Experimental Biology and Medicine

We have measured adipose tissue and plasma FFA and glycerol concentrations in order to investigate the mechanism by which FFA turnover is dramatically decreased during burn shock in the guinea pig. These data, in conjunction with previous work demonstrating acidosis and elevated plasma glucose, lactate, catecholamines, and glucagon, suggest that metabolic alterations are a major factor contributing to the decrease in release of FFA from adipose tissue. Whether or not the alterations in adipose tissue metabolism are related to decreased tissue perfusion remains unknown.

Iodine has been found to be virucidal against rhinovirus on human skin. The kinetics of in vitro rhinovirus inactivation by iodine and the residual virucidal activity of iodine on skin were examined in this study. Maximum inactivation of rhinovirus in vitro occurred within 3 sec with aqueous iodine concentrations of <100 μg/ml. Aqueous iodine concentrations of ≥10 μg/ml were ineffective. Inactivation of rhinovirus on the skin of the hands was accomplished by applying iodine in concentrations of 20,000 μg (2%) or 10,000 μg (1%) iodine/ml. Viral inactivation took place after 3 min of exposure to skin treated with the higher concentration, but took up to 20 min with the 1% solution. Viral inactivation with 1% iodine followed first-order kinetics. The virucidal activity of 2% iodine on hands persisted for up to 2 hr. Iodine will be used to attempt to interrupt hand contact transmission of rhinovirus infection.

The content of norepinephrine, its release, and subsequent disposition was studied in unlabeled and [³H]norepinephrine-labeled strips of canine coronary artery using standard superfusion techniques. Endogenous norepinephrine was isolated from superfusate and vessel by column chromatography then measured using high-pressure liquid chromatography with electrochemical detection. Release of NE was studied in both unlabeled and radiolabeled preparations. The relative importance of neuronal and extraneuronal dispositions of norepinephrine was determined by quantitation of the efflux of the ³H metabolites of norepinephrine, known to arise by neuronal and extraneuronal metabolism, during basal conditions and during increased efflux of norepinephrine by electric stimulation. Similar amounts of endogenous norepinephrine were present in segments of left (0.36 μg NE/g) and right (0.42 μg NE/g) coronary arteries. Levels were not significantly different after 15 min of continuous electric stimulation (0.37 and 0.41 μg NE/g in right and left arteries, respectively). This was apparent even though the fractional loss with electric stimulation seemed high; approximately three times greater from the coronary artery than from a segment of the peripheral vasculature. Of the quantity of norepinephrine released during electrical stimulation, 37% overflows in the superfusate, 43% is taken up in the sympathetic neurons and 20% is metabolized extraneuronally. Of that entering neurons about one-half is metabolized to 3,4-dihydroxyphenylglycol and the remainder is stored in the vesicles. By comparison with the saphenous vein from the same animal, the amount of NE in the vessels is less and a greater proportion of that released reenters the neurons and presumably helps to maintain the stores of transmitter.

Hamsters hypophysectomized at 1300 hr of proestrus were immediately injected ip with various doses of highly purified ovine FSH or LH. The FSH preparation was more potent than LH in inducing ovulation. The results cannot be attributed to significant LH contamination in the injected FSH. Despite the ability of FSH to induce ovulation, the resultant corpora lutea did not secrete progesterone on the morning of estrus.

As a possible step to evaluate the role of tumor promoters in normal cells, the effects of phorbol and its ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on normal mammary gland function and pituitary secretion of growth hormone (GH) and prolactin, were examined in lactating C3H/He mice. Daily subcutaneous injections of 400 μg phorbol or 4 μg TPA during Days 12 and 14 of lactation resulted in a significant decline in litter growth rate and the ratio of RNA synthesis: DNA synthesis of mammary glands associated with an increase in mammary gland DNA synthesis. On the other hand, pituitary synthesis and release of GH and prolactin, the pattern of estrous cycles and ovarian structures were affected little by these treatments. All findings strongly suggest that these tumor promoters can directly inhibit lactation by shifting mammary gland cells from a functional state to a mitotic state.

A factor which impairs the capacity of normal recipient mice to resist Pseudomonas infection has been isolated from the ascitic fluids of tumor-bearing mice. This factor was dialyzable (molecular weight between 4000 and 10,000), showed potent suppressive activity when as little as 10 μg was given ip and was resistant to heating at 56 for °30 min. The factor in the ascitic fluids was detectable rather late after tumor implantation and the production of the factor in the ascitic fluids was closely related to the tumor growth.

