Experimental Biology and Medicine

The effects of 0.8 ppm ozone on the capacity of the tracheal epithelium and alveolar macrophages of mice to produce interferon in vitro was studied. Exposure of mice to ozone for a period of 11 days or more affected the capacity of the tracheal epithelial cells in vitro to produce interferon. The inability of the tracheal epithelium in vitro to produce interferon was not due to the inhibition in the release of intracellular interferon but to an inhibition in the production of interferon. There was a complete recovery of the ability of tracheal epithelium to respond to interferon inducers after the mice were returned to ambient air 24 days post ozone exposure. However, ozone did not seem to have any affect on the capacity of the alveolar macrophages to produce interferon in vitro.

LPS stimulation of mouse spleen cells in the presence of Hu resulted in almost total suppression of [³H]thymidine incorporation without affecting the percentage of cells induced to produce IgM. Utilizing a method which permitted the simultaneous measurement of IgM production and [³H]thymidine incorporation in individual cells, it was demonstrated directly that LPS stimulation of IgM production can occur in the absence of DNA synthesis.

Radiolabeled ¹²⁵I preparations of canine IgG, IgM, and F(ab')₂ fragments of IgG were injected intramuscularly into normal dogs to quantitate the transfer of immunoglobulins from the intravascular space into the lower respiratory tract and to observe the clearance of these proteins from lung secretions. Respiratory fluids were sampled by serial broncho-alveolar lavages during a 3–4 week interval. Only a small portion (0.15%) of the passively administered IgG was recovered in respiratory specimens indicating that minimal transfer of humoral IgG occurs under normal conditions.

Alteration of the IgG molecule by removal of the Fc portion almost eliminates its penetration into lung secretions. Furthermore, IgM, which is not a prominent component of normal lung secretions, is not detected in these secretions following passive intramuscular injection.

The authors appreciate the fine assistance of Mr. Edward W. Harvey in these studies and the help with statistics given by Dr. David W. Alling. We thank Dr. Charles H. Kirkpatrick for his review of the manuscript.

A congenic strain of C₃HSS mice, which is histocompatible with C₃H mice but differs from them in susceptibility to mouse hepatitis virus (MHV), has been developed by introducing the gene for susceptibility to the MHV-PRI virus from the PRI mice. This was accomplished by continual back-crossing of the hybrids to the C₃H mice, but at the same time by selection of susceptibility by use of macrophage culture tests. After 20 back-crosses, a strain homozygous for susceptibility was produced by brother-sister mating of individual mice whose potential for carrying the recessive gene for resistance was tested in progeny. Since the original choice of mice for breeding was based on in vitro macrophage susceptibility, and since highly susceptible mice were developed on the same basis, it seems evident that macrophage susceptibility is an integral aspect of mouse susceptibility.

The continued production of almost 50% susceptible mice in the back-crosses is further evidence of the dominant one-locus explanation of genetic susceptibility to this agent. Incomplete penetrance may also be present in 8 and 9 week old mice of the C₃HSS strain since there was a sharp decrease in susceptibility of these mice even though their macrophages in culture maintained full susceptibility.

We wish to thank Dr. Ilan Shif for his contribution in testing for susceptibility.

Blood clotting and fibrinolytic systems were studied in the plasma of a sei whale (Balaenoptera borealis). The sei whale belongs to the suborder baleen whales of the order Cetacea. Whale plasma had a greatly prolonged kaolin-activated partial thromboplastin time and was deficient in Hageman factor (factor XII), Fletcher factor (a plasma prekallikrein), and PTA (factor XI). All other clotting factor activities were present in amounts comparable to that of normal human plasma. Whale plasminogen was activated by human urokinase, but not by streptokinase. Whale plasma contained inhibitory activities against thrombin, activated Stuart factor, activated PTA, activated Fletcher factor, and plasmin.

We are grateful to Dr. James Mead of the Smithsonian Institution, Washington, D. C., who imported the whale plasma. Ms. Ellen Strecker and Miss Marilyn Burke provided invaluable technical help.

Immunologic tolerance to simple hapten bound to nonimmunogenic carriers has gained wide acceptance to study the mechanism of tolerance (1–6). Of particular interest is autologous IgG as a tolerance-inducing carrier because it is a physiologic protein naturally tolerated by the host (5). Although this phenomenon has been observed in several strains of mice (7), it is not known whether this is a general property of autologous IgG. For this reason, we have examined the tolerogenic properties of autologous IgG in a different system, the anti-fluorescein response of rabbits (8). This particular system was chosen for two reasons. Firstly, the rabbit anti-fluorescein (anti-Fl) response to an immunogenic hapten carrier conjugate (Fl-KLH) (Fl-keyhole limpet haemocyanin) has already been extensively characterized in this laboratory (8). Secondly, if one can induced tolerance to a fluorescent hapten it will offer distinct advantages to follow the fate of the tolerogen in vivo.

Materials and methods. Preparation of hapten carrier conjugates. Keyhole limpet haemocyanin (KLH) and purified isologous rabbit gamma globulin (RGG) were obtained commercially from Schwartz-Mann. Autologous RGG was purified from the serum of individual rabbits by starch block electrophoresis. Fluorescein isothiocyanate (Sigma Chemical Co., St. Louis, Mo.) was conjugated in different molar ratios to KLH, RGG (homologous), and RGG (autologous) by previously described methods (8).

