assistance of Mrs. P. Buzzerd and Mr. R. Varga is gratefully acknowledged.

The effects of intra-arterial infusion of dopamine (3 μg/kg/min) and isoproterenol (0.15 μg/kg/min) on renal blood flow (RBF) and renal venous plasma renin activity (RVR) were studied in eight anesthetized dogs, before and after propranolol. Dopamine caused an increase in RBF from 201±21 to 238±39 ml/min (p < .01); and a decrease in RVR from 470±130 to 333±115 ng/100 ml (p > .05). Isoproterenol increased RBF from 202±28 to 214±39 ml/min (p < .05) and augmented RVR from 470±130 to 1192±395 ng/100 ml (p < .01). Propranolol did not alter the effects of dopamine on RBF and RVR, but effectively blocked the isoproterenol-induced vasodilation and renin secretion.

This demonstrates the unique action of dopamine on renin secretion. The presently available evidence suggests that there probably are no specific adrenergic receptors directly mediating renin secretion by the kidney.

The applicability of rapid decompression as a method for homogenization of liver in the preparation of a microsomal fraction has been studied. It was shown that this method is more efficient and probably less variable than homogenization with a Teflon pestle and glass tube. Increased efficiency was related to more complete cell disruption and probably to the production of smaller microsomal particles. The specific content and activity of components of the mixed function oxidase system of the microsomal fraction were similar to those produced with pestle and tube.

Supported by Grant No. HD-03074, Institute of Child Health and Human Development, NIH.

Polyriboinosinic·polyribocytidylic acid [poly(I)·poly(C)] complexed with poly-D-lysine was studied as a prophylactic and therapeutic material in mice inoculated with herpes simplex virus (HSV). With the treatment as described, a higher proportion of mice treated with poly (I)·Poly (C) [poly-D-lysine] survived than when treatment consisted of poly (I)·poly (C) alone, or phosphate buffered saline (PBS) alone, following subcutaneous (sc) inoculation with HSV. This enhanced protection was still detectable if treatment was begun as late as 4 days following sc inoculation of HSV, at which time an occasional mouse was showing signs of early central nervous system illness. The treatment regimen did not appear to adversely alter the immunological responsiveness of sc infected animals as judged by subsequent ic reehallenge with HSV. No mortality was observed as a result of repeated injections of 100 μg of poly (I)·poly (C) complexed with poly-D-lysine.

To obtain information on inhibition of adenovirus transformation by AAV, cultures of hamster embryo cells were inoculated simultaneously with constant amounts of simian adenoviruses SA7 or SV11, or with human adenovirus type 12 and with varying multiplicities of AAV-1. Induction of transformed cell foci by SV11 or Ad12 was inhibited by more than 80% when 9 to 10 plaque inhibiting units (PIU)/cell of AAV-1 were added. The amount of AAV-1 necessary to reduce transformed cell foci by 50% ranged from 1.5 to 2.5 PIU/cell for SV11 and Ad12 to 5 to 7 PIU/cell for SA7. Analysis of the dose-response data indicates that 1 unit of AAV-1/cell is sufficient to inhibit transformation by Ad 12 and SV11. The relative resistance of SA7 transformation to inhibition seems best explained as a requirement for more than 1 inhibiting unit/cell of AAV-1.

Crude extracts of normal human prostate glands obtained after papain digestion contained 4.83±4.04 mg of hexuronic acid/g of dry tissue. Aliquots of the extracts were fractionated on columns of Dowex-1 ×2 with solutions of increasing NaCl concentrations. Electrophoresis of the chromatographic fractions showed the presence of a band migrating like hyaluronic acid (HA) in Fractions III to ×. The component of this band in Fractions III and IV A is considered to be HA because it was depolymerized by streptococcal hyaluronidase. The corresponding band in Fraction IV B to × is referred to the HA-like component because it was depolymerized by testicular hyaluronidase but not by streptococcal hyaluronidase.

Additional aliquots of the crude extracts were separated into fibrous-like (FP) and flocculent (FPS) precipitates by the addition of ethanol. FPS contained 5 times more GAG than FP. In both precipitates, dermatan sulfate was the most abundant GAG. Spectrophotometric measurements indicated the presence of protein and nucleic acid in FP and FPS.

We are indebted to Dr. Nicola Di Ferrante for his advice in the preparation of this manuscript and for the determination of sulfoaminohexose. The participation of Victor Vlahakos during the initial phase of this work is appreciated.

SDS (5 mM) removes histologically detectable cells from skin previously freed of ground substance with isotonic saline. Such extracts correspond in composition to those produced by trypsin.

The staff of the Surgical Pathology Service, Vancouver General Hospital, assisted in the collection of specimens. Mrs. B. Barkocsy prepared the histological sections.

The immunogenicity of three different conjugates with DNP as hapten and BSA as carrier were compared. Guinea pigs immunized with DNP coupled to amino groups or carboxyl groups responded with high anti-DNP and low anti-BSA antibody production, while those immunized with DNP on tyrosyl residues gave the reverse response. DNP coupled to amino and carboxyl groups produced much more drastic effects on native BSA determinants as measured by precipitin test and delayed sensitivity reactions. It is suggested that haptens coupled to these groups are on the exterior surface of the protein where they are more likely to contact receptors of immunocompetent cells and more likely to alter surface configurations of the native molecule.

This investigation was supported by a grant from the U.S. National Institutes of Health.

Different populations of guinea pig lymphoid cells demonstrate marked heterogeneity in their ability to elaborate an in vitro chemical mediator of cellular immunity, migration inhibition factor. A concentration of 2.5% lymph node cells obtained from animals demonstrating cutaneous reactivity to tuberculin protein effectively inhibited the migration of peritoneal exudate cells from normal donors. Thymic and bone marrow cells were relatively ineffective in this assay; no significant reduction in the area of migration was observed with cell concentrations as high as 20%. The presence of a second but numerically small population of immunologically active cells in both these central lymphoid organs was suggested by the inhibition of migration in assays using 25% cells from sensitized donors. Results of these in vitro assays were further confirmed by in vivo cell transfer experiments.

It appears that vitamin B₁₂ binders in normal rhesus monkey serum differ from human TCII in chromatographic behavior on a DEAE-cellulose column and from MPPB and TCI in molecular size, as determined by gel filtration on a Sephadex G-200 column.

Fig. 3. Radioactive profile obtained by gel filtration on Sephadex G-200 column of fraction eluted from rhesus monkey serum from DEAE-cellulose column with 0.4 M sodium phosphate buffer. Arrow indicates elution volume of TCI from normal human serum.