The tissue concentrations of the imidazole-containing compounds, anserine, carnosine, and free histidine, were determined in male chickens and rats suffering from acute or chronic infections. Either type of infection resulted in a decrease in tissue carnosine concentration. The tissue anserine concentration was unaltered by either the acute or the chronic infection. The tissue free-histidine concentration increased significantly during chronic infections, but remained unaffected during the acute infections. These results were consistent with the hypothesis that carnosine acts as a tissue reservoir for histidine, ultimately serving, when called upon by the stress of an infection, as a precursor of histamine.

I strain mice have a deficiency of muscle phosphorylase b kinase. Their skeletal muscle shows a three- to fivefold elevation in glycogen concentration and a decreased rate of glycogen breakdown. In addition, I strain mice accumulate less fat with age or high fat diets than control mice. The purpose of this study was to determine whether phosphorylase b kinase deficiency and decreased fat accumulation of I strain mice are related. Body weight, epididymal fat pad size, adipose cell number, and size were determined in male I, C3H, and hybrid (IxC3H) mice. Since the phosphorylase kinase deficiency is sex-linked, the male offspring of the I♂ xC3H♀ cross were used. As expected, phosphorylase b kinase activity and glycogen content in I and IxC3H skeletal muscle are similar. Body weight at 16 weeks of age in IxC3H mice resembles the body weight of I mice rather than C3H mice. Epididymal fat pad weight is not significantly different in IxC3H and I mice. Epididymal fat pad weight is significantly lower in I and IxC3H mice compared to that in C3H mice. However, adipose cell number is intermediate in IxC3H mice compared to I and C3H mice. Adipose cell size is not significantly different in the three strains. These results suggest that the decreased fat accumulation in I mice is genetically expressed with phosphorylase kinase deficiency. The inability of I mice to accumulate fat may be due to decreased adipose cell number.

Guinea pigs were sensitized with elastin peptides dissolved in complete Freund's adjuvant, and with complete Freund's adjuvant alone. Four weeks after sensitization, the guinea pigs were challenged intradermally with elastin peptides. Only guinea pigs sensitized and challenged with elastin peptides produced erythema and induration. Histological studies of the indurated skin showed heavy infiltration of mononuclear cells. Migration of peritoneal exudate cells of those guinea pigs was strongly inhibited in the presence of elastin peptides, whereas other proteins such as serum albumin did not inhibit the migration. These observations suggest that the reaction is specific for elastin peptides, although a slight but significant effect was shown by peptides derived from other connective tissue proteins of human lung, i.e., microfibrils and collagen. Cell-mediated immunity could also be induced in normal guinea pigs by the transfer of spleen cells obtained from guinea pigs sensitized and challenged with elastin peptides.

There are several different types of receptors which mediate responses by the cardiovascular system to serotonin. In this study we utilized 16 anesthetized dogs to compare the effects of cyproheptadine and methysergide upon two different cardiovascular responses to serotonin. Injection of serotonin (50-200 μg) into the right atrium produced pulmonary vasoconstriction during which the mean pulmonary artery pressure increased from 14 (±2) to 20 (±2) mm Hg (P < 0.05). As the same serotonin later reached the coronary circulation, a cardiogenic hypertensive chemoreflex is activated, causing systemic hypertension in which the mean aortic pressure promptly increases from 104 (±7) to 179 (±10) mm Hg. Administration of either cyproheptadine or methysergide eliminated the pulmonary hypertension, but only cyproheptadine was effective in blocking the cardiogenic hypertensive chemoreflex. The differential blocking effectiveness of these two serotonin antagonists suggests an important difference in physiological characteristics of the serotonin receptors involved.

The influence of oral sodium salicylate (NaSal) or ethanol (ETOH) on glucosamine synthetase (GmS) specific activity in rat fundic and antral mucosae and liver was measured. Sodium salicylate, 32 mg/kg in 0.1 N HCl, caused a 46% decrease in GmS specific activity in only 5 min. Recovery to 179% of control was complete by 30 min. Larger doses of Nasal caused more severe damage to the gastric mucosa and more persistent decreases in fundic GmS specific activity and also decreased liver GmS activity. ETOH, 20% (v:v) in 0.1 N HCI, 1.6 g/kg, caused a 29% decrease in GmS activity in the fundic mucosa in 15 min. Recovery, to 157% of control was complete in 30 min. We conclude that specific inhibition of hexosamine synthesis, not attributable to necrosis or general metabolic decline occurs during Nasal- and ETOH-induced gastric damage and may be involved jointly with acid not only in the development but also in the delayed healing of severe damage.

