Experimental Biology and Medicine

Chick blastoderms at H & H stage 8-8⁺ were cultured in vitro on 1 ml aliquots of medium containing either 50 or 100 μg of lysergic acid diethylamide. Following 20 hr incubation, drug-treated embryos failed to demonstrate any increase in mortality or growth-depressing effects. At both dose levels, however, disturbances were observed in fusion of the neural folds. A significant reduction in the mean number of somites per embryo was observed in embryos exposed to 50 μg of LSD.

The in vitro release of lymphotoxin by human peripheral blood lymphocytes from noncancer patients, patients with cancer, and patients with Hodgkin's disease were studied. Noncancer patients and patients with cancer other than Hodgkin's disease released no lymphotoxin under unstimulated conditions while stimulation with phytohemagglutinin induced in vitro release of lymphotoxin. Lymphocytes from patients with Hodgkin's disease released significantly less lymphotoxin following stimulation with phytohemagglutinin but also released measurable amounts of a lymphotoxin-like substance in the unstimulated state. Hodgkin's disease lymphocytes appear to differ from the lymphocytes of noncancer patients and patients with cancer other than Hodgkin's disease by both lower in vitro lymphotoxin release following phytohemagglutinin stimulation and spontaneous release of lymphotoxin by unstimulated lymphocytes.

Two nonmetabolized amino acid analogues, α-aminoisobutyric acid (AIB) and 2-aminobicyclo |2,2,1| heptane-2-carboxylic acid (BCH) stimulated immunoreactive insulin (IRI) release from cultured fetal rat pancreas incubated in vitro. AIB was effective in the presence of either caffeine or glucose; whereas BCH was only active in the presence of the methylxanthine. AIB-induced IRI release together with glucose was inhibited by oligomycin or incubation at a low temperature. The uptake of ³H-AIB was demonstrated in this preparation. With changing experimental conditions, IRI release induced by AIB paralleled changes in AIB uptake (i.e., its active transport). The results suggest the existence of a relationship between the two phenomena.

Chemical composition of rectus sheath from normal, and indirect and direct inguinal hernia patients has indicated that in inguinal hernia the rectus sheath contains decreased amount of collagen as compared to controls. Total hexose, uronic acid, and hexosamine were not significantly altered.

Neutral proteolytic activity in the liver exists in an active and an inactive form which can be reactivated by the addition of ATP. The acute stress elicited by complete body restraint produced a significant decrease in the active form of the proteinases conceivably reducing the rate of protein catabolism at neutral pH. Full levels of proteolytic activity could be restored by the addition of exogenous ATP to the homogenates. The fact that stress situations produce a large release of steroids able to increase the rate of protein synthesis in the liver has suggested that the decreased proteolytic activity could be due to a decreased availability of the proteinase activator, at this point believe to be the aspartyl adenylate.

An immunosuppressive antilymphocyte serum prolonged the survival of skin allografts and suppressed the systemic graft-versus-host reactivity of lymph node cells from treated mice. Those same lymph node cells, however, were highly reactive when tested in the skin of the X-irradiated hamster, and that reactivity was dependent, at least in part, upon the presence of viable, histoin-compatible cell populations. We conclude that cells from the ALS-treated mice react well in that local assay while showing suppressed reactivity in the systemic assays of immunocompetence.

In cats with Pavlov pouches antrectomy increased maximal acid secretion in response to histamine (40% increase) and to pentagastrin (30% increase). The mechanism of this effect is not known.

The local Shwartzman reaction was produced in the Syrian and Armenian hamster following preparation and provocation by R. coli endotoxin. The Syrian hamster was also shown to manifest a systemic reaction. The Chinese hamster failed to respond by either a local or systemic reaction under identical experimental conditions.

CELO virus, from infected allantoic fluids, was treated with 1.0% sodium deoxycholate and 0.01% trypsin and centri-fuged to equilibrium in a rubidium chloride gradient. Two bands, at densities of approximately 1.34 and 1.29 g/cm³, were consistently found. Plaque titrations indicated that the band at 1.34 g/cm³contained the majority of infectious virus. Both hemagglutinating and CF activities were associated with these bands; however, soluble CF antigens were also present in the less dense regions of the gradients. The infectivity of purified CELO virions was heat stable and the virions did not contain the adenovirus group antigen.

A study of porpoise blood has shown the presence of “naturally-occurring” agglutinins in the serum which demonstrate three blood groups. These blood groups influence the survival of transfused cells, and thus have clinical significance. Before transfusion, donor bloods must be cross-matched with the recipient, even when the recipient has had no prior transfusions.

Daily variations in fattening responses to prolactin may be phased or driven by injections of adrenal steroids in a fish, Fundulus grandis, a lizard, Anolis carolinensis, and a pigeon, Columba livia. Daily injections of prolactin about 24 hr after injections of adrenocortical hormones favor the accumulation of fat stores; whereas daily injections of prolactin 6 hr after the adrenal steroids cause losses in fat. An experiment on the fish indicated that the rhythm of fat responsiveness to prolactin is capable of self-sustaining circadian oscillations on continuous light following initial synchronization by exogenous hydrocortisone. An experiment on the pigeon indicated that a daily rhythm of cropsac sensitivity to prolactin may also be driven by daily injections of corticosterone, but the temporal pattern of the adrenal steroid and prolactin that favors cropsac proliferation is not in the same phase relations with those that favor accumulation of body fat, liver fat, and increases in intestinal weight. It is concluded that a temporal synergism of prolactin and the adrenal steroids is an important organizational unit in the vertebrate system.

Experiments were completed in isolated strips of moderately large arteries from skin and skeletal muscle. The strips were situated in a reservoir through which blood from a dog was continually circulated by means of an extracorporeal system; tension was recorded by means of Statham force transducers and a Grass recorder. The response of the artery strips from both skin and muscle was always an increase in tension during serotonin infusion, even when the same strips relaxed during acetylcholine or histamine administration. Since in situ skin vessels always constrict during serotonin administration, while in situ medium to large skeletal muscle arteries may exhibit little response to serotonin, this agent may cause some indirect action in the muscle vascular bed.

Hepatomas were induced in approximately 4% of inbred LSH/LAK hamsters inoculated subcutaneously with CELO virus. The sera from these animals stained the CELO T antigen found in lytically infected chick kidney cells. The tumors contained hamster types A and C virus particles. The relationship between these indigenous hamster viruses and tumor induction by CELO virus is being investigated.

PHA-stimulated mouse lymph node lymphocytes were capable of supporting the replication of VSV and mengovirus. Other viruses failed to replicate. The PHA response of the lymphocytes was inhibited by viruses which caused a decrease in cell viability.

