Experimental Biology and Medicine

Visual function of vitamin A. Somatic functions of vitamin A: a. differentiation, b. growth. Reproduction. Membrane phenomena. Conclusions. Addendum.

The chronic effect of TSH on thyroidal cAMP concentrations and release of thyroid hormones was investigated using human thyroid tissue in organ culture. Normal human thyroid slices were placed in HAM's F-10 synthetic culture medium in Falcon organ tissue culture dishes, and incubated at 37° in a humidified atmosphere of 5% CO₂ in air. Medium was changed everyday and daily T₃ or T₄ release was determined using concentration of T₃ or T₄ in the medium. After incubation, slices were transferred to the medium containing 10 mM theophylline and incubated without TSH for an additional 30 min to determine thyroidal cAMP concentrations. Thyroidal cAMP concentrations in slices incubated with 10 mU/ml of TSH increased significantly at 2, 6, and 24 hr and even on the 6th day of incubation. Daily T₃ release was significantly increased above control from the 3rd day and daily T₄ release from the 4th day to the 11th day of incubation with 10 mU/ml of TSH. Histologically, almost all follicles were structurally maintained even on the 11th day of incubation. These results suggest that both thyroidal cAMP concentrations and release of thyroid hormones are stimulated chronically by TSH. This organ culture system is useful for investigating chronic effects of various materials on human thyroid tissue.

Plasma levels of calcium (Ca), phosphate (P), calcitonin (CT), parathyroid hormone (PTH), and prolactin (PRL) were measured at 8, 13, and 17 hr during the 4-day estrous cycle of the rat. Ca levels fell throughout the day during proestrus (PE) and estrus (E). In contrast Ca rose transiently during diestrus (D1, D2). P levels fluctuated inconsistently at all stages of the cycle with the exception of E where P levels were significantly higher at 13 hr. CT levels showed an increase during D1 and D2 and fell to their lowest values during E, at 13 hr. Daily fluctuations in each stage were also recorded. Variations of PTH levels during the estrous cycle were minor. PRL levels increased sharply during PE. No direct relationship between PRL secretion and CT secretion could be established. These results indicate that CT but not PTH varies specifically in relation to the estrous cycle. They suggest that there is a link between sexual hormones and CT, apparently independent of plasma calcium levels.

The present study tested the hypothesis that the combination of parathyroid hormone and phosphate infusion would be phosphaturic in phosphate-deprived rats. Clearance experiments were performed in Sprague-Dawley rats fed a low-phosphate diet for 4 days. The animals were first given a phosphate infusion and then administered parathyroid hormone. The reverse experiment was also performed in a second group of rats by giving the hormone first followed by a phosphate infusion. There was no increase in phosphate excretion in response to phosphate infusion or parathyroid hormone given alone. However, the combination of parathyroid hormone and phosphate infusions, given in either order, was phosphaturic in phosphate-deprived rats.

Broad-Breasted White turkeys were randomized into four treatment groups at 4 weeks of age in each of three trials. Group 1, males, and Group 2, females, were fed an unsup-plemented (control) diet from 4 to 10 weeks of age. Group 3, males, and Group 4, females, were fed the control diet supplemented with 0.07% β-aminopropionitrile (BAPN). The experiments were terminated at 10 weeks of age. There was no mortality among male and female turkeys fed the control diet or significant differences in blood pressure, heart rate, aortic tensile strength, or aortic hydroxyproline between these males and females. Sixty-five percent of the males and 21% of the females fed BAPN died of dissecting aneurysms; blood pressure, heart rate, and dp/dt max were not altered as a result of feeding BAPN. Aortic tensile strength was higher in control turkeys than those fed BAPN, but males fed BAPN had the lowest value. Males fed BAPN also had the lowest aortic hydroxyproline content. Ultrastructural alterations of aortic elastic and collagen fibers were more severe in males than females fed BAPN. Aortic changes did not occur in control turkeys.

Matrix vesicles, small extracellular membranous structures, are known to be the initial loci of calcification of cartilage, bone, and dentin. Calcification is an important complication of atherosclerosis. Using histologic, ultrastructural, and cytochemical techniques, the present study has demonstrated that matrix vesicle-like structures are involved in the calcification of atherosclerotic lesions, as well as in arterial medial calcification. In aortas from autopsied humans and from rabbits and chickens on atherogenic diets, the matrix vesicles appear to be derived from intimal and medial cellular components, mainly smooth muscle cells.