Bovine amniotic fluid or fetal calf serum markedly reduced the cell alterations which were produced in Wistar/Furth rat embryo cell cultures by a rat C-type virus (BV-1) isolated from chemically-induced mammary tumors. At the same time, higher titers of infectious BV-1 were produced as early as 2 days post-infection in cultures maintained on medium supplemented with bovine amniotic fluid or fetal calf serum as compared to cultures on medium supplemented with newborn calf serum. A similar stimulatory effect on the growth of BV-1 in these cultures was exerted by estrogen (95% natural) which was dose dependent, 1.0-2.5 μg/ml being the most effective dose under the conditions employed. Progesterone had no stimulatory effect on the replication of BV-1 and appeared to be suppressive at higher dose levels.

The technical assistance of Hannelore Asmussen and Margaret F. Peek is gratefully acknowledged.

Test tube cultures of normal mouse spleen cells set up as for mixed leukocyte culture, but with antigenic RBC, and fed once with additional medium without disturbing the button of cells, were found to contain antibody producing cells, as indicated by the ability of such cells to produce hemolytic antibody plaques when plated against the RBC. In the case of sheep RBC as antigen, the number of PFC increased to day 7, when a level of 90-110 per 10⁶ starting spleen cells was reached, in comparison with 18-24 PFC in control cultures, without antigen. With burro RBC such results were also obtained, with somewhat lower levels of PFC produced in antigen-containing cultures, but substantially lower background levels. Since dispersion of the cells at re-feeding caused failure of appearance of the PFC, a condition for the production of these was presumably the formation of a microenvironment within the button of cells in the bottom of the tube.

RES cells were infected with the RES₁₆ variant of the WSN strain of influenza A₀ virus and the culture medium changed at frequent intervals; five lines of cells were established in which infection was sustained for periods of 126 to 146 days. Each line of persistently-infected cells showed recurring cycles of cell degeneration and repopulation; these cultures did not remain in equilibrium with the virus indefinitely, however, and ultimately degenerated.

The presence of virus in the RES culture fluids was easily demonstrated for periods of 27 to 105 days in various cell lines using as the indicator intraallantoic inoculation of eggs with hemagglutination by the allantoic fluids. Subsequently this method failed to demonstrate virus, although the continued presence of CPE suggested that the cultures were still infected. By using as indicator of the presence of virus the production of CPE in normal RES or PS cultures inoculated with fluids from the infected lines, it was found that influenza A₀ virus was still present in the fluids, and that it was able to propagate in eggs without producing detectable hemagglutinins. Only modest infectivity titers were found in the nonhemagglutinating allantoic fluids; however the original WSN and WSN/RES₁₆ variants at the same infectivity titers readily produced detectable hemagglutination. This fact suggests that the virus had not simply lost its ability to multiply in eggs to titers sufficient to produce hemagglutination, but that at least part of the infectious virus did not hemagglutinate. Spontaneous reversion to the original hemag-glutinating state was noted in one infected line (VL-V) during the last days of infection, just before complete degeneration of the culture, and in two cases reversion followed serial passages in eggs (VL-V-63) and in eggs and PS cells (VL-IV-106).

A study was undertaken in dogs to determine the role of posture on regional pulmonary arterial perfusion and to establish a set of normal values for lobar blood flow in various body positions. Relative flow through each of the seven lobes was measured by in vitro tissue counting in five body positions following intravenous injection of albumin macroaggregates. In each position a definite flow gradient was found which favored the dependent lobes. Quantitative results are presented as are values for lung and lobar weights.

The technical assistance of Mrs. Mary Lee Griswold is acknowledged.

Butylated hydroxytoluene was administered intraperitoneally to two strains of mice in the dose range 0.004-2.5 g/kg. Histological examination of the lung tissue revealed thickened interalveolar septa, congestion and a general pattern of tissue disorganization at doses of 0.25 g/kg and above. The observed effects are apparently reversible and have a threshold in the vicinity of 0.04 g/kg.

The effects of prostaglandin (PG)E1, dibutyryl cyclic AMP, 8-benzyl-thio-cyclic AMP, inhibitors of phosphodiesterase [papaverine and 4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone], and X-irradiation on two neuroblastoma clones, NBA2(1) and NBDB^- were studied. In the NBDB^-clone PGE1 was much more effective in causing morphological differentiation than any other differentiating agents. In the NBA₂(1) clone PGE1 was as effective as the other agents. Adenylate cyclase of both clones may be sensitive to PGE1. The phosphodiesterase level in clone NBA2(1) is probably inhibited by other differentiating agents but this enzyme level in clone NBDB^- either is too low to be of any significance or is insensitive to phosphodiesterase inhibitors. In clone NBDB^- the biological property of the cell may be more important in initiating the morphological differentiation than the interaction between the cell and the surface of the culture dish.

The chromosome analysis of three continuous cell lines derived from mosquitos showed that A. aegypti (Singh) and A. albopictus lines had a complement formed by three pairs of metacentric chromosomes. Moreover, both lines exhibited chromatid discontinuities ranging from secondary constrictions to chromosome breakages with occcasional chromosomal rearrangements. These discontinuities were nonrandomly located and probably produced at a steady rate in every cell generation. On the other hand, the A. aegypti (Peleg) line had a complement formed by 7 chromosomes in all the metaphases analyzed. Chromosome morphology frequently varied from cell to cell and occasionally the homologue identification was difficult. The analysis of somatic pairing figures showed that the chromosome complement of this line is probably the result of chromosomal breakages and rearrangements involving different chromosomes.

Acetazolamide sodium, when incorporated in the diet at concentrations of 0.05 and 0.2% and pair-fed to female Car-worth rats for 20 days, had no significant effects on plasma Ca, Na, and K levels, the defatted dry weights of the humerus, or the renal Ca, Mg, K, Zn, and P levels. Growth rate was moderately and progressively inhibited with increasing dosage. At the 0.5% level, which other studies have shown to prevent the development of disuse osteoporosis in rats, only two parameters, plasma Ca and renal Ca, were affected. Plasma Ca was increased from 9.7 to 10.6 mg/100 ml (p < .001); renal Ca content was increased from 0.31 to 0.58 mg/g of dry weight of kidney (p < .025). It is concluded that acetazolamide fed to female rats for a 3-week period has no major toxicity related to mineral metabolism at dietary levels equal to and below that level which has been shown to prevent disuse osteoporosis in rats. These findings further enhance the potential of acetazolamide as an orally effective therapeutic agent for the control of osteoporosis of disuse in man.

The author acknowledges the technical assistance of Richard Lane.