Inhibition of C hemolytic activity by administration of fumaropimaric acid resulted in development of immune response to tolerogenic deaggregated HGG. Mice equally treated but without FPA regularly developed immunological tolerance. From these and prior studies the conclusion can be drawn that C activity of blood is involved in some way in development of immunologic tolerance.

SQ-18,506, furazolidone, and nitrofurazone are metabolized by at least two enzyme systems in the microsomal and soluble fractions of rat liver. Under anaerobic conditions, the reduction in liver microsomes is mediated by an NADPH-dependent, carbon monoxide insensitive enzyme, presumably NADPH-cytochrome c reductase. Parallel increases in the rates of reduction of the nitrofuran compounds and cytochrome c were noted during the solubilization and partial purification of NADPH-cytochrome c reductase. In the soluble fraction, rat liver xanthine oxidase was able to reduce these compounds. Moreover, this reaction was blocked by allopurinol, an inhibitor of xanthine oxidase, and catalyzed by milk xanthine oxidase.

White blood cells, mainly lymphocytes, in culture from patients with cystic fibrosis, showed a diminution of the activity of beta-glucuronidase. The mean activity of this enzyme in cystic fibrosis was 30% lower than in the controls at 5 days of culture and 35% lower than the controls at 7 days. Acid phosphatase activity in cystic fibrosis cultures was not significantly different from control cultures, while beta-acetylglucosaminase activity was significantly decreased in cystic fibrosis cultures of 7 days compared to control cultures.

Nonlethal infection of adult (4-5 month old) MF-1 mice with coxsackievirus B₃ results in greater amounts of infective virus in their hearts and pancreases and more extensive lesions in these organs than are observed in adolescent (2 month old) animals. While significant differences in neutralizing antibody response to coxsackievirus infection were not demonstrated, adult animals produced relatively less interferon in response to infection and absolutely less in response to administration of Newcastle disease virus than did the younger animals.

Present evidence is inconclusive that differences in interferon formation account for the age-related difference in viral susceptibility observed in this model system.

The findings of the present study are in accord with clinical and epidemiologic evidence that the severity of viral infection increases with advancing age, if the contributions to resistance of the neonatal state or previously acquired specific immunity be excluded from consideration.

Lipogenesis, as measured by the incorporation of glucose-U-¹⁴C into fatty acids by adipose tissue slices was determined periodically in 8 pigs from the age of 35 days (9.0 kg) to 180 days (90.0 kg). In the suckling pig, the capacity for fatty acid synthesis was very low, presumably due to the high-fat content of sow milk. Upon ingestion of the high-carbohydrate diet, fatty acid synthesis increased rapidly and reached a peak at 67 days of age. From 67 days of age there was a gradual decrease in the capacity for fatty acid synthesis with advancing age. Changes in the activities of citrate-cleavage enzyme and malic enzyme occurred in parallel with the changes in fatty acid synthesis, supporting the participation of these enzymes in lipogenesis in pig adipose tissue.

Very low (5 × 10^-12 to 10^-10 M), as well as higher (10^-6 to 5 × 10^-6 M) concentrations of cyclic guanosine 3', 5'-monophosphate (cyclic GMP) stimulate the proliferation of rat thymic lymphocytes in vitro, but intermediate concentrations (10^-9 to 10^-7 M) of the cyclic nucleotide do not affect cell proliferation. Other guanosine monophosphates such as guanosine 5'-monophosphate (5'-GMP) and cyclic guanosine 2',3'-monophosphate (cyclic 2',3'-GMP) do not affect thymocyte proliferation.

The maximally mitogenic concentrations (5 × 10^-11 and 5 × 10^-6 M) of cyclic GMP cause rapid (within 10 to 20 min) rises in the cellular cyclic AMP content, but nonmitogenic, intermediate cyclic GMP concentrations do not affect the cellular cyclic AMP content. The higher cyclic GMP levels probably cause the cellular cyclic AMP level to rise by reducing the cyclic AMP-degrading activity of cyclic AMP-phosphodiesterase. The nature of the cyclic AMP-elevating action of the very low cyclic GMP levels is unknown. It is concluded that cyclic GMP can stimulate thymocyte proliferation by two distinct mechanisms both of which are mediated by cyclic AMP.

The rates of urine flow and the plasma fluoride concentrations were measured in anesthetized rats receiving continuous infusions of graded levels of fluoride in isotonic solutions. Infusion of 500 nmoles of fluoride/min produced the maximum diuresis, three times the mean control urine flow rate, and a plasma fluoride concentration of 272 μM. The 100 nmoles/min fluoride infusion rate produced an 80% increase in urine flow rate with a plasma fluoride concentration of 56 μM. These findings are consistent with estimates made for man and with the suggestion that the nephrotoxicity following methoxyflurane anesthesia is at least in part due to metabolism of methoxyflurane to inorganic fluoride.

Sarcomas induced by benzo[α] pyrene in the mature offspring of hamsters injected sc on the second day of pregnancy with Freund's incomplete adjuvant appeared earlier and grew more rapidly than did similarly induced tumors in the offspring of untreated mothers. Though females were intrinsically more susceptible than males to BP tumorigenesis, the enhancing effect of the adjuvant was approximately equal in male and female offspring.

Displacement radioimmunoassay of fructose-1,6-diphosphatase is described, capable of measuring quantitatively 100 mμg of enzyme in biological fluids. The preparation of radioiodinated FDPase with high specific activity, a necessary prerequisite for the radioimmunoassay, was complicated by the lability of the enzyme and was, therefore, investigated in detail. Low specific activities of radioiodination in the range of 1-8 mCi/mg of protein were obtained by the IC1 method. Using chloramine-T as an oxidant resulted in radioiodinated FDPase with specific activities in the range of 20-80 mCi/mg of protein. However, the enzyme proved to be sensitive against exposure to chloramine-T; best yields were obtained at short reaction times of less than 10 sec.

Prolonged treatment with poly I·Poly C was effective in suppressing the development of virus-induced murine sarcoma and the resultant mortality in infected mice. However, treatment at the dosage used was ineffective in preventing the infection of these mice by the murine leukemia virus present in excess in stocks of murine sarcoma virus. The poly I·Poly C treated virus-infected mice eventually developed lymphoma, and their spleens contained both murine leukemia, group-specific, complement-fixing viral antigens and infectious murine leukemia virus. These experiments suggest that poly I·Poly C treatment may not be effective against Friend and Rauscher leukemia viruses at high challenge doses.