We investigated the effect of the concentration of type-specific antibody to pneumococcal polysaccharide (PPS) on opsonic requirements for phagocytosis in vitro of Streptococcus pneumonia types 7 and 19. We measured the uptake by human neutrophils of radiolabeled S. pneumoniae opsonized with either complement-intact, complement-depleted (by heat inactivation), or alternative complement pathway-activated (by magnesium dichloride-ethylene glycol tetraacetic acid (MgEGTA) chelation) immune sera with varying concentrations of antibody from individuals immunized with polyvalent PPS vaccine. Increased opsonization was found with increasing concentrations of type-specific antibody in the sera. Higher concentrations of antibody were required to opsonize type 7 than type 19 bacteria, both in the presence and absence of complement activity. Type 19 bacteria were more efficiently opsonized via the alternative complement pathway than type 7. For both types, antibody and the alternative complement pathway provided most of the opsonic activity in sera with lower concentrations of type-specific antibody. At high antibody concentrations, effective opsonization occurred in heat-activated sera, indicating the requirement for complement could be overcome with sufficient amounts of antibody alone.

Gluconeogenesis and ketogenesis from a variety of substrates by isolated hepatocytes from thyroxine-treated and control 40-hr starved BHE rats were determined. Thyroid hormone treatment stimulated glucose production from lactate and inhibited glucose production from alanine, lactate plus NH₄Cl, or glutamate. β-Hydroxybutyrate production by cells incubated with glycerol was decreased in the cells from thyroxine-treated rats. Glycerol had a greater antiketogenic effect in the thyroxine-treated rats than in the control rats. Acetoacetate production from 10:1 lactate:pyruvate, lactate plus NH₄Cl, lactate plus lysine, and lactate plus ethanol was also less in the hepatocytes from the thyroxine-treated rats. Acetoacetate production from alanine was increased by thyroxine treatment. From these results it can be suggested that the metabolic abnormalities previously reported in the BHE rats can, in part, be attributable to abnormalities in endogenous thyroid hormone action and in part due to some as yet unknown regulatory process within the liver cell.

The cardiovascular effects of isoprenaline, methacholine, and sodium nitroprusside were studied in spontaneously hypertensive (SHR) and in Wistar-Kyoto normotensive (WKY) rats. In conscious rats with chronic indwelling arterial cannulae, methacholine caused a much greater fall in mean arterial blood pressure (MAP) in SHR rats, abolishing the initial 30-40 mm Hg difference in MAP at a dose of 2.5 μg/kg. The hypotensive response to methacholine was accompanied by reflex tachycardia in WKY rats but by a dose-related bradycardia in SHR rats. Isoprenaline increased heart rate and reduced blood pressure to a similar extent in rats of both strains. Administration of sodium nitroprusside resulted in a hypotensive response that was of greater duration in SHR rats. In addition, the reflex tachycardia was more marked and of greater duration in WKY compared to SHR rats. This demonstrates that the baroreflex response to vasodilation is suppressed in SHR rats and that this may contribute to the increased vasodepressor response of these animals to methacholine. However, when baroreflexes but not initial MAP were suppressed with pentobarbital, methacholine caused a similar degree of bradycardia in SHR and WKY rats but a greater vasodepressor response in SHR rats. With isoprenaline, the MAP difference between strains was maintained at all doses up to 200 ng/kg. Although it is not yet clear what is responsible for the increased vascular constriction of SHR rats, our findings suggest that it can be removed by muscarinic receptor activation with methacholine but not by stimulation of β-adrenoceptors.

The proteins of Long strain RSV and three temperature-sensitive (ts) mutants of the A2 strain were compared by pulse labeling virus-infected cells with [³⁵S]methionine and [³H]glucosamine followed by analysis of the cell lysates by polyacrylamide gel electrophoresis. At the permissive temperature (30°) proteins ranging in molecular weight from 24,000 to 50,000 (VP24, VP27, VP33, VP44) could be identified. Immunoprecipitation of viral lysates by immune rabbit serum demonstrated antigenic similarity with VP27, VP44, VP50, and VP67 in all ts mutants and Long strain RSV. [³H]Glucosamine labeling demonstrated glycoproteins of 90,000 (GP90) and 50,000 (GP50) in Long strain and GP90 in the ts mutants.