Three purified Parvoviruses (H-1, H-3 and RV), treated with various proteolytic enzymes, showed chemically-induced changes in their hemagglutination pattern only after exposure to papain which enhanced the values found. This increase was probably due to a dual effect of the papain: one on the virus itself by removing bound contaminants and, two, on the red cell membrane, probably by reducing the negative charge. Although the HA titer of virus was altered by papain treatment, no change in the SDS-acrylamide electrophoretic pattern of the capsid proteins or in infectivity was observed.

The authors thank Miss Barbara Madden for technical assistance.

Daily indirect systolic pressure was measured, without anesthesia, for 3 months to determine the relationship between variability and height of pressure in individual normotensive and spontaneously hypertensive (SHR) rats. Average systolic pressure correlated with its standard deviation indicating the higher the pressure the greater its variability. The empiric dividing line for hypertension in the rat of 150 mm Hg was reexamined and found appropriate. No SHR rat had an average weekly pressure less than 150 mm Hg and 28% of the controls had at least 1 weekly average above 150 mm Hg; we have termed these rats as “borderline hypertensives.” Daily variability of pressure of borderline hypertensives was greater than the remaining controls. Therefore, borderline hypertensives have greater lability of arterial pressure.

The authors gratefully acknowledge the statistical advice of Drs. John McCoy (of the Veterans Administration Southern Research Support Center, Little Rock, AR) and Donald Parker (of the University of Oklahoma Medical Center), the technical assistance of Mrs. Janice Pfeffer and Mr. Philip Barrett, and the secretarial work of Mrs. Irene Smith.

Intravenous injections of SQ 20, 881, a competitive inhibitor of the angioten-sin-converting enzyme, suppressed the pressor and reduced renal blood flow responses initiated by the infusion of angiotensin I (50 to 100 ng/kg/min) into anesthetized dogs. Both the degree and duration of inhibition by SQ 20,881 were proportional to the dose of SQ 20,881 up to 300 μg/kg; this dose produced a maximal inhibition of about 80%, and an inhibition of at least 25% that lasted for approximately 2 hr.

Characteristics of a model for the study of diffusional movement in human red blood cells have been examined. Tracer influx of sodium was accelerated in red cells with high internal sodium content. This occurred both in fresh and stored human red blood cells. These results were not due to an alteration in electrical potential across the red blood cell membrane and uptake of a non-polar tracer was not enhanced by high internal sodium content. The data seem best explained by an exchange diffusion process.

I am grateful to Professor R. Whittam for suggesting the model and to both Professor Whittam and Dr. Neal S. Bricker for reviewing this manuscript. Mr. Foster Harris provided valuable technical assistance. This work was supported by U.S. Public Health Service Grant No. AM 14586-01.

Studies were undertaken to examine the effects of zinc on the repair of the wound in the epithelium. Biopsy specimens obtained from zinc-deficient rats showed a reduced uptake of ³H-thymidine when compared with controls, as shown by liquid scintillation and autoradiography. With wounding, proliferation as indicated by the labeling index increased about twice normal for zinc-supplemented rats, but 3.5 times for zinc-deficient rats. However, despite the increased proliferative rate, repair of wounds in deficient rats was always slower than in zinc-supplemented animals.

Concentrations of luteinizing hormone (LH) were determined in serum samples collected from the femoral vein of rhesus monkeys at 5-15-min-intervals following administration of ovine stalk-median eminence extract (SMEE). Following iv injections of .02-.32 eq SMEE, a dose-related response was observed in serum LH. A marked increase in LH of 3-50 × baseline occurred within 5 min of SMEE injection. This rise in serum LH returned to baseline within 60-90 min. Chronic iv infusion of SMEE for 5-6.5 hr elicited an immediate rise in serum LH and a plateau which lasted until the infusion ceased. The increased concentrations of serum LH prompted by equivalent dosages of SMEE given at Days 5-7, 9-13, or 19-23 of the menstrual cycle or following injection of 0.5 mg of progesterone sc on days 6-12 were comparable. Antiestrogen, U-11, 100A injected sc at 0.5 mg daily on days 7-12, significantly reduced the LH released by SMEE injections. The decreased response to SMEE stimulus following antiestrogen, but not progesterone injections, suggest these compounds influence LH release via different mechanisms. In control, progesterone- and antiestro-gen-treated monkeys the iv injection of SMEE did not induce ovulation, whereas two of three “progesterone-blocked” females did ovulate following a 5-6.5 hr chronic infusion indicating the duration of LH elevation was critical.

CG 603 is a cyclic imide. Some cyclic amides like thalidomide are central nervous system depressants (1). Barbiturates block ovulation in the rat (2, 3), and recently this laboratory reported that a single injection of Na pentobarbital (Pb) at 1 pm on the day of proestrus prevented the pronounced rise in serum LH on the late afternoon of proestrus (4). Serum prolactin was elevated 30 min after the Pb injection, but the late afternoon surge of serum prolactin was blocked completely. It was of interest, therefore, to determine the effects of a single injection of CG 603 given on the early afternoon of the day of proestrus on serum prolactin levels.

Materials and Methods. Mature, 3 to 4 month old virgin female Sprague-Dawley rats (Carworth Farms, Portage, MI), weighing 210-245 g each, were housed in a temperature controlled (25±1°) and artificially illuminated (lights on from 7 am to 9 pm daily) animal room. They were given Wayne Lab Blox pellets and water ad libitum. At least two consecutive estrous cycles were followed on all rats by examining daily vaginal smears, and only rats with regular 4 or 5 day cycles were used.

Individual blood samples (.5 to 1.0 ml) were removed by heart puncture under light ether anesthesia at 12 noon on the day of proestrus. This method of blood collection was found not to alter normal serum prolactin values in rats (4). A dose of 5, 10 or 25 mg CG 603 2 /100 g body wt was injected ip in .85% NaCl at 12:30 am, and the rats were bled at 1, 2, 3 and 5 pm. Control rats were injected only with .85% NaCl. The animals were killed by guillotine immediately after the 5 pm bleeding and their pituitaries were removed and weighed.

A study was made of the effects of ergocornine methanesulfonate (ECO) administered to normal cycling rats on the afternoon (16.30 hr) of D₂ (day before PE) and on the morning (10.30 hr) of PE on prolactin content and concentration of the pituitary gland and on ovulation.

Pituitary prolactin content and concentration on the afternoon of PE in the animals treated with ECO were about 3 times higher than those obtained in the saline-treated control animals. When animals treated with ECO were killed on the morning of estrus, the level of prolactin content and concentration was very high and the level was not significantly different from that obtained in the saline-treated control animals killed at the same stages of the estrous cycle.

Ovulation occurred in 5 out of 6 animals treated with ECO. No significant difference was obtained in the average number of ova shed between the ECO treated animals and the saline-treated controls.