The leg or hock abnormality of zinc-deficient chicks fed isolated soybean protein-based diets has previously been shown to be largely prevented by any of the following supplements: zinc, histidine, histamine, or any of several antiarthritic agents (aspirin, cortisone, phenylbutazone or indomethacin). Only zinc corrected all signs of deficiency while the other agents alleviated only the leg deformity. To better understand the mechanisms involved, the present experiments tested the effects on the leg abnormality of zinc-deficient chicks of certain vasoactive agents, compounds affecting histamine metabolism, certain vitamins required for normal hock development, and certain additional drugs which are known to influence experimental or human arthritis. Zinc, histamine, and chloro-quine, an antiarthritic agent, were shown to alleviate the hock abnormality. The condition was not influenced by the dietary addition of the other vasoactive amines, serotonin, norepinephrine, and epinephrine, or by two vitamins required for normal hock development, niacin and p-aminobenzoic acid. Likewise, dietary addition of pyridoxine or injection of heparin, agents which could influence histamine metabolism, was without effect. The injection of gold sodium thiosulfate, in contrast to all other antiarthritic agents tested to date, was without effect. The injection of mechlorethamine, an immunosuppressive agent, or statolon, an antiviral agent, had only slight and questionable beneficial effects.

A single intraperitoneal injection of L-dopa, the precursor of dopamine, into female rats significantly reduced serum prolactin concentration at 30 min, 1 hr, and 2 hr after injection compared to pretreatment levels or control rats not given this drug. A single injection of each of 3 monoamine oxidase inhibitors, pargyline, iproniazid or Lilly-15641, also significantly decreased serum prolactin below pretreatment values. Injection of L-dopa and pargyline together was more effective than either alone in lowering serum prolactin. Each of the above drugs is believed to reduce pituitary prolactin release because it increases catecholamine activity in the hypothalamus. By contrast, a single injection of methyldopa, which inhibits catecholamine synthesis, increased serum prolactin many fold over pretreatment levels. A single injection of d-amphetamine, a sympatheticomimetic drug, also greatly increased serum prolactin concentration.

Addendum

The hypothalami of the rats treated with saline (controls), iproniazid, pargyline, L-dopa or pargyline and L-dopa together, were assayed for PIF activity by our standard in vitro procedure. All drugs increased hypothalamic PIF activity and the combination of pargyline and L-dopa was more effective than either alone. These observations suggest that the increase in hypothalamic catecholamines produced by these drugs results in increased hypothalamic PIF activity, and this in turn depresses prolactin release by the pituitary.

Samples of renal venous plasma were obtained from dogs in which a lung from another dog was interposed in the renal artery. The lung was ventilated with various gas mixtures. In this way it was possible to locally alter the oxygen tension and flow rate of the perfusing blood. Reduction of oxygen tension to 6 mm Hg for 17 min was without effect on renin activity. Reduction of blood flow raised renin activity and superimposition of near anoxia failed to alter this changed activity. Therefore, these studies do not provide evidence for an acute local effect of hypoxia on renin secretion.

The ability of pure growth hormone (GH)-releasing hormone (GH-RH) to deplete pituitary GH content and simultaneously to increase plasma GH-like activity was investigated in the rat using the “tibia test” method. GH-RH (350 ng) induced a significant fall in pituitary GH content which was accompanied by increased GH-like activity in plasma. GH-RH administered at two dose levels, 80 and 400 ng, induced a dose-related fall in pituitary GH. There was a concomitant rise in plasma GH-like activity of rats given the larger dose. Plasma GH-like activity in rats injected with saline was not detectable. These results indicate that the pituitary depletion of GH content in rats caused by GH-RH reflects true release of the hormone.

The administration of 100 μg of MTX to newborn mice resulted in fatal intoxication of 50% of untreated mothers. The mortality rate of foster mothers when foster nursing was begun 24 hr after treatment of the newborns was 40%. None of the foster mothers died, however, when the foster nursing began 48 hr after the treatment. MTX is rapidly excreted, largely in the urine, within from 24 to 48 hr after administration. It is assumed that the antifolate entered the mothers' circulatory systems via the excreted urine of the newborns. Both natural and foster mothers died after a maximum of 24 μg/g, which is a manyfold lower dose than that tolerated in adults (LD₅₀ = 300 μg/g per os). It is thought that this striking deviation in tolerance reflects the folic acid deficient state in the immediate postpartum period in mice.

The effects of infection with mycoplasma (strain HT, mycoplasma fer-mentans) on cell morphology and growth were different for primary amnion cells and for SV40 transformed amnion cells before and after “crisis.” Post-“crisis” cells showed distinctly more growth reduction, cytopathic changes, and cell destruction than pre-“crisis” cells, although cell-associated mycoplasma was present in similar, high amounts. Pronounced resistance of primary amnion cells was correlated with a very low amount of cell-associated mycoplasma, even at high concentrations of free mycoplasma. The presence of the primary cells supported mycoplasma propagation, as determined in the culture fluid, much less than either pre- or post-acrisis” cells. There is indication that the primary cells may be responsible for a mechanism of inhibition of mycoplasma propagation. It is suggested that the differences in mycoplasma susceptibility among the three cell types are related to their different state of in vitro malignancy, and may serve as useful criteria in further studies of the changes occurring after the virus transformation.

Various methods for inducing AA in rats were examined using different rat strains, adjuvant preparations, and sites of injection. The time course of the development of arthritis in the injected and nonin-jected extremity was determined and methods for inducing a disease with limited variability and a high incidence was demonstrated. Standard compounds were tested in two models of AA with special reference to denning a method for discriminating between inhibition due to immunosuppression (IS) and that due to anti-inflammatory (AI) effects. 12 daily doses of IS agents, started on the day of adjuvant injection, totally prevented the disease in the noninjected extremity when measurements were made on day 21. Standard AI agents were ineffective. This test (nonestablished arthritis) was therefore capable of detecting compounds with immunosuppressive potential. The same standard compounds were administered daily for 12 days after the peak of the disease had been reached (established arthritis). In this test the IS compounds were ineffective in reducing the disease in the injected extremity, whereas the anti-inflammatory agents inhibited the disease in a dose related manner. The results indicate that with the use of both models of AA it was possible to detect compounds which were selectively either AI or IS.

C3H/Bi weanling mice were grafted with skin taken from 4-week-old preleukemia, syngeneic mice (GPA virus system) which had been inoculated at birth with Gross passage A virus. Careful analysis of the tissues of the grafted weanlings (carried out after natural death of the animals) revealed a high incidence of thymus enlargement and a 100% incidence of disseminated lymphoma. This indicates that leukemogenesis was induced by means of skin transplantation in 100% of the weanling animals by the skin grafting of preleukemia skin.

Changes in the rat plasma concentration of cholesterol were effected by lecithin infusion, biliary obstruction, clofibrate administration, and by induction of hypothyroidism. The extent of incorporation of ³²P into hepatic phospholipid was altered (i.e., decreased) in only those rats subjected to lecithin infusion. However, changes in the plasma content of labeled phospholipid were observed only in those animals subjected to biliary obstruction (increase) or clofibrate (decrease) administration.