Studies were designed to determine if permeability of adapted (remnant) small bowel mucosa to polyethylene glycol (PEG) was altered after major intestinal resection. Rats underwent 50% small bowel resection with preservation of duodenum and terminal ileum. Sham-operated animals served as controls. Two and four weeks later we cannulated the portal vein and measured mucosal permeability to luminal [³H]PEG and [¹⁴C]PEG in isotonic Ringer solution in remnant proximal or distal in situ closed intestinal loops. A lumen-to-portal blood gradient of at least 1000/1 persisted throughout the one-hour experimental period in both resected and sham-operated animals. Thus the adapted remnant intestinal mucosa was highly impermeable to luminal radiotracer PEG. In separate experiments 2 and 4 weeks after 70% small bowel resection or sham operation, in vivo segments of proximal and distal small intestinal were perfused through the lumen for one hour with hypertonic (800 mOsm) mannitol or NaCl solution containing [³H]PEG. There was equal and almost total recovery of [³H]PEG at the end of the experimental period in resected and control animals. The combined data of all experiments indicate that radiotracer PEG may be confidently used as a luminal water phase marker in transport studies of remnant bowel following intestinal resection.

The angiogenic capability of PGE₂ was tested by implanting pellets of an ethylene vinyl acetate slow release polymer containing PGE₂ on the chorioallantoic membrane of 8-day-old chicken embryos. Elvax pellets releasing approximately 0.2, 2.0, or 20 ng/day PGE₂ were found to induce neovascular responses. In contrast, pellets releasing 2.0 or 20 ng/day of either PGA₂, PGF₂, or TXB₂ did not appear to be angiogenic when compared with PGE₂. These release rates of PGE₂ are similar to those reported for a variety of tumors, activated macrophages, inflammatory exudates, and rheumatoid synovia, suggesting that PGE₂ may be a key factor in various neovascular reactions.

Prolactin dissociates more readily from rat liver than from rabbit mammary prolactin receptors. The rate of dissociation is dependent in the time of association. In rat liver, prolactin dissociates from receptors at the cell periphery (plasma membrane, PM) more rapidly than those of microsomes, a major component of which is the intracellular Golgi membranes. The dissociation curves following 1 hr of association can be resolved into a fast and slow component by logarithmic transformation, with a greater than twofold increase in the fast componment of the dissociation rate constant (k ₂) in PM compared to microsomal membranes. With longer association times (10 hr), plasma membranes develop a more slowly dissociable component, with dissociation characteristics (rate constants) similar to those observed in microsomes following 1 hr of association. Another means of visualizing this difference is by a Scatchard plot of prolactin binding to PM, microsomal, and Golgi membranes. The affinity constants in the microsomal and Golgi fractions are identical whereas that for the PM fractions more than twofold lower. The decreased affinity in the PM would allow prolactin to more readily dissociate from its receptor than from receptors with higher affinity. Although the differences between PM and Golgi receptors observed are small, they are confirmed by direct measurements of affinity constants and dissociation rate constants. Therefore, it appears that receptors with lower affinity at the cell periphery are those involved in the initial mechanism of action of prolactin.

Tetrabenazine is considered to be a reserpine-like drug because of its ability to block dopamine storage in presynaptic vesicles. We used two methods to determine that tetrabenazine is also a dopamine antagonist. Tetrabenazine displaced the specific [³H]spiperone binding to the dopamine receptors of the anterior pituitary, the corpus striatum, and a transplantable rat pituitary tumor with values for 50% displacement (IC₅₀) of about 15 μM. Under in vitro conditions, 0.5 to 10 μM tetrabenazine blocked dopaminergic inhibition of prolactin secretion from rat anterior pituitary glands. One, four, and twenty-four hours after a single tetrabenazine injection (30 mg/kg, ip), the serum prolactin changed from 22 ± 9 ng/ml initially, to 450 ± 52, 254.7 ± 10.4, and 9.3 ± 1.1 ng/ml, respectively. Pituitary glands of the treated rats incubated in vitro were refractory to dopaminergic inhibition of prolactin release to an extent that was maximal at one hour but inapparent by 24 hours after injection. In vivo and in vitro, tetrabenazine induces biological responses characteristic of a dopamine antagonist. These actions are independent of the reserpine-like properties of tetrabenazine. The unusual ability of tetrabenazine both to antagonize dopamine and to block presynaptic dopamine storage may provide a new tool for understanding the physiology of dopaminergic systems.