A possible physiological role of prolactin release around the critical period was discussed.

The effect of L-dopa and isoproterenol in DNA, RNA, and protein synthesis was measured in the salivary glands, brain, and liver of the mouse. These data indicate that L-dopa does not cause gross changes in nucleic acid and protein synthesis.

We thank Dr. George C. Cotzias for stimulating this work and Miss Roberta Klimaski for technical assistance.

In the liver of castrated rats a significant decrease of total folate coenzymes and, in particular, of 10-HCO-H₄folate and H₄folate was observed. Treatment with testosterone normalizes the folate content of these structures. The decrease in folate coenzymes is evident in the prostate but here too, hormone administration restores the normal folate content. Castration does not significantly alter the content or distribution of folate coenzymes in seminal vesicles.

We are indebted to Mr. Roberto Laffi for excellent technical assistance.

Phthalyl amino acid esters of fluorescein were synthesized and characterized. They were tested (in vitro) for use as possible fluorogenic substrates for LAP. The rates of enzymatic hydrolysis of these compounds were compared with those of some commercially available fluorescein esters.

The lactogenic activity of human chorionic somatomammotropin (HCS) 2 has been assessed in mature, nonpregnant female Rhesus monkeys. When HCS (25 mg twice daily) was administered alone no changes in mammary gland morphology were observed. However, when HCS was administered for 2 weeks to animals previously treated for 2 weeks with 17β-estradiol (5 mg/day) plus progesterone (20 mg/day), histologic changes in mammary tissue suggestive of milk production were observed in the mammary glands. In addition, HCS alone neither altered mammary gland morphology in monkeys with well-established lactation nor affected the rate of weight gain in their suckling monkeys. Thus, in primates, HCS secretion during pregnancy appears to facilitate progesterone and/or estrogens in preparing mammary tissue for lactation.

The author wishes to thank Mrs. Sally A. Wells and Mrs. Susan Ohaver for their technical assistance.

Terminal chorionic villi (CVI) were prepared from the rami chorii of the middle zone of the placenta, and were purified by forcing them through an 80-mesh followed by washing through a 325-mesh wire cloth. Trophoblast basement membrane (TBM) was obtained from CVI pellets by sonication in 1 M saline followed by extraction with 0.3 M acetic acid, and treatment with deoxyribo- and ribonuclease. The final preparation, on electron microscopy, was free of connective tissue contaminants and of plasma membranes.

Explants were taken from central and peripheral regions of the anterior pituitary, cut into small (1 × 1 × 2 mm) or large (1 × 1 × 10 mm) pieces, and incubated by continuous superfusion in TC-199 with 0.035 or 0.224% NaHCO₃ for 6 hr. Explants from cows released 137% more LH than those from steers but 65% less prolactin. Average LH concentration 2 and 6 hr postculture was 49% greater in explants from cows. Prolactin release but not LH release was greater into medium containing 0.224% than 0.035% NaHCO₃ and postculture concentration in the pituitary tissue inversely reflected this pattern. Release and postculture tissue concentration of LH and prolactin was greatest for explants from the central and peripheral pituitary regions, respectively. Explant size did not influence release of prolactin or LH but release of both hormones was increased significantly during the incubation period.

Fibroblasts readily adhere to polymerizing fibrin, but not to fibrinogen or fully polymerized fibrin. Conditions for optimal interaction are similar to those known for the platelet-fibrin interaction. Fibroblasts retract fibrin formed by thrombin but not by reptilase. Fibrin retraction induced by fibroblasts is inhibited by agents inhibiting clot retraction induced by platelets.

We thank Dr. L. Prevec for generous supplies of fibroblasts and Messrs. A. F. Senyi, K. L. Wong and T. Bistricki for skilled technical assistance.

The formation of a new sinusoidal marrow in a heterotopic autologous marrow implant requires the intermediation of bone. This conclusion follows from the intimate association of developing marrow tissue and newly formed bone in the implantation site. It has been shown that there is a sigmoidal relationship between the evolution of a medullary cavity and ossicle volume. Moreover, the mean relative medullary cavity size and ossicle volume for each stage of marrow development from hemocytoblasts to a sinusoidal structure conforms to this relationship. Hematopoietic foci emerge during the period of rapid bone resorption. A fully developed marrow may occur in a small or large ossicle provided that the medullary cavity represents a critical fraction of the total volume. It is suggested that processes of bone resorption may contribute to the formation of an appropriate microenvironment and/or provide a source of appropriately modulated progenitor cells. Significantly, bone plays a necessary role in the genesis of an organized marrow in an evacuated medullary cavity as well as in a heterotopic marrow implant.

The authors thank Miss G. Dunn and Mrs. M. Miller for technical assistance.

The results of our experiments indicate that tritiated thymidine, after penetrating the inner shell membrane of the chick egg, is incorporated into those embryonic cells which are synthesizing DNA. The amount of radioactive tracer absorbed is a function of the age of the embryo, the concentration of the isotope dose and the length of the embryos' exposure to the isotope. The uptake of isotope by the embryo may be reproducibly determined and thus the process may be used to determine the effect of various agents on the DNA synthesis of embryonic chick cells. Using this system we are presently studying the effect of ionizing radiation on developing chick embryos.

We gratefully acknowledge the constructive criticism and advice of Dr. T. C. Evans.

Bilateral adrenalectomy of 300±20 g male Holtzman rats resulted in extreme sensitization to endotoxin shock produced by iv Salmonella enteritidis Boivin lipopolysaccharide. Within 60 min after adrenalectomy, resistance to endotoxin as reflected in LD₅₀ values decreased from 800 μg/100 g to 0.59 μg/100 g and by 168 hr after adrenalectomy the LD₅₀ had diminished to 0.25 μg/100 g. Within 15 minutes after adrenalectomy endotoxin resistance had diminished to a LD₅₀ of 50 μg/100 g. Liver homogenates from either acute (60 min) or chronic (168 hr) adrenalectomized and sham-operated control rats were assessed for endotoxin inactivating ability using a lead-sensitized rat bioassay. The ability of liver to detoxify endotoxin was not significantly altered by either acute or chronic adrenalectomy. Endotoxin inactivating ability also was measured in livers removed from either control or chronic adrenalectomized rats in the terminal phase of endotoxin shock; no appreciable loss of hepatic endotoxin inactivating ability was manifested in either group as compared to control or chronic adrenalectomized rats which were not exposed to endotoxin shock. The data suggest that the defect in host resistance to endotoxin imposed by adrenalectomy is probably not due to an impairment of the hepatic intracellular system for endotoxin inactivation.