Cell cultures grown as monolayers conventionally may be removed from the surface of the culture vessel by physical or by chemical methods. Physical methods, such as scraping the cells from their growth substrate, generally yield heterogenous suspensions ranging from single cells to cell aggregates of varying size to monolayer membranes. Furthermore, many cell types, such as human fibroblasts, do not yield readily to removal by physical means. Most chemical methods for the removal of cell monolayers employ proteolytic enzymes. Although chemical techniques tend to produce more uniform single cell suspensions, the possibility exists that repeated exposure of cell cultures to proteolytic enzymes may have deleterious effects upon cell membranes. However, it is also possible that cell membranes are effectively “rejuvenated” with each division; in which case one could consider that individual cells are exposed to proteolytic enzymes only once and that membrane damage therefore might not be cumulative.

Here we introduce a new physical method for subculturing monolayer cell cultures, one in which the cells are not removed from occupied space, but the occupied space is removed and replaced with available and accessible space.

Materials and Methods. Cell cultures of human fibroblasts were initiated by trypsinization of neonatal foreskins obtained at the time of circumcision. They were maintained at 37° and 5% CO₂. Cells were fed twice a week using Eagle's minimum essential medium fortified with calf serum, penicillin, streptomycin, and Mycostatin. Cultures were doubled when the cell monolayers became confluent.

The standard enzymatic method for subculturing monolayers is as follows: spent culture medium is aspirated from plastic tissue culture dishes (Falcon No. 3002) as completely as possible and the cultures are washed briefly with 2 ml of phosphate buffered saline. The saline is aspirated and 1 ml of 0.25% trypsin in calcium- and magnesium-free Hanks' balanced salt solution is added to the dish.

In midcollicular decerebrate cats, four benzodiazepine analogues, Ro 5-4023, Ro 5-3059 (nitrazepam), Ro 5-3350, Ro 5-6901 (Dalmane) were found to be very effective in blocking spinal polysynaptic reflexes. Subsequent to spinal cord transection during the period of drug-induced depression, these reflexes returned and became highly resistant to depression by these drugs. With stimulation experiments, these four agents depressed both the reticular facilitatory and inhibitory effects on the knee jerk. It was concluded that these drugs act mainly on supraspinal structures, most likely in the brainstem reticular system.

U-29,409 [2,3-dihydro-2-(1-naphthyl)-4(1H)-quinazolinone] is an antifertility compound in the rat. Antifertility is due to inhibition of spermatogenesis—which is accomplished by sc, ip, or oral treatment administered as single or multiple doses. Lowered fertility commences in the second week and may continue, depending upon severity of the dose, through the tenth week. Libido, as measured by ability and desire to mate, is not altered. U-29,409 affects spermatogenesis with a massive exfoliation of immature germinal cells which then migrate from the testis to the excurrent ducts.

The Randall—Selitto assay, measuring the reaction threshold of the inflamed rat paw to pressure, was modified to obtain quantal data. Inclusion of maximum “cutoff pressures” in the calculation of mean responses is not necessary and relative analgetic potencies of drugs may be determined graphically by a probit plot.

Rabbit corpora lutea were incubated with estradiol 17-β to verify a direct stimulatory role of estrogen on progesterone biosynthesis. A significant increase in net progesterone and 20α-ol synthesis was obtained with no concurrent alteration in free and esterified cholesterol content of luteal tissue. Pretreatment with aminoglutethimide in vivo resulted in accumulation of cholesterol in luteal tissue, but estrogen failed to stimulate progesterone synthesis above control levels.

These results demonstrate a direct effect of estradiol on rabbit corpora lutea in vitro, but suggest the possibility of a unique sterjo-dogenic mechanism for the species.

The effects of an estrogen (mestranol), a progestational agent (norethindrone), and a combination of the two hormones on plasma zinc concentration, growth, and zinc-65 uptake in selected tissues were investigated in female rats fed a zinc-sufficient diet. Mestranol significantly (p<0.02) lowered plasma zinc concentration whereas norethindrone had no effect. Mestranol caused a significant increase in the uptake of a single injected dose of zinc-65 in the liver, spleen, adrenals, and uterus. Mestranol also caused a significant decrease in the growth.

Intravenous injection of tolbutamide (15.0 mg/kg) caused a significant decrease of plasma FFA and BS concentration in geese and ducks, but not in owls. The decrease of plasma FFA was significant 5 min after the injection whereas the decrease of BS became significant first at 15 min of injection. The decrease of plasma FFA produced by tolbutamide in geese and ducks whose plasma FFA had been elevated by continuous infusion of glucagon (0.2 μg/kg/min) was not greater than that observed in intact birds injected with the same dose of the drug. Glucagon infusion in geese previously injected with tolbutamide caused an elevation of plasma FFA comparable to that observed when glucagon was infused at the same rate in intact birds. These results indicate that tolbutamide does not inhibit the adipokinetic effect of glucagon in vivo.

Pyran copolymer markedly sensitizes mice to the lethal effects of endotoxin. The sensitization occurs throughout both phases of RES activity. Pyran action on microsomal enzymes necessary for the metabolism of hexobarbital or antipyrine is not responsible for the sensitization because reversal of this activity with chlorcyclizine does not alter endotoxin sensitization. Preheparinization of pyran-treated mice only slightly prolonged the survival time, but did prevent the kidney damage.

The relationship in mice between immune status to polyinosinic·polycytidylic acid (poly I·Poly C) and interferon induction by poly I·Poly C was studied in vivo and in vitro. Circulating antibody to poly I·Poly C reduced the in vivo interferon response to poly I·Poly C. A similar but less profound inhibitory effect of specific antibody upon interferon induction was demonstrated in vitro. The findings imply that antibody-poly I·Poly C immune complexes are less potent interferon inducers than poly I·Poly C without antibody. Immunological tolerance to poly I·Poly C did not alter interferon induction by poly I·Poly C indicating dissociation between immunological status and interferon induction with this synthetic RNA in vivo.

The serum of man and other animals has been found to contain a growth factor of protein nature, and chemical modifiers of its biological activity. The growth factor, in one of its two readily interconvertible forms, will support the growth and reproduction of nematodes in defined culture; the other form is without growth promoting activity in the cultures. Distinct materials have been separated from serum which either convert the growth factor into the activated form or prevent its conversion. These have been called activator and inhibitor, respectively. Although growth factor has growth promoting activity in tissue cells in culture as well as nematodes, nematodes provide the most convenient quantitative assay of growth factor and of its chemical state of activation. Its biological activity in all systems depends on which form it is in. Specific chemical interaction has been demonstrated between growth factor and its natural modifiers. The balance of physiological activator and inhibitor appears to regulate its biological effectiveness.