Immunizations. Randomly bred New Zealand white rabbits (Gloucester Rabbitry) were used throughout these studies. The rabbits were injected subcutaneously (in the rear footpads) with 3–5 mg of hapten carrier conjugate in complete Freund's adjuvant (CFA), or intravenously (in the marginal ear vein) with 30–50 mg of the hapten carrier conjugate in phosphate-buffered saline (PBS). For iv injections the molar ratio of hapten to carrier was 8–10:1 based on molecular weights of 900,000 for KLH and 150,000 for RGG.

The rat eosinophil granule possesses a major basic protein (MBP) similar to that previously isolated from human and guinea pig eosinophils. This conclusion is based on demonstration that the major protein of the rat eosinophil granule is a low molecular weight, highly basic material with an amino acid composition similar to that of the human and guinea pig MBP.

The authors wish to thank Dr. Kenneth Mann for use of his amino acid analyzer and Dr. Jon Lewis for performing the electron microscopic examination of the granule preparation.

A radioimmunoassay for serum calcitonin in the rat is described. Materials for assay of human calcitonin are employed. The sensitivity of the assay is 3 pg and corresponds to a lower limit of detection of 60 pg/ml for peripheral serum. The normal range is up to 650 pg/ml in rats weighing between 125 and 200 g. Serum calcitonin is not detectable in thyroparathy-roidectomized animals. It increases and varies directly with the serum calcium after intraperitoneal administration of the ion. Values for serum calcitonin in male and female rats are comparable under basal conditions and after calcium. The radioimmunoassay should allow the further characterization of calcitonin secretion in response to physiologic and pathophysiologic stimuli in this species.

These studies were supported by research funds from the Veterans Administration. The technical assistance of Mr. Jack Kimble and Mrs. Sarah Larson is gratefully acknowledged.

The present studies were conducted to determine whether inherited muscular dystrophy in the 129/ReJ-dy mouse was associated with differences in specific activity, substrate availability, or apparent K _m of glutathione peroxidase. The results indicate that glutathione peroxidase is elevated in skeletal muscle of mice with genetic muscular dystrophy when the activity is expressed on a protein basis. This elevation precedes the development of severe paralysis since muscles from the fore legs showed increased enzyme activity as early as the more severely affected hind legs. There was no difference in glutathione peroxidase activity in tissues other than skeletal muscle. GSH concentration was elevated in muscle and normal in other tissues of dystrophic mice, showing that adequate substrate was available to the enzyme. The apparent K _m for cumene hydroperoxide was also similar for muscle of normal and dystrophic mice. This report provides further evidence that mice with dystrophia muscularis have a functional glutathione peroxidase system in all tissues including skeletal muscle, and that a defect in this in vivo protective system is apparently not a contributing factor in the pathology of the disease.

Seroepidemiologic studies were made of normal subjects in populations in the United States and Costa Rica and in family outbreaks of hepatitis in Costa Rica. Hepatitis A affected a majority of children of very young age in Costa Rica, while such experience in persons of high socioeconomic status in the United States did not occur before middle life. Persons of low socioeconomic status (paid plasma donors) and residents and attendants of institutions for the mentally retarded showed a far greater incidence of hepatitis A antibody than did their counterparts in the open community. Hepatitis A and B epidemics occurred in families in Costa Rica with rapid spread to other susceptible members of the group. The disease was clinically apparent in roughly half the cases, whether the responsible agent be hepatitis A or B. Five cases of nonhepatitis A or B (hypothetical hepatitis C) were found and all but one of them were subclinical.

The authors are indebted to M. A. Chacón, D. Jimenez E., and A. Rivas E. and to P. Giesa, L. Hoover, W. P. M. Fisher, and M. Johnston for valuable technical assistance.

Past studies reported by this laboratory have documented myocardial dysfunction and progressively developing hypoglycemia in canine endotoxin shock. The purpose of the present study was to determine the effects of glucose concentrations and insulin infusions on myocardial performance following endotoxin administration. Experiments were carried out on isolated, working canine left ventricular heart preparations exchanging blood with intact dogs. Myocardial function was evaluated following endotoxin and correlated with concentrations of glucose and effects of insulin infusion. Cardiac dysfunction occurred within 2–4 hr postendotoxin and the degree of malfunction was not related to arterial blood glucose concentrations. Maintaining blood glucose at control, preshock, levels by infusion of 50% glucose did not prevent myocardial dysfunction as evidenced by elevations of left ventricular end diastolic pressure, and depressed power. Infusions of insulin reversed cardiac failure and maintained normal performance in spite of wide ranges in glucose concentration (5-120 mg%). Findings suggest that myocardial dysfunction is not precipitated or enhanced by the hypoglycemia of endotoxin shock. The beneficial actions of infused insulin on cardiac performance appear to be elicited on the basis of mechanisms other than myocardial glucose transport.

Appreciation is expressed to Beverly Beller for technical assistance.