A microhemadsorption method is described for measuring the interaction between concanavalin A and single transformed or untransformed cells without removing them from the substrate. When indicator red blood cells were coated with Con A, their adherence to SV3T3 cells was only 2-4-fold greater than to 3T3 cells. On the other hand, when the two cell lines were coated with Con A, there was a very marked difference in the ability of the lectin on each to adsorb uncoated red cells.

Positive free-water clearance, signifying production of hypotonic urine, is commonly observed in well-hydrated owl monkeys following transfusion of shed blood after prolonged hemorrhagic hypotension. This investigation has examined the nature of this derangement of the urinary concentrating mechanism by experimental reduction of glomerular filtration rate by partial aortic constriction during this phase of experimental shock, and in turn, diminution of solute and water load to the nephron. When the resulting reduced volume was delivered to the final concentrating segment, removal of only a small portion of the residual volume was necessary for the elaboration of a urine of higher solute concentration, approaching that of plasma, so that U/P _osm which was well below 1.0 immediately after transfusion was restored close to unity.

Using swimming stress to evaluate the effects of experimental conditions, the maximal oxygen consumption seems to be a preferable measurement to swim time to exhaustion. Mice swimming to exhaustion with 7% weight load in 36° water exhibited similar Vo_2max values for their first and second exposures. However, swim time more than doubled for the second exposure. Almost no correlation existed between Vo_2max and swim time. This suggests that swim time is dependent on several factors, both physiological and psychological, other than aerobic power.

Mean maximal Vo₂ for mice with 0 and 5% loads was about 140 ml/kg/min. This response was achieved well within the 5-min duration of the test and was consistent for three trials. At 7% load, Vo_2max was lower as difficulty was encountered remaining at the surface. This Vo₂ test appears suited for experimental designs which require groups of mice to be tested several times. The duration of the test is relatively short, and no previous training is necessary. In addition, because of the high relative Vo₂ of mice, they seem to be well suited for experiments which may alter the ability to pick up, deliver and consume oxygen.

The authors express their appreciation to Mr. Joseph F. Pitt for his assistance.

In the isolated, blood-perfused dog heart daunomycin (50 mg) or adriamy-cin (50 mg) induced significant increases in coronary perfusion pressure. This effect appeared to be related to the conversion of the parent compounds to aglycone metabolites similar to daunomycinone. Pretreatment of the heart with agents blocking the vasoactive actions of epinephrine, histamine, serotonin or acetylcholine did not prevent the alteration in coronary vascular resistance. In contrast, 100 mg of either EDTA or ICRF 159 prevented the marked increase in perfusion pressure and reduced the formation of aglycone metabolites. Chelation is a common property of the 2 agents suggesting that removal of certain cations may alter metabolism and reduce the toxicity of adriamycin and daunomycin.

Seven synthetic peptides that correspond to compounds found in the venom of Bothrops jararaca were studied in normo-tensive, urethanized rats infused with pen-tolinium. When administered stat iv, the peptides inhibited pressor responses induced by angiotensin I but not those induced by angiotensin II. This activity was most likely the result of inhibition of angiotensin-converting enzyme. The peptides appeared to differ from each other with respect to potency and duration of action. The 3 test peptides injected im and sc displayed rapid, effective absorption by those routes.

Three peptides tested stat iv in normoten-sive; urethanized rats augmented the vasodepressor effect of bradykinin to different degrees.

A method was developed that permits rate zonal and isopycnic centrifugation in a zonal rotor in a single run. This procedure effected a high degree of purification of enveloped viruses in a relatively short time.

The authors thank Mr. G. Göttlicher for his assistance. The work was supported by the Sonder-forschungsbereich 47, Virologie.

Binding tritiated pteroylglutam-ic acid to cow or to goat milk protein was associated with a substantial reduction in its absorption from rat jejunum, while the ileal absorption increased markedly. There was no difference in the amount of folate absorbed whether it was bound to cow milk or to goat milk. Despite the fourfold higher folate binding capacity of goat milk than of cow milk, there was no difference in the amount of folate absorbed after their administration. Folate depletion or folate overload had no effect on the amount of the milk-bound folate absorbed, while such treatments decreased the absorption of free folate.

Turtle and duck pituitaries were fractionated and preparations of purified growth hormone (GH) were obtained. Phenylalanine and leucine are present as N-terminal residues in both species of GH and the amino acid composition of both is similar. Studies by agar-gel diffusion and radioimmunoassay employing antirat GH serum and antiturtle GH serum showed that the purified GHs were identical to the immunoreactive material in pituitary extracts.

We are grateful to Miss Eleanor Lasky, Miss Laurie Hidekawa, and Mr. Dan Viele for their skilled technical assistance. We thank Dr. Stanley Ellis for making available the highly purified rat and rabbit GH and rat prolactin preparations; the Endocrinology Study Section of the National Institute of Arthritis and Metabolic Diseases, for supplying us with ovine prolactin. Some of the turtle pituitaries were made available to us by Dr. Paul Licht. We are indebted to Doctors Selna Kaplan and Melvin Grumbach of the Department of Pediatrics for the iodinations of rat GH.

Functional interaction between histone and RNA was studied using Nelson mouse ascites cells. Liver RNA was capable of stimulating RNA synthesis and arginine-rich histone inhibited it. The potency of low molecular weight fraction was superior to the high and whole RNAs, but inferior to the nucleolar RNA. Poly I-C was unable to lift the RNA synthesis to a level above the controls.

Intracellular distribution of ¹²⁵I-histone was traced by high resolution autoradiography. The predominant nuclear uptake of histone coupled with early work on RNA uptake provided evidence for the in situ action of histone and RNA. The RNA-stimulated RNA synthesis was sensitive to actinomycin D. From studies on the action of RNA versus histone, it was concluded that the action of RNA was mediated by arginine-rich histone.

Fig. 10. The response of the histone pretreated cells (H) to RNA (HR) and RNAse-digested RNA (RNAse). The concentration of histone and RNA is same as Fig. 9 (³H-uridine triphosphate as precursor).

The effect of antiarthritic drugs on a sulfhydryl-disulfide interchange reaction between rat serum sulfhydryl groups and dithiobisnitrobenzoic acid was determined. Gold sodium thiomalate and gold thioglucose produced potent, dose dependent inhibition of the interchange reaction. Thus, the interchange reaction utilizing rat serum appears to be a sensitive and quantitative procedure to measure the sulfhydryl group reactivity of potential antiarthritic agents.

Nonsteroidal antiinflammatory drugs (phenylbutazone, indomethacin and acetyl-salicylic acid) administered in vitro or in vivo, accelerated the interchange reaction in diluted rat serum. The mechanism by which these agents produce this effect and its relevance to their therapeutic activity is not known at the present time. In contrast, the in vivo administration of Prednisolone or chlo-roquine diphosphate did not significantly alter sulfhydryl group reactivity in rat serum.