Three days after splenectomy rats unable to form normal hemolysin titers to sheep erythrocytes will produce normal titers when they are injected with leucogenenol as well as sheep erythrocytes. Normal rats do not respond with an increase in hemolysin titers when they are injected with leucogenenol as well as sheep erythrocytes. The injection of leucogenenol apparently stimulates cells that are potentially capable of antibody synthesis.

The results of these studies show that in the pig the intestine contributes only about 4% to cholesterol synthesis from acetate-1-¹⁴C while liver and adipose tissue contribute 67 and 29%, respectively. Each of the proximal three segments of the intestine contributed more to cholesterol synthesis than did the distal segment. Fatty acids were synthesized primarily in the adipose tissue of the pig when pyruvate-3-¹⁴C was the substrate while the intestine and liver together contributed only about 9% to overall fatty acid synthesis. Fatty acid synthesis from acetate-1-¹⁴C in pig intestine was of minor importance when compared with liver or adipose tissue fatty acid synthesis.

Following intravenous injection of ⁵⁷Co vitamin B₁₂, clearance rates of en-dogenously labeled TCII and TCI were delayed in patients with C.M.L., compared to normal subjects and cases of P.A. If ⁵⁷Co B₁₂ from TCII reappeared bound to TCI, it occurred at a rate slower than that of plasma disappearance of ⁵⁷Co B₁₂ bound to TCI.

Testicular metabolism was examined after adult rats were adrenalectom-ized and/or treated with corticosterone (0.7S, 1.5, and 3.0 mg/day) for 5 days. The weight and DNA and RNA concentrations of testes were not affected by either adrenalectomy or corticosterone replacement but corticosterone (3.0 mg) reduced (p<0.05) testis protein and prevented the loss of testis carbohydrate noted after adrenalectomy. Anaerobic glucose catabolism (cell-free preparations) increased 12% (p<0.05) after adrenalectomy and decreased 13% (p<0.05) after corticosterone (3.0 mg) therapy but neither of these treatments affected aerobic glucose catabolism. Corticosterone (3.0 mg) depressed (p<0.05) the oxidation of glucose-1-¹⁴C and glucose-6-¹⁴C to ¹⁴CO₂; whereas adrenalectomy showed a tendency to increase the activity of ¹⁴CO₂. Testosterone synthesis dropped (p<0.05) after corticosterone (3.0 mg) therapy but was not altered by adrenalectomy. The results suggest that adrenalectomy modifies testicular glucose ca-tabolism and that corticosterone may affect the testis by reducing glucose utilization and testosterone synthesis.

A quantitative hemagglutination-inhibition test was developed for the determination of endotoxin (hapten) in serum of mice. When the blood sera of conventional mice given a minimal or severe burn were tested for circulating endotoxin, significantly elevated titers of E. coli endotoxin (or hapten) were found in the sera. There is as yet no conclusive evidence, however, that the circulating endotoxin is biologically active.

These studies demonstrate a major effect of age of animals on the effect of (poly rI)·(poly rC) on Moloney sarcoma virus-induced tumors. In 4-6-day-old mice, tumors appeared later in the treated than in the control group, whereas in 20-day-old mice, treatment enhanced tumor formation.

The T-1 cell line, derived from the human kidney, responds to two active thyroxine analogs, dijodothyropropionic acid (DTP) and triiodothyroacetic acid (TTA), qualitatively paralleling findings made with other systems. Compared to thyroxine, DTP is about equivalent in stimulating cellular proliferation, but possesses only 0.1 its potency in augmenting colony formation by these T-1 cells; TTA is at least 10 times more potent. These observations extend the usefulness of the T-1 cell line as an in vitro model for characterizing the peripheral response to the iodinated thyroidal hormones.

Hepatic disposition of DiBSP was studied in adult and newborn guinea pigs from days 2 through 16 of life after intravenous administration of DiBSP in doses sufficient to achieve maximal rates of dye excretion into bile. Neonatal guinea pigs showed a significant reduction in the maximal rate of dye excretion into bile when compared to adult animals. A minimal estimate of hepatic uptake of DiBSP showed that uptake was similar in neonatal and adult animals and was significantly greater than the maximal rates of excretion into bile. Hence hepatic uptake did not limit hepatic disposition of DiBSP. With aging, injected DiBSP was excreted at progressively more rapid rates reaching adult levels of excretion at the beginning of the second week of life. This study demonstrates the presence of a defect in the excretory process by which DiBSP is transported from liver cells into bile in the neonatal guinea pig.

Potassium iodide (KI) pretreatment; which has been reported to increase the clinical effectiveness of cyclophosphamide, increased the lethality of cyclophosphamide for mice. However, KI pretreatment failed to increase the experimental therapeutic effectiveness of cyclophosphamide for mice inoculated with LI210 leukemia. KI pre treatment also failed to alter hexobarbital sleeping times. There appears to be no significant synergistic action of KI on the therapeutic effects of cyclophosphamide investigated.

Inbred LSH and MHA hamsters were compared with random bred LVG hamsters for susceptibility to induction of tumor by SV₄₀, adenovirus 7, or adenovirus 12 viruses and for appearance of tumor after homologous transplantation. The LSH inbred strain proved superior to the MHA inbred line in trems of susceptibility to induction of tumor by SV₄₀ and adenovirus 7 virus and showed a shorter latent period after transplant with homologous SV₄₀ virus tumor. The LSH animals were also somewhat better than MHA hamsters from the point of view of husbandry. The susceptibilities of the inbred LSH and random bred LVG animals to induction of tumor by viruses were similar and the transplantation characteristics of tumors evolved in the two breeds of animals were approximately the same. The inbred LSH hamster provides a satisfactory model for induction of tumor by virus and for investigations of transplant tumor immunology employing a truly syngeneic system.

Actomyosin concentration, its viscosity response to ATP, and total phosphorus levels in subcellular fractions were determined in fasted (1-5d) and alloxan diabetic (3-210d) rats. No alterations in these parameters were detected as a result of fasting. However, during alloxan diabetes cardiac actomyosin ATPase activity was reduced for at least 7 days following alloxan administration and returned to normal levels 14 days after alloxan injection. These transitory toxic effects on cardiac muscle indicate that alloxan is not specifically beta cytotoxic.

The template activity of mouse liver chromatin is elevated when examined 5 hr after infection of the animal with D. pneumoniae. At 14 hr, the activity falls below control values. These changes demonstrate an early influence of generalized infection on the control of transcription in the liver. Infected, adrenalectomized mice do not respond with the early rise in chromatin template activity, suggesting the role of glucocorticoid mediation. It is proposed that these changes in transcription characteristics are related to changes known to occur in protein synthesis during the course of bacterial infection.