The human reovirus-like (HRVL) agent, Nebraska calf diarrhea virus (NCDV), epizootic diarrhea of infant mice (EDIM) virus, simian agent (SA)-11, and the “O” (offal) agent were found to be similar, if not identical, in reciprocal complement fixation (CF) tests employing hyperimmune animal sera. In addition, in CF tests with paired sera from 35 diarrhea patients who shed the HRVL agent, 74% developed serologic evidence of infection with the HRVL antigen, 43% with NCDV, 51% with EDIM virus, 57% with SA-11, and 71% with the “O” agent. Thus, in addition to the NCDV, which had previously been described as a suitable substitute CF antigen for the HRVL agent, the SA-11, “O,” and EDIM viruses may also be utilized as substitute antigens for the HRVL agent. However, the “O” agent appears to be the most efficient of the four substitute CF antigens and thus should be used preferentially when the HRVL agent is not available. The “O” agent was about as efficient as the HRVL agent and significantly more efficient than the NCDV for detecting seroresponses. The greatest efficiency for detecting infection with the HRVL agent resulted when sera were tested with both the HRVL and “O” agents as 31 (89%) of the patients developed serologic evidence of infection with one or both antigens. The finding of additional substitute CF antigens for the HRVL agent may have implications in the immuno-prophylaxis against human disease.

We thank Dr. D. W. Alling for assistance in statistical analyses and Mr. C. E. Banks, Mr. E. Williams, Mr. B. Andrews, Mr. J. P. Jackson, Miss D. M. Bertran, Mrs. S. J. Kelly, Mr. Robert P. Chames, Mrs. L. P. Kendrick, Mr. C. S. Osborne, Mrs. B. V. Armiger, and Mrs. C. L. Armiger for assistance in these studies.

Serial measurements of plasma “true glucagon” (PG) and of glucagon-like immunoreactive materials (GLI) were carried out during and after total resection of the pancreas in a 62-year-old man with calcified pancreatitis. The postoperative course of this patient was uneventful and diabetes was well controlled. PG disappeared from the blood within 30 min after resection of the pancreas. In spite of the evidence that no pancreatic tissue was present in the abdomen, PG was detected again in the blood from 18 hr after total pancreatectomy until the ninth postoperative day. However, plasma PG did not rise following infusion of arginine during the fourteenth postoperative week. After an initial decrease, plasma GLI rose abruptly on the second postoperative day and remained elevated thereafter. The fluctuations of plasma PG and GLI were not parallel.

Murine spleen cells were cultured in vitro to study the induction of committed granulopoietic stem cell (CFU-C) proliferation and maturation. Marbrook-type diffusion cultures were established with and without the addition of colony-stimulating activity (CSA) and harvested at intervals up to 14 days for viable and differential cell counts, [³H]TdR autoradiography, and quantitation of CFU-C by the agar plate method. Without CSA there was poor cell viability and little proliferative capacity. In CSA-stimulated cultures there was a prominent rise in viable cell counts and [³H]TdR labeling indices rose from a mean of 2% at 0 time to 47% after 5 days in vitro. CFU-C increased by 70-fold in these cultures. Peak numbers of CFU-C, immature cells, and [³H]TdR-labeled cells occurred at about 7 days. Thereafter, mature granulocytes and macrophages predominated in culture. Because the liquid spleen cell culture system begins in a resting state and undergoes a wave of proliferative activity in response to CSA, it can provide a useful model system for studying phenomena associated with stem cell activation and differentiation in vitro.

The authors thank Ann Sullivan for technical assistance.

It has been found that damage to a tissue of a rabbit or a rat, such as results from a skin incision or an incision through the skin and muscles into the abdominal cavity, is followed 24 hr later by a significant increase in the concentration of leucogenenol in the animal's serum. Likewise, loss of approximately one-quarter to one-half of the blood in the circulation of rabbits or rats causes an increase 24 hr later in the animals'serum leucogenenol concentration.

The stability of Rauscher leukemia virus (RLV) was investigated under certain laboratory conditions. The half life of the virus at 37° was 7 hr, and considerably longer at lower temperatures. RNA dependent DNA polymerase activity was more stable than infectivity at all temperatures. Air dried virus had a half life of approximately 1 hr, but was rapidly inactivated by uv light or 70% alcohol.

We are indebted to Drs. W. Emmett Barkley and Herbert K. Oie for advice and encouragement, and Edward Russell for technical assistance.

We have previously reported the capability of SCT to produce an acute diuresis and natriuresis in the saline loaded rat (1), as well as its ability to reduce the paw edema or inflammatory phase associated with the chronic disease state known as rat adjuvant arthritis (12). In addition, we have noted a significant decrease in the heart weight of normal rats given high doses of salmon calcitonin for 7 days (unpublished). We thus became interested in determining what effects SCT might have in a chronic hypertensive state such as that produced in rats by administration of DOCA in combination with a high salt intake.

Methods. Two separate experiments were performed in which female Sprague-Dawley rats with initial weights of 50–70 g were treated sc for 5 weeks with 5 or 25 mg/kg/ dose of DOCA given b.i.d. with 1% NaCl in the drinking water. Synthetic salmon calcitonin (4000 MRC u/mg), purified porcine calcitonin (100 MRC u/mg), or synthetic human calcitonin (80 MRC u/mg) was administered sc at a dosage of 5 MRC u/kg/ dose b.i.d. in a 16% gelatin vehicle concomitantly with DOCA but at a different injection site. At the end of the treatment period direct measurements of aortic blood pressure were recorded in the unanesthetized state over at least a 5-min period following cannula insertion and pressure stabilization. Systolic, diastolic, mean blood pressure, and heart rate were recorded. The animals were then sacrificed and measurements made of heart and kidney weights. Appropriate control groups receiving distilled water or 1% NaCl to drink and injected with vehicle were included. Levels of statistical significance for group differences were determined using Student's t test.