The results of our investigation suggest that the sulfhydryl-disulfide interchange reaction between rat serum sulfhydryl groups and dithiobisnitrobenzoic acid is a useful procedure in establishing the profile of potential antiarthritic agents.

The authors express their appreciation to Sandra Davidheiser, Michele DeCoste, Joseph Flagiello, Jr., and Sophronia Morgan for valuable technical assistance.

The ability of a variety of inhibitors and substrates to stabilize liver microsomal glucose-6-phosphatase (D-glucose-6-P phosphohydrolase, EC 3.1.3.9) against thermal inactivation has been investigated. Studies were carried out by incubating microsomal suspensions in sodium cacodylate buffer at pH 6.5 for 20 min at 40°, either in the absence or presence of various concentrations of certain inhibitors or substrates, cooling; and then assaying for enzymic activity. Significant protection was afforded by the inhibitors citrate, bicarbonate, P₁, and ammonium molybdate, and by the phosphoryl substrates glucose-6-P, PP₁, ADP, and ATP. d-Glucose had little effect, and inhibition by 1,10-phenanthroline was irreversible. These observations support the reaction mechanism previously suggested for this multifunctional catalyst, and indicate the advisability of including phosphoryl substrates or certain competitive inhibitors as stabilizers of the enzyme preparations during future attempts at purification.

Electrocardiograms obtained during exercise on a treadmill and on a bicycle ergometer have shown that the relation between the QT and RR intervals can be represented by the equation QT = K ⁺ 0.5 RR when K has a value of 70 msec. This equation was the same in trained and untrained subjects. During recovery from exercise the RR intervals lengthened much faster than the QT intervals.

The alveolar pCO₂ increased during the exercise while QT shortened. Immediately after exercise the alveolar pCO₂ continued to increase at a time when the QT was no longer shortening.

While precise patterns of efferent innervation of the heart have been extensively investigated, a large fraction of the studies have been made in the dog, cat, rabbit and a few other domestic animals with little or no information upon the primate. We have recently described the anatomical distribution of the autonomic cardiac nerves, together with functional responses to their electrical excitation in the baboon (1, 2). The emphasis of these reports, however was almost exclusively upon epicardial innervation. In subsequent experiments, after developing techniques for studies of endocardial structures in the dog (3, 4), strain gauge arches and/or intramyocardial pressure transducers were applied to the interventricular septum and papillary muscles in the intact baboon heart. The present report illustrates and compares changes in contractile force and in intramyocardial pressure development within the septum and epicardial portions of the left ventricle. To our knowledge, these data represent the first such direct expeirimental approaches to determination of endocardial functions in the primate heart.

Methods. Walton-Brodie strain gauge arches were carefully sutured to the endocardial surfaces of the interventricular septum and/or to the longitudinal surfaces of the papillary muscles of the left ventricle through left atriotomy, while the animal was on compete cardiac bypass and the heart was in fibrillation, in seven baboons (Papio anubis). After installation of the transducers, the animals were removed from the bypass pump with defibrillation and restoration of in vivo circulation. The small branchings of both sympathetic and parasympathetic cardiac nerves distal to the stellate and middle cervical ganglia and the cervical vagosympathetic trunk were carefully isolated and electrically stimulated using either a Nuclear Chicago constant current stimulator (model 7151) or a Grass S5 square wave voltage generator (2). Recordings were made upon either a Model R Beckman rectilinear or Offner Model R curvilinear recorder.

Residual red cell, plasma, and blood volumes were measured in the small vessels of nine major organs of the developing beagle, ranging in age from 4 hr to 7 months. The animals were sacrificed by bleeding. The tissues analyzed were the heart, lung, kidney, spleen, liver, stomach, intestines, muscle, and skin. The data showed that these organs contain a significantly larger volume of the total blood volume in the newborn than in the 3- to 7-month-old dogs. In all age groups the muscle, skin, and liver contained the largest amount of blood, approximately 70% of the total organ blood volume. With the exception of the spleen, and in some cases the liver, the organ hematocrits for a particular age group were lower than the corresponding total body and the large vessel hematocrits.

Many investigators have studied the effects of trypsin and chymoltrypsin inhibitors on the size, enzyme content and enzyme secretion of the pancreas of rats and chicks. In general, these investigations showed that the pancreases of animals fed diets containing sources of proteolytic inhibitors had greater quantities of enzymes after a fast than comparable fasted animals fed control diets (1-5). When pancreatic enzyme levels were measured after animals were fed, those receiving sources of proteolytic inhibitors generally had lower levels of all pancreatic enzymes than animals not receiving the inhibitor (2-4, 6-8). Thus dietary proteolytic inhibitars appear to cause a general stimulation of the pancreas, resulting in both an increased synthesis and a greater secretion of pancreatic enzymes.

Trypsin inhibitors from the soybean (6, 9), the navy bean (10), egg white (5, 9), a synthetic inhibitor, p-aminobenzamidine (11), tannins from carobs (12) or peanut skins (13) and a chymotrypsin inhibitor from potatoes (14) all have been shown to stimulate the pancreas. These are chemically very diverse and have in common only the ability to inhibit proteolysis by interfering with one or more of the pancreatic enzymes.

The physiological mechanism whereby these inhibitors stimulate the pancreas is not well understood. Since inhibitors from widely varying sources are able to cause the effects, it appears that the inhibition of proteolysis itself may be the signal to the pancreas rather than some property of the inhibitor molecule per se. Snook (5) has proposed that the gastrointestinal hormone, pancreozymin, mediates the response to trypsin inhibitor. Khayambashi and Lyman (15) showed that a factor present in plasma of rats receiving a diet containing crude trypsin inhibitor stimulated pancreatic enzyme secretion. Rothman and Wells (16) showed that repeated administration of pancreozymin caused pancreatic enlargement and increased enzyme synthesis rats.

The intestine of the rat can be used as a cholera model. The amount of fluid production in in vivo intestinal loops was dependent on the amount of toxin present. With increased fluid production HCO₃ and Cl concentrations approached those in plasma. Acetazolamide did not minimize fluid production while it was uncertain whether cycloheximide had any effect. In in vitro everted sacs, prepared from intestines which were secreting fluid in response to cholera toxin in vivo, there was a net transport of fluid from the mucosal to serosal side.