The subcutaneous or oral administration of melatonin to immature rats which had received PMSG to induce follicular growth and ovulation caused a significant reduction in the incidence of ovulation. Furthermore, the administration of HCG concurrently with melatonin overrode the inhibitory effect of melatonin. These observations support the concept that the pineal gland exerts control over reproductive functions via an inhibitory action of melatonin on the release of hypophyseal gonadotropins.

Chlorite-oxidized oxyamylose (COAM), a polyacetal carboxylic acid, enhances the formation of antibodies against sheep red blood cells and influenza virus hemagglutinin. The effect was present only when the polymer was included in the immunizing antigen injection (intraperitoneally or intravenously), not when it was given at a time interval before the antigen or by a different administration route. This indicates that an interaction occurs at the site of injection between antigen and polymer, or between polymer and host cells which are involved in antigen processing.

Blood flow and heat loss characteristics of peripheral areas, such as the hand, change with continuous exposure to cold. In man, blood flow and heat loss from the hand in general indicate less vasoconstriction and greater heat loss at temperatures of 1-5° in cold-adapted persons (1). In the rabbit ear, it has been shown that the relation of thermal conductance to blood flow is temperature dependent (2); that the cold exposures alter the response of the ear circulation to norepinephrine (less change in blood flow and ear temperature) (3), although this effect appears only at higher skin or blood temperatures (5); and that the precooling of arterial blood during its flow from the interior to the ear surface in a cold environment is more marked in cold-adapted animals than warm-adapted (4). The investigation described in this paper is concerned with the heat transfer from the rabbit ear when local blood temperature is changed.

Methods. Male New Zealand, California white and Dutch-belted rabbits were used. The rabbits were kept at 3-5° for 8 weeks or more (cold-adapted, CA) and at 25° (warm-adapted, WA). Body weight and food consumption were checked twice a week. Food and water were given ad libitum.

The surgical and perfusion procedures in the experimental preparation have been described previously (5). Perfusion pressure was monitored. Blood flow was set to give an approximate blood flow of 20 ml/min·100 ml of tissue. The blood flow was measured with a drop counter.

The magnesium deficient rats developed red ears and were unable to gain weight at a normal rate after 5 days on the magnesium deficient diet. The plasma magnesium concentration in animals on a magnesium deficient diet for 1 week was approximately 20% of control values and dropped further, although insignificantly, during the following 3 weeks.

The plasma alkaline phosphatase activity in magnesium deficient animals was depressed to approximately 50% of control values by 1 week and remained at this level over the next 3 weeks. A significant decline in the erythrocyte pyrophosphatase activity in magnesium deficient rats did not occur until the second week and remained constant during the following 2 weeks. The slower change in erythrocyte pyrophosphatase activity, when compared with plasma alkaline phosphatase activity, was related to a slower decrease in erythrocyte magnesium content. The whole blood ATP content was significantly lower in the magnesium deficient animals after 2 weeks.

The number of erythrocytes per unit volume of whole blood progressively decreased with time in magnesium deficient animals when compared with control values. The magnesium deficient animals had a macrocytic, normochromic anemia. Microscopically, the erythrocytes were spherocytic in magnesium concentration in magnesium repleted animals returned to control values within 1 week. The erythrocyte factors, namely, erythrocyte pyrophosphatase activity, erythrocyte magnesium concentration, and hemoglobin, in magnesium repleted animals progressed toward the control values, but were still below control values at the end of 2 weeks. Pair-fed magnesium deficient and control animals exhibited essentially the same changes as animals provided the respective diets ad libitum at 28 days.

From these observations, we conclude:

1. Fragments of kidney, implanted in subcutaneous tissue undergo coagulation necrosis similar to necrosis which supervenes after ligation of the renal artery. The necrosis may result from failure of the kidney to elicit a prompt vascular response in the surrounding tissue within the implant. Scattered fragments of pelvic transitional epithelium remain viable.

2. Osteogenesis is observed in more than one-third of the implants as early as 14 days after implantation; yet the size of bone tissue is small at this time and a single section through the tissue may not disclose it.

3. implantation of cortex alone results in no bone formation. The likely stimulus to bone formation is the transitional pelvic epithelium which is implanted along with the medulla.

4. Implantation of kidney fragments in subcutaneous tissue seems to unify two previous models frequently used to study heterotopic osteogenesis; i.e., ligation of renal artery and transplanation of bladder epithelium.

Washed suspensions of group A, C, and G streptococci and group A L-forms possess phosphatase, esterase, and N-acetylglucosaminidase, respectively. Cell-free extracts, obtained from streptococci and Informs by mechanical disintegration or by treatment of group A streptococci with phage-associated lysin, possess, in addition to these enzymes, an adenosine triphosphatase (ATPase). Over 90% of the total ATPase and NAGAase and 50% of phosphatase and esterase activities were solubilized by cell breakage, indicating that the latter 2 enzymes are membrane-bound.

A partial separation between the phosphatase, NAGAase, and ATPase was achieved by gel filtration of cell-free extracts on Sephadex G-200. Phosphatase was eluted with high molecular weight material excluded from the column. NAGAase and ATPase were associated with much lower molecular weight fractions, while esterase activity was present in both high and low molecular weight fractions. Studies on substrate specificity showed that fractions which split PNP-phosphate also split PNP-acetate and PNP-propionate fractions which split ATPase also split CTP, ITP, and GTP but to a lesser extent. Fractions which were active against β-napthylacetate also split β-naphthyl butyrate (15% efficiency); no activity against longer fatty acid esters of PNP or β-naphthyl derivatives was present.

The activation and inhibition of the enzyme activities by di- and trivalent cations and by sugars is also reported.

The in vitro incorporation of L-(³H)-phenylalanine into proteins of the APG, and hypothalamus was measured in female rats given androgens soon after birth and compared with control animals during sexual cycle. Incorporation of labeled phenylalanine into proteins of the hypothalamus and APG were higher during proestrus and estrus than during diestrus and metaestrus. Protein synthesis in the hypothalamus and pituitary of androgenized rats was the same as in the normal group during diestrus and metaestrus, and always lower than in controls during proestrus and estrus.

Coxsackie virus B₄ has been shown to produce significant lesions in the large and small blood vessels of 2-day-old and 12-day-old mice. The Coxsackie virus B₄-infected mice developed focal endothelial cell degeneration and necrosis with subsequent desquamation and denudation of intima of the aorta, coronary arteries, large veins, and other blood vessels.