Chlorazol fast pink is representative of a group of azo dyes that are anticoagulant both in vitro and in vivo. At a concentration of 2.5 mg/ml, it completely inhibited the thrombin time, prothrombin time, and partial thromboplastin time of human plasma, and the thrombin time and Reptilase time of purified human fibrinogen. Among modes of action considered, it was found to inhibit the cleavage of fibrinopeptides A and B from fibrinogen, and the augmentation of the coagulant action of antihemophilic factor (factor VIII) by thrombin. It did not retard the aggregation of fibrin monomers, but the clots that formed were fragmentary. Clot formation returned to normal after removal of all but tightly bound dye from fibrinogen and thrombin. This and similar experiments suggested that the dye interfered reversibly with the interaction of thrombin and fibrinogen to a degree that accounted for much of its anticoagulant activity. The studies suggest that chlorazol fast pink may be an unusual tool for clarifying the steps involved in the formation of a fibrin clot.

Ouabain-sensitive ⁸⁶rubidium uptake was used to estimate sodium-potassium pump activity in the tail arteries of Dahl salt-sensitive and Dahl salt-resistant rats on normal and high oral intakes of salt. Uptake was increased in the salt-sensitive strain relative to the resistant strain at a given salt intake. It was also increased in a given strain when the salt intake was increased. In each case the increased ouabain-sensitive uptake was associated with increased ouabain-insensitive uptake which in part reflects the permeability of the cell membrane to rubidium. The results suggest that the increased pump activity is a secondary compensatory response to increased passive penetration of sodium. In this respect, the Dahl salt-sensitive rat is similar to SHR, another genetic model, but different from the other low-renin, presumably, volume-expanded models of hypertension we have studied.

The use of platelets labeled with ¹¹¹In-oxine for in vivo studies of thrombosis was examined. Platelets were separated from the blood of rabbits, labeled in vitro with ¹¹¹In, and returned to the animals by intravenous injection. In some animals repeated blood samples showed that the ¹¹¹In platelets gradually declined in the blood reaching half their initial concentration in 48 hr. In other rabbits, inflammatory lesions were produced in the skin of the back by the intradermal injection of killed Escherichia coli, or endotoxin, producing a local Shwartzman reaction. One hour after the iv injection of 111In platelets the animal was killed, the inflammatory lesions were removed, and the radioactivity in them was determined. ¹¹¹Indium accumulated in E. coli lesions within 1 hr after the injection of bacteria. The maximum rate of accumulation occurred in lesions which were 3 −4 hr old, but some ¹¹¹In platelets continued to accumulate in lesions that were 8-24 hr of age. The local Shwartzman reaction produced extensive thrombosis. This was demonstrated by a large accumulation of ¹¹¹In-labeled platelets, and by the observation of numerous platelet-containing thrombi in histological sections of the lesions. The findings demonstrate that ¹¹¹In can be used to label platelets for the in vivo quantitation of thrombosis in inflammatory tissue.

To ascertain the influence of carbon tetrachloride on the ability of the liver to remove circulating endotoxin, groups of rats were injected ip with endotoxin obtained from Escherichia coli 026 and also received carbon tetrachloride orally. The serum concentrations of endotoxin at different time intervals were measured by an immunoradiometric assay specific for the endotoxin used. It was shown that carbon tetrachloride delayed the removal of endotoxin by the liver. The effect was dependent on the dose of carbon tetrachloride given and was seen as early as 1 hr after the endotoxin injection.

Rats fed either a soybean protein-based diet or a casein-based diet supplemented with picolinic acid gained significantly more weight than rats fed unsupplemented diets. The zinc concentration of the kidneys from the rats fed the diets supplemented with picolinic acid was significantly greater than that of the rats fed the unsupplemented diets. Rats fed a pyridoxine-free diet formulated with vitamin-free casein and supplemented with picolinic acid gained significantly more weight than rats fed an unsupplemented pyridoxine-deficient diet. The zinc concentration of the kidneys from the rats fed the pyridoxine-deficient diet supplemented with picolinic acid was significantly greater than that of the rats fed the unsupplemented diet. Dietary zinc absorption and food efficiency were increased when rats were fed supplemental picolinic acid. The results suggest that picolinic acid may increase the bioavailability of dietary zinc and perhaps other divalent cations which results in increased food efficiency.