The induction of delayed hyper-sensitivity in mice to purified acidic (anionic) proteins by injecting these subcutaneously in incomplete Freund adjuvant appears to require the aid of phosphate ions in the aqueous phase of this adjuvant. Arthus hypersensitivity to these antigens is readily induced by such injections whether phosphate ions are present or not. Phosphate may function by affecting host cell-antigen associations in the earliest stages of induction. These experiments suggest that a more detailed investigation of the functions of the aqueous phase of Freund adjuvants is needed, and also that for at least some models of delayed hypersensitivity incomplete Freund adjuvant can be used in place of complete Freund adjuvant if phosphate ions are present.

Polymorphonuclear leukocytes (PMN) from adult human beings showed enhanced phagocytosis of S. aureus following in vitro exposure to staphylococcal antigens. The enhanced phagocytic response by stimulated PMN appeared to be an acquired characteristic.

We thank Dr. John Sugg for reading and commenting on this manuscript.

Endotoxin causes an increase in vascular resistance and the release of some lysosomal enzymes in the circulation of the isolated lobe of the dog lung perfused in situ. These effects of endotoxin were prevented by the administration of PGE₁. When PGE₁ was infused after endotoxin was given, the increase in the pulmonary perfusion pressure was greatly reduced. Thus similar to the previously tested anti-inflammatory drugs, PGE₁ can block some of the effects of endotoxin in the circulation.

We are grateful for the skillful technical assistance of Mrs. P. Downs. Prostaglandin was kindly supplied by the Upjohn Company and by Dr. J. Nakano of O. U. Medical Center.

The presence of chronic, experimentally induced hypercholesterolemia in rhesus monkeys led to atypical patterns of lipid response during acute pneumococcal infection. When compared to infection in previously normal monkeys, hypercholesterolemic monkeys showed an impaired synthesis of cholesterol with accumulation of squalene in the plasma, an increased conversion of newly formed cholesterol to an esterified form, and an impaired utilization of triglycerides.

Mestranol in a wide range of concentrations has no direct effect upon triglyceride release from livers obtained from normal rats. The hypertriglyceridemia seen with mestranol, probably results from a stimulation by the steroid of the release of various endocrine factors which in turn may stimulate the liver to release more trigly-ceride.

This investigation was supported in part by Grant GRS FLU362-09 and TO1 AM05670-01DM from the National Institutes of Health. We are indebted to Dr. E. Forchielli from the Syntex Corporation, Palo Alto, CA, for supplying mestranol, and to Mrs. L. Andiric for her excellent technical assistance.

In the rat prolactin has been shown to exent a luteotropic action an newly formed corpora lutea and a lutmlytic effect on older corpora lutea (1). During each estrous cycle of the rat, luteolysis of corpora lutea from previous cycles occurs (2, 3). Recent work from our laboratory has shown that the proestrous surge of prolactin during the estrous cycle is responsible for luteolysis of the old corpora lutea (4). Billeter and Fluckiger (5) also reported a luteolytic action by prolactin when injected daily in cycling rats with an ergot drug (CB 154) for 21 days.

In the mouse prolactin produces a luteotropic action on the corpora lutm (2), but a luteolytic action for prolactin has not been described. A brief report by Kwa and Verhofstad (6) states that during the estrous cycle of the mouse, blood prolactin rises beginning late in proestrus and reaches a peak early in estrus. It was of interest therefore to determine in the mouse whether inhibition of prolactin secretion by the drug ergocornine (EC) during proestrus and estrus could prevent luteolysis of the old corpora lutea, and whether concurrent administration of prolactin could induce luteolysis. EC previously was reported to decrease pituitary prolactin levels in the mouse (7) as well as in the rat (879).

A DEAE-cellulose column chromatographic technique was used for separation of TCII, MPPB and TCI in 1 ml of human serum. The percentage of vitamin bound to each binder remained practically constant at levels of added vitamin varying from 250 to 5000 pg ⁵⁷Co B₁₂/ml of serum.

The percentage ⁵⁷Co B₁₂ bound to MPPB and TCI in chronic myelocytic leukemia serum was significantly higher than in normal serum.

Coagulation processes are considered important in the pathogenesis of several forms of human and experimental renal disease. The possibility was examined that anticoagulation could modify the course of acute serum sickness nephritis in rabbits.

Bovine serum albumin (BSA) was given iv to 28 rabbits. Beginning on the sixth to eighth day 15 rabbits were anticoagulated with iv warfarin sodium. Renal biopsies done prior to BSA injection and at time of BSA elimination from the serum were examined by light and immunofluorescent microscopy. Most animals in both the nonanticoagulated and anticoagulated groups had significant glomerulonephritis and no differences between the groups were detected.

Although the evidence is convincing that the coagulation process is operative in the pathogenesis of nephrotoxic serum nephritis–a condition due to antiglomerular basement membrane antibodies–our data suggest that this mechanism does not play a significant role in the pathogenesis of antigen-antibody complex nephritis of the acute serum sickness type.

To determine the effect of hydronephrosis on treatment of pyelonephritis, 85 Slonaker-Addis rats with congenital right unilateral partial ureteral obstruction and 120 with two normal kidneys were infected intravenously with Streptococcus jaecalis. Two weeks after infection one-half of each group was treated with 25,000 units penicillin G twice daily for 3 weeks. Measurement of renal microbial population after therapy ended indicated that the efficacy of treatment was significantly impaired not only in the hydronephrotic (right) kidney, but also in the contralateral normal (left) kidney.

Mice injected with syngeneic lymphoma cells are able to mount an immu-nological reaction against the tumor cells. This results in marked morphological changes in the spleen and the appearance of circulating antibodies demonstrable by immu-nofluorescent techniques. The lymph nodes and particularly the regional node do not exhibit evidence of an immune response. This may indicate that in certain cases the draining nodes may not participate in the immune response of the host against tumor antigens.

The authors express their appreciation to Mrs. Shirley Fullen in helping prepare this manuscript.

Relatively few investigations have dealt with the pathogenesis of the intestinal phase of trichinosis. This is especially true in human infection which is usually diagnosed only after the disease has progressed into the extraintestinal stage. In guinea pigs administered graded doses of Trichinella spiralis larvae the gut phase is accompanied by diarrhea and weight loss, the intensity of which is directly dependent on the size of the worm burden (1).

Interestingly, the small intestine of animals with these symptoms is characterized by histopathology similar to that reported for humans with malabsorption syndromes, and the capacity of the gut to transport glucose actively is greatly reduced (2). Since the transport of glucose is mediated through the brush border of the gut epithelium, it is probable that other physiologic functions associated with this region, including the activity of digestive enzymes such as disaccharidases (3) are adversely affected and may contribute to the clinical picture.