Results. In Table I are shown the results of the blood pressure measurements in the two experiments after 5 weeks of treatment.

Cellophane perinephritis hypertension was produced in four dogs, while five additional dogs served as normotensive controls. A competitive antagonist of angiotensin II, 1-sarcosine-8-alanine angiotensin II, was infused iv into these conscious dogs at a rate of 6 μg/min/kg of body weight for 45 min. Arterial pressure averaged 170 ± 11 (SEM) mm Hg in the dogs with perinephritic hypertension, and was not altered significantly during infusion of the angiotensin antagonist. In the normal dogs the arterial pressure averaged 100 ± 10 mm Hg and likewise, did not change during administration of the angiotensin analog. Plasma renin activity values were essentially the same in these two groups of dogs and did not change during infusion of the angiotensin antagonist. These studies provide strong evidence that the renin-angiotensin system is not involved in maintaining the elevated arterial pressure in dogs with chronic hypertension produced by cellophane perinephritis.

The authors gratefully thank Dr. A. W. Castellion of Norwich Pharmacal Company for supplying the 1-sarcosine-8-alanine angiotensin II used in these studies. The cellophane used to produce perinephritis hypertension was supplied by E. I. DuPont de Nemours and Company. Mr. Charles Payne assisted in the surgical procedures and the assays for plasma renin activity were performed by Ms. Jane Green.

Calcium directly stimulates renin release from rat kidney slices previously treated with calcium-free medium. The stimulant effect of calcium (0.5 or 6.0 mM) is not seen without the period of calcium depletion. The stimulant effect of calcium is still present in sodium-free medium but is reduced when the incubation is performed at 20° instead of 37°. The results suggest that the underlying mechanism of renin release may be comparable to that of catecholamine release, involving calcium-dependent and energy-dependent steps.

We acknowledge the valuable technical assistance of Mr. John Gillespie and Mrs. Roselle Poisner.

In 1957 Schwarz and Foltz (1) described an association between dietary selenium deficiency and hepatic necrosis. However, although the dietary requirement for selenium is established, its possible role in the maintenance of normal hepatic function remains speculative. Diplock et al. (2, 3) have recently suggested that selenium may be incorporated into a nonheme iron containing microsomal protein active in the NADPH-dependent electron transfer chain. It is conceivable, therefore, that the integrity of the hepatic microsomal mixed-function oxidase system may in part be dependent on an adequate dietary supply of selenium.

We have recently described an impaired inducibility by phenobarbital of cytochrome P ₄₅₀ and cytochrome b ₅ in the selenium deficient rat (4). The normal inducibility of the flavoprotein component of the mixed function oxidase system, NADPH cytochrome c reductase, in these animals suggested that the impaired response in these selenium deficient rats may be limited to the hemecontaining microsomal proteins. Preliminary observations (4) indicated that the apoprotein component of cytochrome P ⁴⁵⁰ was induced by phenobarbital in the selenium deficient rats. The present study is a continuing investigation of the possible discoordinated synthesis of heme and apoprotein in the selenium deficient phenobarbital-stimulated rat.

Materials and methods. Male Holtzman rats weighing 60–90 g were placed on a selenium deficient torula yeast diet (5) and water ad lib. Two dietary groups were formed by the addition of supplemental selenium, 0.5 mg/kg as Na₂SeO₃, to the diet of one group of animals. Thus, a “selenium replete” and “selenium deficient” group was formed; the animals were maintained on this diet for 3 months, at which stage animals in both groups weighed 300–350 g.

Four animals from each group were then subdivided into two further groups. Within each dietary group two animals received a daily intraperitoneal injection of phenobarbital (80 mg/kg in 0.9 NaCl) for 5 days, the remaining two animals receiving only the vehicle.

Leukocytic endogenous mediator (LEM) was shown to produce a significant increase in the intestinal absorption of zinc within 7 hr after its administration to normal rats. Consistent with earlier studies, LEM also produced a significant decrease in the plasma zinc concentration and an increased hepatic uptake of this metal which further demonstrates that LEM may be a key intermediate in altering zinc homeostasis during inflammation.

Tissue factor apoprotein and relipidated tissue factor preparations extensively hydrolyze bradykinin, Lys-bradykinin, Met-Lys-bradykinin, substance P, [Asp 1 ,Ile⁵-angiotensin II, [Asp 1 , Ile⁵]-angiotensin I, and human fibrinopeptide A while acting more slowly on [Sar 1 , Ile⁵]-angiotensin II, [Me₂Gly 1 , Ile⁵]-angiotensin II, bradykinin potentiating pentapeptide from B. jararaca, luteinizing hormone-releasing hormone, melanocyte stimulating hormone-release-inhibiting factor (Pro-Leu-Gly-NH₂), and oxytocin. No hydrolysis of thyro-tropin-releasing factor or bradykinin potentiating nonapeptide from B. jararaca is observed. Relipidated and apoprotein tissue factor act at identical rates under the conditions of the assay. Dansylation and chromatography of tissue factor-peptide incubation mixtures further indicate that relipidated and apoprotein tissue factor also hydrolyze peptides by identical mechanisms. No fewer than six bonds are hydrolyzed in bradykinin while the angiotensins and substance P are degraded to constituent amino acids. Only the N-terminal alanine is released from fibrinopeptide A. 2-Mercaptoethanol greatly inhibits the hydrolysis of bradykinin by relipidated tissue factor.