A series of investigations was designed to study the role of cellular immunity, humoral antibody, and interferon in recovery from primary systemic herpes simplex virus type I (HVH) infection in mice. Nude and neonatally thymectomized mice, which have a specific defect in T-cell function, were dramatically more susceptible to primary systemic HVH infection. These immunosuppressed mice did not form neutralizing antibody to HVH, and had pathological lesions involving primarily the liver and brain. Passive transfer of physiologic amounts of neutralizing antibody after infection largely protected these T-cell-deficient HVH-infected mice. The results thus suggest that the formation of anti-HVH antibody in the nonimmunosuppressed mouse is critically important in promoting recovery from systemic HVH infection. The findings do not favor a dominant defensive role for immune cells, but they do not exclude a defensive role for these cells in this experimental primary HVH infection.

Some biological properties of thiocystine(bis[2-amino-2-carboxyethyl]tri-sulfide), a persulfide analog of cystine (l), have been investigated. Thiocystine replaced cystine in supporting growth of young rats fed a low-fat, low-methionine diet. When injected intravenously into adult rats, thiocystine protected against two to three times an LD₅₀ dose of intraperitoneal sodium cyanide. Thiocystine exhibited marginal activity for protecting mice against lethal X-ray irradiation. The biological properties of thiocystine can be attributed to its conversion in vivo to cystine and a persulfide sulfur moiety.

Rats fed a diet supplemented with phenobarbital at a concentration of 0.25% gained less weight than rats fed the unsupplemented diet. The reduced weight gain in the phenobarbital-treated rats accompanied the induction of marked hepatomegaly. Circadian cycling of food consumption in control rats followed a biphasic pattern, with the first feeding episode occurring during the middle of the 12-hr dark phase and the second occurring as the dark phase ended. In rats on phenobarbital, eating activity was confined to the first feeding episode, with the level of intake during this interval increasing to compensate for the absence of significant subsequent feeding, so that the daily levels of food consumption were similar in both groups. Measurements of circadian cycling of deep body temperature showed that ingestion of 0.25% dietary phenobarbital approximately doubled the amplitude of the temperature cycle and advanced the time at which peak temperature was attained by approximately 2 hr. It is suggested that the lower weight gain in rats chronically exposed to 0.25% dietary phenobarbital results primarily from alterations in hepatic metabolism, but phenobarbital-mediated changes in the circadian cycling of food intake and deep-body temperature may also contribute to the growth reduction.

The effect of increasing gestational age on the biochemical composition of porcine placental glycosaminoglycans (pGAG) was investigated. Glycosaminoglycans (GAG) were isolated from placentas obtained surgically at 20-day intervals during the second half of gestation (Days 50-110) and were characterized by anion-exchange chromatography and cellulose acetate electrophoresis. The dried, defatted porcine placenta contained approximately 60 μg GAG/mg. Isolated pGAG contained heparin, chondroitin sulfates-dermatan sulfate, heparan sulfate, and hyaluronic acid. Hyaluronic acid was the predominant pGAG throughout the second half of gestation and increased from 7.3 μg on Day 50 to 10.4 μg uronic acid/mg dried, defatted placenta on Day 110 (P < 0.05) and from 45.6% of total pGAC isolated on Day 50 to 63.9% on Day 110 (P < 0.05). Chondroitin sulfates-dermatan sulfate and heparan sulfate decreased (P < 0.05) both absolutely and on a percentage basis as gestation progressed. Neither the absolute amount nor the percentage of heparin changed with increasing gestational age.

Rats treated with a small dose (0.5 mg P/kg/day) of dichloromethylene diphosphonic acid (C1²MDP) exhibited a marked increase in the shear strength of the proximal tibial epiphyseal plate with no suppression of longitudinal growth. Our results suggest that mechanisms in addition to the absorption of diphosphonate onto hydroxyapatite crystals account for the increase in epiphyseal plate shear strength noted in our experiment. In this regard, this diphosphonate could be valuable in studies of the normal determinants of epiphyseal plate shear strength. In addition, since C1²MDP increased epiphyseal plate strength without suppressing longitudinal growth, the compound may eventually prove to be useful in the treatment of epiphyseal displacements in humans.