Presently, the physiologic basis of diarrhea in trichinosis is not well understood, although diarrhea as a clinical entity may be caused by various intestinal malfunctions, including deranged absorption and impaired digestive processes (4). Despite the general assumption that the failure of parasitized animals to gah weight at normal rates is related to anorexia, weight loss cannot be attributed entirely to this factor (1). To establish the possibility that helminth-induced diarrhea and weight loss are related, in part, to impaired digestive function, the activity of maltase, sucrase, trehalase, and palatinase in the small intestine of control guinea pigs was compared to that of animals infected with Trichinella.

Antisera prepared to HCG/GEF (abbreviated AS) inhibited the uterotropic response of immature mice when HCG was assayed in sera diluted 1:120. The response to HCG dissolved in AS diluted 1:2400 or 1:4800 was enhanced. Enhancement was also produced by normal rabbit serum similarly employed at all dilutions tested, 1:120 to 1:4800. Anti-HCG serum, powerfully inhibitory at all dilutions tested as a solvent for the hormone, revealed enhancing activity when subjected to 60 min of heat at 92-95°. The latter treatment also abolished HCG antagonism by AS, although the respective antibodies of both the latter and the former sera were stable to treatment at 65° for 1 hr. These combined data suggest that each of the antisera studied contained not only their respective antibody species, which inhibited the response to HCG, but also possessed some degree of gonadotropin-enhancing potency. Thus, the results obtained can be interpreted as the resultant of superimposition of an inhibitory influence upon enhancing activity, both present simultaneously in the sera.

The technical assistance of Mrs. Lucille M. Nickerson is gratefully acknowledged.

Sympathetic denervation of the eye in experimental mammals, either by surgical means (1, 2) , or pharmacologically (3), has been reported to cause a decrease in intraocular pressure (IOP), an increase in outflow facility (C) (1, 2), no change (4) or a decrease (5) in aqueous secretion, no change in episckral venoaq pressure (4, mydriasis (6) or miosis (7) , suparsensitivity to alphaadrenergic drugs (7), decreased vascular tone and increased blood flow to the anterior segment of the eye (4, 8). Many of these effects are transient, the peak response occurring about 1 day after anterior cervical ganglionectomy. This “24-hr ganglionectomy effect” has been attributed to the release of stored catecholamines, primarily norepinephrine, from degenerating postganglionic nerve terminals (1, 9). This hypothesis was strengthened by Eakins and Eakins (10), and by Sears et al. (11), who showed that catecholamine stores in the weal tissues were depleted over a time period which coincided with postganglionectomy changes in IOP and C.

There are conflicting reports regarding the long-term effects of ganglionectomy on the eye: a recturn to normal may occur within 3 days (2), especially after unilateral rather than bilateral ganglionectomy. However, Sears et al. (11) reported that, following a transient rise, outflow facility was greatly reduced for almost 4 weeks after unilateral ganglionectomy, and C did not return to normal until 12 weeks after bilateral sympathetic dmervation. Langham (7) found that supersensitivity to norepinephrine was still evident 8 days after unilateral ganglionectomy, and in the experiments of Holland and Mimhls (3), miosis during the first 48 hr was followed by mydriasis lasting as long as 5 weeks in chemically sympathectomized monkeys. From the reduced IOP and increased C, these authors postulated that postganglionectomy effects were accoqpanied by reduced intraocular pressure and miosis (3).

Treatment of adult female rats with medroxyprogesterone acetate caused a marked reduction in size of prolactin cells. Growth hormone cells were enlarged and increased in relative number. Gonadotropic cells responded variably: in some rats certain of them were enlarged and more intensely stained. Corticotropic cells were not affected significantly.

The effects of changes in the extracellular as well as the intracellular Na concentrations on the movement of leucine into or out of isolated intestinal epithelial cells was determined. The influx of leucine was optimal at an initial extracellular Na⁺ concentration of 118 mM despite variations in the intracellular Na⁺ concentration over the range 8-122 mM. The influx of leucine was decreased 40% when the initial extracellular Na⁺ concentration was zero and the magnitude of the reduced influx was not affected by initial intracellular Na⁺ concentrations over the range 57-122 mM. Leucine movement into or out of leucine-loaded cells followed the direction of the Na⁺-gradient across the cell membrane. These results indicate that extracellular rather than intracellular Na⁺ is the determining factor in the Na⁺ dependence of leucine transport, a finding consistent with the Na⁺-gradient hypothesis for amino acid transport.

The authors thank Loutitia Ferdinandus and William Heaton for their valuable technical assistance.

Systemic hypersensitivity to tuberculin in mice is compared with anaphylaxis and shock reactions following the injection of histamine-serotonin or endotoxin. Time of occurrence, symptomatology and cardiovascular parameters demonstrate a striking similarity between tuberculin shock and reactivity to bacterial endotoxin in tuberculous mice and clearly differentiate these reactions from anaphylaxis and from shock caused by histamine-serotonin or by endotoxin administered to nontuberculous mice. The findings are compatible with the view that nonimmunological mechanisms may underlie the pathogenesis of systemic tuberculin reactivity. A greatly increased susceptibility to bacterial lipopoly-saccharides in mycobacterial infections and the fact that endotoxins are common contaminants of tuberculin preparations may play a decisive role.

The skilled technical assistance of Mrs. H. Scherrer, Miss M. Gut, Mr. N. Kaufmann and Mr. L. Criscione is gratefully acknowledged.

A strain of vaccinia, V1-010, was discovered which is defective in surface antigen production as detected by mixed agglutination. Evidence for existence of major and minor surface antigens on vaccinia-infected cells was obtained. Minor surface antigen could be distinguished from the major antigen serologically as well as by the pattern of mixed agglutination reactions. V1-010 was capable of producing minor surface antigen and hemagglutinin.

Sensitivity of three assay techniques for the detection of murine leukemia viruses were compared. These techniques were the mouse antibody production test, appearance of new surface antigen(s) on JLS-V9 cells, and production of plaques on D56 cells. The sensitivity of the three assays in the detection of murine leukemia virus is similar. The plaque assay on D56 cells offers certain advantages over the other tests since smaller quantities of leukemia virus could be detected on day 5 after infection by using this technique. The antibody production test requires 9 weeks for completion. For viruses with a differing host range which will induce neither plaque nor membrane antigens on D56 cells, the assay of JS-V9 cells may be the preferred method.

The authors thank Dr. Robert H. Bassin, Viral Leukemia and Lymphoma Branch, NCI, Bethesda, MD, for providing the D56 cell line. These investigations were conducted under Contract No. NIH-69-2005 within the special Virus Cancer Program of the National Cancer Institute, National Institutes of Health, U.S. Public Health Service; the Jane Coffin Childs Memorial Fund for Medical Research, Project No. 287; and grants from the Swedish Cancer Society. The expert technical assistance of Mrs. Britt Salven is gratefully acknowledged.