The authors are grateful to Dr. Maurice Manning of the Medical College of Ohio at Toledo for the generous gift of MIF, LH-RH, and oxytocin. They also thank Dr. M. C. Khosla of the Cleveland Clinic Foundation for his generous contribution of angiotensin II analogs.

This research was supported by a grant from the Northwest Ohio Chapter, Inc., of the American Heart Association.

A cloned cell line H-11 (HP) derived from an MSV-induced neoplasm (a hemangiosarcoma) was found to possess virus-specific tumor rejection antigen(s). The specific nature of the immune response was established through the use of a polyoma virus-induced neoplasm #89 both in cross immunization and cross challenge experiments. Virus antigens or surface antigens associated with virus production are not responsible for tumor rejection since virus production could not be detected, nor were any viral fingerprints of MSV found by the several assays used.

Control and streptozotocin diabetic rats were studied at 5 and 12 days after induction of diabetes. Strontium absorption was measured by in situ perfusion of duodenum and ileum. Duodenal absorptive capacity (absorption per unit length) and absorptive specific activity (absorption per gram of dry weight mucosa) were depressed. Depression was present both at 5 days, when mucosal growth is similar in controls and diabetics, and at 12 days, when mucosal growth is 50% greater in diabetics. Effects of diabetes on ileal absorption were minimal in comparison with effects on duodenum. This depression of duodenal strontium absorption in the diabetic rat is analogous to effects of diabetes on calcium absorption and may be mediated by abnormal vitamin D metabolism.

The present study was undertaken to examine some factors which lead to early-appearing resistance in human cells treated with interferon. It was previously shown that two conditions required for rapid development of resistance were continuous maintenance of cultures at 37° and use of more than 10 units/ml of interferon. The decay kinetics of the established resistance appear to be approximately the same whether the resistance was induced under conditions favoring rapid or slow development. With the use of actinomycin D it was shown that the mRNA for the antiviral protein is produced between 30 and 45 min after the first contact with interferon. Ruled out were the possibilities that a priming action of interferon and a newly synthesized intermediary protein were necessary for rapid development of resistance.

Many normal human nasal secretions contain an inhibitor of human fibroblast IF. This inhibitor had no effect on human leukocyte IF. The amount of inhibition of fibroblast IF increased with increasing quantities of nasal secretions. Also, the inhibition could be overcome with increasing concentrations of IF.

Freshly isolated fetal liver explants in organ culture did not convert L-[¹⁴C]alanine or L-[¹⁴C]lactate to carbohydrate, but L-[¹⁴C]serine and D-[¹⁴C]glycerol were both transformed. When explants were subjected to 42 hr of preliminary incubation without supplements, followed by transfer to fresh medium with added precursor, all four substrates underwent gluconeogenic transformation. It was concluded that the ability of fetal rat liver in organ culture to convert alanine and lactate to carbohydrate evolves slowly, but the conversion of glycerol, and to a lesser extent serine, to glucose and glycogen is initiated immediately.

This work was supported by U.S. Public Health Service Grants AM 09006, HD 09715 and CA 14194 and the General Research Support Grant RR 5589.

An inhibitor for lutropin receptor site binding (LH-RBI), which strongly inhibited the binding of ¹²⁵I-labeled ovine lutropin ([¹²⁵I]oLH) to ovarian LH receptors, did not inhibit the [¹²⁵I]oLH binding to testicular LH receptors. Preincubation of the LH-RBI with [¹²⁵I]oLH did not affect the binding of preincubated [¹²⁵I]oLH to ovarian LH receptors. No inhibition of [¹²⁵I]oLH binding to testicular LH receptors was observed even when the concentration of LH-RBI was significantly increased or when the testicular LH receptors were first incubated with LH-RBI prior to the addition of [¹²⁵I]oLH and a second incubation. Scatchard analysis revealed that the dissociation constant of [¹²⁵I]oLH binding was essentially the same in the presence or absence of LH-RBI. The results suggest that: (i) the lutropin receptor of ovaries, but not of testes, has a specific LH-RBI binding site in addition to the lutropin binding site, and (ii) the binding of the LH-RBI produces an “allosteric” type of inhibition to the binding of lutropin at the lutropin binding site.

We thank Miss Kathleen Harris for her excellent technical assistance and Miss Debra Starr for typing the manuscript.

This study was supported in part by the following: American Cancer Society Grant No. PDT-41D; USPHS, NIH Grant No. CAO5831-15 and AM-09801; Contract No. HD-9-2221; and Robert A. Welch Foundation (G-147).

The dietary and hormonal regulation of the level of glutathione-insulin transhydrogenase in rat liver was investigated in these studies. In order to make valid comparisons, the assay of glutathione-insulin transhydrogenase was performed at near zero-order kinetics wherein enzyme rate was proportional to enzyme amount. Changing the protein content of the diet or administration of glucagon or cortisone did not significantly affect the specific activity of glutathione-insulin transhydrogenase in microsomes from rat liver. However, the V_max and K_m of this enzyme in the livers of adrenalectomized rats were increased three- and fourfold over these values in microsomes from normal liver. Administration of cortisone resulted in a return to the normal kinetic constants of microsomal GIT within 4 hr.

In adult female or male rats subjected to hypophysectomy and auto-transplantation of the pituitary beneath the renal capsule, the levels of prolactin in the renal vein of the pituitary-bearing kidney were not significantly different from those found either in the renal vein of the contralateral kidney or in the jugular vein. LH levels were slightly but highly significantly higher than those found in hypophysectomized rats; the levels found in the two renal veins and the jugular vein were also not significantly different from one another.