The effect of streptozotocin-induced diabetes in pregnant rats, on subsequent maternal and neonatal bile acid metabolism was investigated. Plasma glucose levels (mg%) of diabetic pregnant (DP) rats was significantly greater (p < 0.05) in comparison to control pregnant (CP) rats. Total bile acid pool (mg/100g body wt) in DP was significantly higher (p < 0.05) when compared to CP (DP, 29.42 ± 4.44 vs 8.14 ± 1.13 in CP) with a marked increase in cholic acid pool size in DP (DP, 22.65 ± 3.33 vs 3.50 ± 0.48 CP, p < 0.05). The fecal excretion of bile acid was significantly lower (p < 0.05) in DP rats when compared to CP rats. Thin-layer chromatographic analysis of bile acid conjugates at postpartum revealed a significant increase in glycine conjugates in bile from DP rats. Examination of the cholic acid pool (μg/100 g body wt) in neonates (2 days old) derived from the two groups of rats indicated a strikingly reduced pool of cholic acid in neonates derived from diabetic mothers (neonates from DP 853.45 μ 213.98 vs 3690.86 μ 797.26 in CP,p < 0.05). Plasma cholesterol levels (mg%) in neonates of diabetic mothers was also significantly (p < 0.05) reduced. This study demonstrates that the diabetic state during pregnancy not only alters maternal bile acid metabolism, but also affects fetal bile acid metabolism in utero such that the effects may persist into neonatal life.

Tissue copper levels in the small intestine of neonatal rats increased rapidly following birth, reaching a maximum value of 302.6 ± 22.2 μg/g dry wt on Day 7 and then steadily declined to 6.4 ± 0.5 μg/g dry wt on Day 21. When subjected to gel filtration on Sephadex G-75, high-speed supernatant prepared from the small intestine of rats aged 0 to 21 days eluted as two copper-containing fractions corresponding to molecular weights of 60,000 and 9000 daltons. The copper contents of both fractions varied with age in a manner that correlated significantly with the variations observed in the tissue copper levels. However, orally administered ⁶⁴Cu was found to be associated primarily with the low-molecular-weight fraction in 5-day-old rats. Furthermore, the amount of ⁶⁴Cu in the low-molecular-weight fraction did not appear to turnover during the 42 hr following administration of the dose. These results suggest that a portion of the copper ingested by the neonatal rat is initially sequestered in the small intestine by low (9000 daltons) −molecular-weight cystolic proteins either as a mechanism for detoxifying excess copper or as a pool for the slow release of copper and maintenance of copper homeostasis during a period of rapid growth and development.

The contribution of chylomicron cholesterol to total milk cholesterol was studied in the lactating rat. Pregnant rats were fed a high-fat, high-cholesterol diet or standard rat diet. On the 13th day of lactation, dams were intonated with 25 μCi [³H]cholesterol (25 μCi/μmole). Milk was collected at 5 and 10 hr following intubation; plasma was collected at 5, 8, and 10 hr following intubation. There was a lower specific activity of chylomicron cholesterol in dams fed the high-fat, high-cholesterol diet compared with control animals at 5, 8, and 10 hr after intubation (P < 0.05) with radiolabeled cholesterol. There was no difference in the specific activity of milk cholesterol between both groups at 5 hr. These data indicate that chylomicron cholesterol does not significantly contribute to milk cholesterol levels in the lactating rat. At 8 and 10 hr following intubation with radiolabeled cholesterol, the specific activity of plasma cholesterol from dams fed the high-fat, high-cholesterol diet was lower than that from control animals (P < 0.05). The specific activity of milk cholesterol at 10 hr was also lower in experimental animals (P < 0.05). Taken together, these data suggest that plasma lipoproteins other than chylomicrons contribute significantly to milk cholesterol levels.

Bioassayable PGE² like material released from isolated perfused rabbit kidneys was assayed in superfusion cascade, and the response to bradykinin and angiotensin II quantitated in a dose-dependent manner. Chronic high-sodium intakes produced qualitatively and quantitatively similar responses compared to normal diets when kidneys were stimulated with BK or A¹¹. Low Na⁺ diets increased sensitivity to BK or A¹¹ with a shift of the dose —response curves (DRC) to the left with the same maximum response. No difference in in vitro metabolism of [¹⁴C]arachidonic acid by microsomal preparations of cortex and medulla from Na′-loaded and Na+-depleted rabbits could be detected. These results suggest that the shift of the DRC to the left for angiotensin II and BK may be related to increased sensitivity for arachidonic acid release in response to the peptides and may be responsible for the observed increase in PGE² urinary excretion observed in chronic salt depletion in rabbits.