Viral infections in man are common, but infection of the great blood vessels remains little studied. Viral lesions are suggested as an initiating factor in the production of vascular diseases in man, possibly including atherosclerosis.

RMI 10,393 is a new compound which inhibits aggregation of human platelets in plasma. It is effective against aggregation induced by ADP, thrombin, collagen, epinephrine, and serotonin. In the effective concentration range it also inhibits clot retraction, but does not cause activation of platelet factor 3. In an attenuated system activation of a platelet-derived procoagulant effect occurs, but this is not seen in the standard clotting system. RMI 10,393 has no effect on soluble clotting factors. A compound with such activity may have potential as an antithrombotic agent.

Three groups of male weanling rats of the BHE, Wistar, and inbred BHE strains were fed a 6% corn oil, 40% protein, 45% carbohydrate diet until they were 100 days old. Half of the animals were killed after a 24-hr fast while the remaining animals were killed without prior fasting. Animals of the BHE and IN-BHE strains had lower levels of IRI and higher blood glucose levels than animals of the Wistar strain. Strain differences in the fasting and nonfasting activities of the hepatic enzymes and in the lipid levels of the blood and liver appeared to be related in part to the insulin status of these animals. However, the data also suggested that different mechanisms of internal control of intermediary metabolism exist for each of these strains.

The colorimetric procedure of Dill et al. (11), originally described for DPH, has been modified to permit assay of both DPH and HPPH in the same sample of plasma or urine. In a normal adult subject receiving a single 250 IV dose of DPH, the maximum plasma level of HPPH occurred about 8 hr after dosing. A direct relationship was found to exist between the plasma levels of HPPH and the rate of urinary excretion of this metabolite.

The extent to which platelets adhere to glass is dependent on fibrinogen concentration. Platelet adhesion increased progressively in a linear fashion when increasing concentrations of purified fibrinogen (0.5-14.0 mg/100 ml) were added to the blood of a patient with congenital afibrinogenemia.

Since the isolation of angiotensin from dog plasma 30 years ago, investigators have devoted considerable effort to elucidating the mechanism of regulation of renin secretion. Initially, attention was directed at the role of renal arterial pressure, or pulse pressure as the regulating factor. However, more recent studies, using a refined technique for measuring renin activity, demonstrate that renin secretion can vary quite independently of arterial (1) or pulse pressure changes (2). A number of reports appear to stress the role of the autonomic nervous system (1, 3-5), body sodium or volume changes (6, 7), and intrarenal mechanisms unrelated to blood pressure changes. Because of their anatomical and functional relation to the renin secreting cells of the juxtaglomerular apparatus, the macula densa cells of the distal tubule have been considered to be involved in the intrarenal regulation of renin secretion (8-11). The sodium concentration of the tubular fluid or sodium reabsorption at this site may in some way affect renin release.

Part I of the present study was designed to examine the role of this intrarenal mechanism, namely, to see the effect of changing distal sodium reabsorption on renin secretion. It was found that the saluresis brought on by administration of chlorothiazide in the dog resulted within 15 min in an increased secretion of renin even when urinary loss of water and salt was replaced.

Part II therefore was undertaken to see whether this same effect of chlorothiazide obtained in man and whether this effect was under the influence of the autonomic nervous system.

The effect of the diuretic on peripheral renin activity was measured before and after neuronal depletion of catecholamines induced by chronic reserpine administration.

Part I. Methods. This study was carried out in 10 mongrel dogs, weighing between 10 and 15 kg.

Binding of dapsone and monoacetyldapsone to human plasma proteins has been investigated by means of an ultrafiltration technique. When binding was studied in vivo or in vitro at therapeutic concentrations of the drugs, dapsone was 70 to 80% bound, and monoacetyldapsone 98 to 100%. Rapid and slow acetylators of dapsone bind both compounds to the same degree. Protein binding of monoacetyldapsone may well account for the limited excretion of this compound in the urine. The contribution of protein binding to the long plasma half-lives of these compounds must be evaluated in the light of future studies.

The ip administration of 1.5 g/kg of D-galactosamine HCl in a single injection resulted in profound histological and biochemical evidence of hepatocellular injury.

Electron microscopy disclosed focal cytoplasmic degeneration 6 hr after injection and this was paralleled by a progressive increase in the serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase. Progressive cellular injury was accompanied by lipid accumulation, hyperplasia of the smooth endoplasmic reticulum, and organelle injury involving almost every cell. Although liver triglyceride levels were normal 6 hr following D-galactosamine administration, at 24 hr significant hepatic triglyceride accumulation was demonstrated. Serum enzyme levels, histologic features of the liver, and hepatic triglyceride content remained normal in animals treated with equimolar D-glucosamine HCl and 2-deoxy galactose.

Specific enzyme activities and isoenzyme patterns were determined in cell-free extracts from uninfected and virus-infected mammalian cultures. Samples were harvested at intervals prior to the appearance of morphologic changes in the cells, Two enzymes of the Embden-Meyerhof pathway, 2 of the tricarboxylic acid cycle, 1 in the hexose monophosphate shunt, and 4 hydrolytic enzymes were tested. Enzyme assays revealed differences in specific activities from infected cells as compared to controls. The main differences occurred during the early stages of contact between virus and cells and at a late time period when viral particles were accumulating in the cells. The malate dehydrogenase isoenzyme patterns from cultures infected with pseudorabies virus and harvested just prior to the appearance of morphologic changes had a different pattern than those of comparable uninfected cultures. The difference involved a predominance of the anodal band with some deletion of the bands that remained closer to the origin.

The phosphomonoesterase and Mg- (Na + K) -adenosine triphosphatase (ATPase) activities were measured in the mucosa of the small intestine of hamsters (Mesocrkelus auratus) exposed to 1,000 rads ⁶⁰Co whole-body irradiation. The enzyme assays were made in irradiated and sham-irradiated hamsters sacrificed at intervals through Day 4 postirradiation. The two enzy-mic activities expressed per unit of tissue weight were significantly higher in the irradiated animals as compared to the sham-irradiated group at Day 2 postirradiation. No changes were found in the protein contents of jejunal mucosa between irradiated and sham-irracliated animals. While the ATPase activity was significantly lowered at Day 4, the phosphomonoesterase activity was observed to be higher at that time. The increase in enzyme activities may represent increased synthesis of these enzymes in the crypt cells that gave origin to the mature cells of the intestinal villi of animals sacrificed at Day 2 postirradiation. The increase in ATPase activity at Day 2 followed by a decrease at Day 4 corresponds to the previously observed alterations in the intestinal glucose absorption by irradiated hamsters.