We express out thanks to Helen Wilk and Rosa Garnett for their assistance with the animals and the preparation of this paper, respectively. The hormones and antibodies used for the assay of LH and prolactin were generously donated by Drs. G. D. Niswender, J. D. Neill, and L. E. Reichert and by the Program Officer, NIH Hormone Distribution Program.

The activities of acid phosphatase and β-glucuronidase were assayed in fetal and neonatal mouse epidermis by microanalytical methods. The level of acid phosphatase activity was low in the epidermis on Day 15 of gestation. Acid phosphatase activity increased 10-fold between Day 15 of gestation and neonatalhood. On the other hand, β-glucuronidase activity was high on Days 14 and 15 of gestation and low after Day 17 of gestation. The relative ratio of acid phosphatase to β-glucuronidase activity appeared to represent a marker for the degree of differentiation in whole epidermis.

Incubation of tritiated 2,5,-2′,5′-tetrachlorobiphenyl with normal monkey liver microsomes in a NADPH-generating system results in the formation of active metabolite(s) of the [³H]2,5,2′,5′-tetrachlorobiphenyl capable of covalently binding to RNA and protein isolated from the incubation mixture. The metabolite is not formed when the control microsomes are held at 100° for 10 min prior to incubation. The addition of microsomal supernate to the solution causes an increase in the binding of the active metabolite to macromolecules while the addition of glutathione to the incubation medium significantly inhibits this increase.

This investigation was supported in part by U.S. Public Health Service Grants ES-00472, ES-00958, and RR-00167 from the National Institutes of Health. This is Primate Center Publication No. 15-019.

Changes in hepatic hexosamine metabolism and serum seromucoid concentrations during postnatal development were investigated in the rat. A relatively low activity of hepatic L-glutamine: D-fructose-6-phosphate aminotransferase (AT) was observed within 24 hr of birth. This rapidly increased to a maximum at about 2 weeks of age, followed by a decline to adult levels after another 2 weeks. The developmental pattern of hepatic UDP-N-acetylglucosamine 2′-epimerase (EP) closely resembled that of AT. It was relatively low in the newborn, increased to a maximum at about 2 weeks and then declined to adult values after another 2 weeks. The serum seromucoid concentration was low in new born rats and then increased gradually with development to adult levels at about 50 days.

The hepatic concentrations of UDP-GlcNAc and CMP-NANA both were relatively high at 24 hr after birth compared to values observed at 50 days. The developmental pattern observed for both over this time period was quite similar and was almost the reciprocal to that for the seromucoid fraction. After 50 days the concentration of UDP-GlcNAc remained constant at adult levels as did that of the seromucoid fraction, but the concentration of CMP-NANA showed a marked increase.

It is suggested that the hepatic concentrations of these amino-sugar nucleotides during development primarily reflect the demand for the hepatic synthesis and secretion of plasma glycoproteins as reflected in the seromucoid fraction.

The authors wish to thank Dr. A. M. Chandler, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, for his assistance in preparation of this manuscript. The authors also express their thanks to Dr. Kazunori Kawamura, The Second Department of Medicine, Miyazaki Medical College, for his advice.

(Des-Asp 1 )-angiotensin I, angiotensin II and III were evaluated for pressor activities in conscious nephrectomized rats and for steroidogenic actions in rat adrenal zona glomerulosa. The pressor effect of this angiotensin nonapeptide was similar to that found with mole-equivalent doses of angiotensin III (one-third as active as angiotensin II) and was significantly attenuated by pretreatment with the B. jararaca nonapeptide converting enzyme inhibitor. Hence, (des-Asp 1 )-angiotensin I is a substrate for converting enzyme in vivo, and the rapid conversion indicates that an alternate pathway for the formation of angiotensin III could exist. (Des-Asp 1 )-angiotensin I possessed only 0.1% of the activity of angiotensin III as a steroidogenic agent in cell suspensions of rat adrenal zona glomerulosa. Angiotensin I was a weak steroidogenic agent in vitro (1%) and was not blocked by an inhibitor of converting enzyme. Adrenal cells dispersed from the outer zone of the cortex would appear to be devoid of significant converting enzyme activity.

The presence of a single cysteine in the sweet-tasting protein monellin was confirmed by titrations with p-hydroxy-mercuribenzoate (PHMB) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). The sulfhydryl group in native monellin reacts very slowly with each of these reagents, indicating that the sulfhydryl is relatively inaccessible. In the presence of either 6 M guanidine-HCl, 8 M urea, or 1% sodium dodecyl sulfate, the rate of reaction of the sulfhydryl group with titrant is dramatically increased. Under a variety of conditions, the presence of 1 mole of sulfhydryl per mole of protein (of molecular weight 10,700) was found.

Reaction of the sulfhydryl by titration with PHMB or DTNB leads to loss of sweetness. The free sulfhydryl is also lost by carboxymethylation of monellin in the presence of guanidine-HCl, yielding a protein that is not sweet. Exposure to air in the presence of denaturant leads to a decrease in the sweetness of monellin. Sweetness of the PHMB-reacted monellin can be recovered upon treatment of the protein with mercaptoethanol, and the partial loss of sweetness that occurs with air exposure is lessened in the presence of mercaptoethanol. It is postulated that alteration of the single sulfhydryl group of monellin leads to a change in the tertiary structure of the protein and hence its sweet taste.

This work was supported in part by Research Contract No. NIH-NIDR-72-2413 from the National Institute of Dental Research, USPHS (to R.H.C.). We thank Mr. H. L. O. Holloway of the Crops Research Institute, Bunso, Ghana, for his continuing cooperation in supplying us with fresh fruits of D. cumminsii. We thank Miss Linda Graham for technical assistance in the purification of monellin.

Penicillin G and related antibiotics have been shown by a number of investigators to inhibit platelet reactions in vitro (1-5). These antibiotics have also been reported to cause excessive bleeding in patients, and to increase the bleeding times in patients and volunteers receiving high doses of these drugs (1, 3-7).

In previous studies ((2), and Cazenave et al. Brit. J. Haematol., in press) we showed that penicillin G, carbenicillin, and cephalothin inhibit the adherence of platelets to a collagen-coated surface and to the damaged aortic surface of rabbits. We also found that penicillin G and cephalothin became associated with the platelets and that their inhibitory effects in vitro persisted after the platelets were washed and resuspended in fresh media.

Although the antibiotics have been shown to affect platelet function in vitro, it has not been established whether these antibiotics prolong the bleeding time in vivo by affecting the vessel wall, the plasma proteins, or the platelets. To investigate this question, we have treated platelets with penicillin G or cephalothin in vitro, washed the platelets, resuspended them in fresh media, reinfused them into thrombocytopenic rabbits, and examined their ability to shorten the prolonged bleeding times of these animals. We have also studied the effect of pretreatment of rabbit platelets with penicillin G, on platelet survival time upon infusion into rabbits.

Materials and methods. Reagents. Penicillin G (USP sodium, 1650 units/mg) was obtained from General Biochemicals, Chagrin Falls, Ohio). Cephalothin (Keflin) was a gift of Dr. R. S. Dolman, Eli Lilly and Co. (Canada), Toronto, Ontario. The antibiotics were dissolved in water at a concentration of 150 mM. Dilutions to the required concentrations were made with modified Tyrode solution (no calcium or magnesium); the pH was adjusted to 7.35 and the osmolarity to 290 mOsm.

Extracellular particles, with a density of 1.18-1.22 g/cm³ in sucrose, were detected in the culture medium of a continuous cell line (JIII) derived from a patient with monocytic leukemia. These particles contained RNA, DNA, and a DNA polymerase. They synthesized DNA with endogenous templates and primers and also used exogenous DNA but not poly(rC) oligo(dG) as a template. Pretreatment with Nonidet P-40 stimulated DNA polymerase activity while treatment with ribonuclease partially inhibited the enzyme activity. Fluorescent antibodies made to the particles stained both JIII and Z-597 cells derived from human leukemias but not other types of human or nonhuman cultured cells tested. The particles do not appear to be oncornaviruses but may be a particulate antigen associated with malignant cells of hemopoietic and lymphoid origin.

Fluorescent antibodies prepared against extracellular particles from a continuous culture of cells derived from a monocytic leukemia stained JIII cells but not cells infected with Rauscher leukemia virus or simian sarcoma virus. These antibodies reacted with 38% of bone marrow preparations from patients with lymphoma, 26% of preparations from patients with nonmalignant blood disorders and 6% of preparations from patients with leukemia. Bone marrow films from patients with lymphoma over the age of 50 stained less frequently than those from patients under 50. These particles released from JIII cells are not antigenically related to two of the commonly studied oncornaviruses, but may be indicative of the etiology or disease process of lymphoma in young patients.

The effects of tryptophan and its metabolites were studied to determine whether the known inhibiting effect of tryptophan on emptying was due to local or systemic effects or due to tryptophan metabolites. In five dogs with chronic gastric fistulas, instillation of 300 ml of dl-kynurenine (5 mM), 5-hydroxytryptophan (5 mM), or 5-hydroxytryptamine (20 mM) into the gut did not slow gastric emptying. Furthermore, iv infusion of l-tryptophan (5, 20, 50 mM), dl-kynurenine (2, 5, 10 mM), 5-hydroxytryptamine (2 mM, 10 mM), and 3-indolepyruvic acid (2, 5, 10 mM) also did not slow gastric emptying.

These studies indicate that tryptophan slows gastric emptying by exciting a receptor in the gut and not by a direct effect on the stomach or brain or via its major metabolites.

This work was supported by Grant AM 15886 from the National Institutes of Health and Veterans Administration Research Funds.

Intestinal blood flow is increased during digestion. This study assesses if the concentration of nutrients and/or osmolality of chyme in the intestinal lumen are factors determining the hyperemia. Six digested food mixtures containing different concentrations of nutrients and/or having different osmolalities were placed into the jejunal lumen, and their effects on local venous outflow compared. The 100% (999 mOsm/kg), 33% (291 mOsm/kg), and 20% (183 mOsm/kg) food mixtures all increased flow, but the 10% food mixture (94 mOsm/kg) did not. The hyperemic effect of 33 and 20% food was similar, but 100% food produced a greater increase in flow than did 33 or 20% food. Luminal placement of a 30% solution of a nonabsorbable substance polyethylene glycol (1000 mOsm/kg) did not alter flow. Also, the vascular effects of 20 or 10% food mixtures were not altered when these mixtures were made isotonic by the addition of NaCl. These studies indicate that lumen osmolality, within a range of 180 to 1000 mOsm/kg, is not a significant factor contributing to the local hyperemia occurring when nutrients are in the gut lumen. However, the concentration of nutrients in the lumen is a factor determining the local hyperemia.