Heart-lung machines can effectively oxygenate blood, but their prolonged use causes difficulties not directly related to the patient's heart condition or the specific procedures used to correct it. In extreme cases a patient may be seriously affected and may develop thromboses, abnormal permeability of the vascular system (particularly in the lungs), blood clots, and occasionally brain damage (1, 2). The results of several studies (3-6) suggest that denaturation of blood proteins at blood-gas interfaces in the heart-lung machine may be responsible for some of the postoperative difficulties.

We have found no specific studies of the effects of the gas/liquid interfaces in the heart-lung machine on individual blood proteins. Therefore, we undertook an investigation of the denaturation of gamma globulin, albumin, and their mixtures in the disc oxygenator. The advantage of studying individual blood proteins is that the results can be interpreted more easily than studies with whole blood. However, data obtained in this simple model system can, at best, tell only part of the story regarding the medical complications of open-heart surgery.

We chose to work with gamma globulin because of its instability at the gas/liquid interface. This instability is easily demonstrated. If a stream of air is passed through a clear solution of gamma globulin, a precipitate of the aggregated protein rapidly forms. Albumin was selected for study because it is one of the principal components of whole blood and because it is often added to solutions of labile proteins as a stabilizer.

The Complement System and Denatured Gamma Globulin. The complement system can be activated by interaction with antigen–antibody complexes or by interaction with heat denatured, aggregated gamma globulin. Activation is known to increase capillary permeability, to enhance phagocytosis, to initiate chemotactic migration of leucocytes, and to damage membranes (7, 8).

The circulatory effects of morphine sulfate were studied in anesthetized dogs in which myocardial infarction had been produced by serial ligation of coronary arteries. In control dogs, morphine sulfate (0.5 mg/kg) produced early and transient decreases in aortic mean pressure, the rate of rise of left ventricular pressure and systemic vascular resistance, without significant changes in left atrial pressure, heart rate, or aortic blood flow. Following myocardial infarction, morphine sulfate (0.5 mg/kg), produced a similar early and transient depression of aortic mean pressure. The rate of rise of left ventricular pressure was more greatly depressed and remained depressed for a longer period of time. Systemic vascular resistance fell initially but was elevated at 15 and 30 min. Left atrial pressure was markedly decreased while heart rate and aortic blood flow were moderately depressed. It is concluded that morphine sulfate has a mild transient depressant effect on the circulation in intact animals, while in animals with myocardial infarction, morphine sulfate has a more pronounced and more persistent depressant effect on the circulation.

The prophylactic and spaced administration of clofazimine was studied. The drug had an effect when given orally for 2 days immediately after infection, but had no effect when given 5 weeks before infection. When given once every 4 weeks from Day 70 to Day 140 after infection, the drug had a distinct effect. It was also active when injected intraperitoneally on the day of infection.

The effect of clofazimine was compared with another, more newly developed rimino-compound, B1912, in M. leprae infections in mice. The two drugs were found to have very similar activity.

Deficient DNA repair in fibroblasts from xeroderma pigmentosum is expressed as markedly reduced survival after low doses of uv. However, the life-span in vitro of XP cells is not reduced below normal age-matched controls. Senescence of normal cells in vitro, measured by decreased plating efficiency, appears before any measurable decline in either the capacity for DNA repair or survival following uv. In no cases was spontaneous transformation to permanent cell lines observed. It is concluded that failure in DNA repair is probably not causal to aging in vitro or in vivo.

The effects of caffeine sodium benzoate on glucose utilization, hexose monophosphate shunt (HMP), and oxygen consumption of polymorphonuclear leukocytes (PMN) during phagocytosis were studed. The stimulation of HMP during phagocytosis was depressed when the dose of caffeine was in the range of 0.5-1.0 mg/ml. Caffeine doses of 1.0 mg/ml, or greater, depressed the HMP shunt significantly during phagocytosis; whereas the glucose utilization was affected less. The stimulation of oxygen consumption by latex particles was terminated with a caffeine dose of 0.5 mg/ml. No stimulation occurred if the same dosage was used before the addition of latex particles. Control experiments with glucose and sodium benzoate did not change the HMP shunt and glucose utilization during phagocytosis. The data presented indicate that the preferential effect of caffeine during phagocytosis by polymorphonuclear leukocytes is the inhibition of the direct oxidation of glucose via the HMP shunt. Caffeine inhibits the stimulation of oxygen consumption during phagocytosis.

Lactic acid production in the presence of high oxygen tensions (aerobic glycolysis) was found to occur in the teleost retina. Forty-two percent more glucose was utilized and 33% more lactic acid was produced under anaerobic than under aerobic conditions. Glycolysis was inhibited 33% by elevated oxygen (Pasteur effect), and it was further demonstrated that glycolysis is dependent upon the integrity of the retinal cells, being almost completely abolished by cellular disruption.

The shift in the oxygen dissociation curve to the right by acid metabolites causes release of oxygen from hemoglobin in the choroid region. This O₂ release explains in part the high PO₂ encountered in the vicinity of the retina. The Pasteur effect may function as a possible component of a negative feedback control loop for the generation of high PO₂ in trout eyes.

Recently several investigators have shown that the immune response of an animal immunized with hapten-carrier conjugates is significantly influenced by the nature of the carrier molecule. Siskind et at. (1) demonstrated in guinea pigs that the carrier to which DNP is coupled influences the amount, type, and functional activity (i.e., affinity) of anti-DNP antibodies, and Fronstin, Sage, and Vazquez (2) have shown that the degree of anti-DNP responsiveness elicited in mice is directly related to the antigenicity of the carrier.

The present study examines the antihapten antibody response of guinea pigs immunized with arsanilic acid conjugates of homologous and heterologous serum protein carriers, and compares the effect of natural and acquired tolerance to the carrier on these responses.

Materials and Methods. Random bred, adult male albino guinea pigs (300-360 g) were used in these experiments.

Reagents. Azobenzenearsonate (ABA) conjugates were made as before (3) by diazoti-zation and coupling in the ratio of 10^-5 moles arsanilic acid to 10 mg protein. Human, bovine, rabbit, and guinea pig serum albumins (HSA, BSA, RSA, GSA) and gamma globulins (HGG, BGG, RGG, GGG), obtained from Pentex Inc., Kankakee, Ind., were utilized as carrier proteins. In addition, arsanilic acid conujgated to egg lysozyme (Worthington Lab.) was used as the test antigen in studies of the antihapten antibody response. All conjugates were dialyzed against 0.01 M borate buffer pH 8.5 before use.

Immunization procedure. Guinea pigs were immunized with 0.1 ml of complete Freund's adjuvant containing 100 fig of ABA-protein distributed equally among the four footpads. The complete Freund's adjuvant was prepared with 8.5 parts light mineral oil: 1.5 parts Arlacel A: 5 mg/ml killed, dried M. tuberculosis.