Peripheral blood lymphocytes from pregnant and nonpregnant females were studied for the presence of the following surface receptors: (i) Receptor for heat-aggregated human IgG (AggIgG); (ii) Receptor(s) for complement components; (iii) Receptor for sheep red blood cells (SRBC).

Absolute numbers of lymphocytes with receptors for complement and those with receptors for SRBC were present in equal numbers in both groups. However, in the pregnant females there was a significantly lower number of lymphocytes with receptors for AggIgG. It is hypothesized that some of the immunological changes which occur during pregnancy may be mediated partly through changes in the total numbers of certain lymphocyte subpopulations.

We gratefully acknowledge the assistance of Dr. R. A. Bashore and Dr. W. Nagata in obtaining patients for this study.

Radioimmunoassay of acid ethanol extracts of the rat intestinal tract showed the presence of glucagon-like immunoreactivity (GLI) from the duodenum to the colon with maximal concentrations in the ileum and colon. Twenty percent glucose instilled in the duodenum at a dose of 2 g/kg body weight stimulated a twofold increase in peripheral plasma GLI concentration. When the instilled glucose load was restricted to only the duodenum and jejunum, or to only the ileum and colon, the increase in peripheral plasma GLI was approximately half that seen when the entire gut was exposed to the glucose. It is concluded that the distal gut as well as the proximal gut releases GLI in the rat.

Phospholipids and their acyl group composition in mammary adenocarcinomas and mammary tissue of the same tumor-bearing animals were investigated. Breast adenocarcinoma induced by dimethylbenz(a)anthracene exhibited a phospholipid pattern which was different from that of the mammary tissue. Tumor phospholipids had higher proportions of diacyl-GPI, diacyl-GPE, and alkenylacyl-GPE and a lower proportion of diacyl-GPC than the controls. The acyl groups of most phospholipids in tumors showed a marked increase in the proportion of 18:1 and a decrease in the proportion of 18:2. The fatty acid composition of plasmalogen and triglyceride, however, remained unchanged. In spite of the decrease in the proportion of 18:2, there was no apparent difference in the proportion of 20:4 in most of the phosphoglycerides; however, a significant decrease in this fatty acid was noted in diacyl-GPI. Results of this study demonstrated that the membrane phospholipids of mammary adenocarcinoma were altered in respect to acyl group composition. Changes in physical properties of the cell membrane, in turn, could lead to abnormal manifestation of membrane regulated events in tumor cells.

The technical assistance of Mrs. H. Winniczek is gratefully acknowledged. This investigation was supported in part by US PHS Research Grant NS-12118 and Grant RR-334 from NIH, Bethesda, Maryland, grants from The American Cancer Society, Oregon Division, and a research grant from the Camach Trust Fund of Oregon.

Restricted to heavy water (99.8%), rats drank it freely for the first day, then they drank progressively less and died within 14 days. When given a choice between distilled water and heavy water (99.8%) rats avoided heavy water, partly by virtue of some deleterious effects of heavy water and partly by virtue of a faint smell of heavy water with which the untoward effects could be associated. Rats did not taste heavy water. Whether a higher viscosity of heavy water plays any part could not be determined. Apparently the low intensity of any untoward effects of heavy water and the faintness of its smell could explain the erratic variability of the reactions of rats to heavy water that put it in a different category from other substances tested so far.

Moderate hypoxia did not influence the pulmonary incorporation of an intravenous dose of [1-¹⁴C]palmitate either in dogs with experimentally produced granulomatous disease or in normal controls. The lung weight in the diseased animals, was, on the average, double that of the controls. There was a proportionate increase in uptake of the radioactive label at 1 hr after infusion in the diseased lungs, hence the specific activity of labeled palmitate (counts per minute per gram of phospholipid) was no different in the two groups. Moreover, half the radioactivity of the phospholipids was recovered in palmitate separated from the phosphatidyl choline fraction in both diseased and normal lungs. Anatomic studies demonstrated increased numbers of Type II pneumocytes lining all alveolar air spaces in the diseased lung. Autoradiographic studies indicated the presence of labeled palmitate in the Type II cells, but not in the inflammatory cells of the granulomata. We conclude that the increased palmitate uptake in this disease in accounted for by the metabolic activity of the Type II pneumocytes.

This study was supported by USPHS Grants HL-02001, HL-05443, Pulmonary SCOR Award HL-15088, and CA-11096. The authors wish to acknowledge the expert technical assistance of Nordal Mantaras, Lourdes Fernandez, Valeria Shishkoff, and Carin Colbjorinsen.

The ACTH-releasing activity of hypothalamic extract and rat plasma was examined with the dispersed rat pituitary cell technique of Swallow and Sayers (8). Although both plasma and serum caused ACTH release which was dose-related, stress did not enhance the ACTH releasing activity. Furthermore, separation studies of plasma using ultrafiltration and gel separation suggest that the CRF activity in plasma is associated with molecules of a molecular weight greater than 15,000.

The hypothalamic extract used in this study was kindly provided by the National Institute of Arthritis, Metabolism and Digestive Diseases (NIAMDD) Rat Pituitary Hormone Distribution Program.