A diurnal rhythm of jejunal sucrase activity has been shown previously to make its developmental appearance in the rat at the time of weaning (Day 22). In this study we found that the rhythm was not present on Day 23 if the onset of feeding was not coordinated with the onset of darkness. Conversely, the sucrase rhythm appeared precociously (Day 19) in pups weaned onto chow on a schedule in which the onset of feeding is coordinated with the onset of the dark period. It is concluded that the normal developmental appearance of the sucrase rhythm is due, at least in part, to the fact that ad libitum feeding becomes nocturnal around Day 22.

There exists a critical period for the development of cervicovaginal lesions in both mice and humans exposed neonatally and antenatally to sex hormones. Mammary glands from year-old female BALB/c mice exposed neonatally to 20 μg estradiol for 5 days commencing at 1 day of age showed the most mammary abnormalities, significantly greater than in controls (P < 0.005). The incidence of abnormalities declined when treatment was begun after Day 1. Treatments begun after Day 3 did not result in this structural pattern. Mice ovariectomized after treatment all had inactive mammary glands with no abnormalities. There is a critical exposure period for the later occurrence of mammary gland abnormalities. However, the aberrant secretory state which accompanies these mammary gland alterations may be a consequence of permanent alteration in ovarian function or its endocrine control.

We attempted to determine whether or not Yersinia enterocolitica could invade two cell lines derived from Burkitt's lymphoma (BL), P3HR-1, and Raji cells, and if the expression of Epstein-Barr virus (EBV) genome could be activated by the invasion of the organisms into the cells. The invasiveness of Y. enterocolitica into BL cells was examined morphologically, using transmission and scanning electron microscopy, and the induction of EBV antigens in the BL cells was examined by indirect immunofluorescence. Y. enterocolitica was clearly observed to invade P3HR-1 and Raji cells within 2 hr incubation at 37°, after bacterial challenge. However, the invaded BL cells did not show significant induction of EBV early antigen after cultivation for 72 hr at 37°. Although the present experiment failed to yield positive findings pertaining to the activation of EBV genome, our experimental system per se may still be a useful model when attempting to assess the effects of invading bacteria on the viral genome persistently carried in the host cells.

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) has been enriched more extensively than previously from fresh rat liver. For this, 10% homogenates in sucrose-phosphate buffer were treated with 0.1% Tween-20 prior to high-speed centrifugation to obtain soluble proteins. Those precipitated by 40% saturation with ammonium sulfate were subjected to stepwise addition of calcium phosphate gel to remove pyrophosphatase, and the remaining synthetase was further enriched by passage through a tricalcium phosphate column. An apparent yield of greater than 70% and purification over 70-fold was achieved from the high-speed supernatant fraction. The synthetase activity in solution at 4° was largely lost within a week unless protected by thiols which could partly restore inactivated enzyme. The pH optimum for synthetase activity is near 7.7 when assayed with suitable concentrations of FMN, ATP, and Mg²⁺. Purified enzyme could be separated into lower (140,000) and higher (325,000) molecular weight components when subjected to molecular sieving on a Sephadex G-200 column.

D-Galactosamine is an amino sugar with unique hepatotoxic properties in animals. Although the mechanism of liver injury by galactosamine remains controversial, a role for bacterial endotoxin has been suggested. In the present study, using New Zealand rabbits, we show that the significant increase in serum glutamic oxaloacetic transaminase which followed the injection of 4.25 mmole/kg of D-galactosamine was completely prevented in animals subjected to resection of small bowel and colon. Using an immunoradiometric assay specific for E. coli 026 endotoxin we showed that after instillation of 50 mg of E. coli into the colon, serum levels of this endotoxin were higher in the animals injected with galactosamine than the controls injected with saline. However, the differences in endotoxin concentration between the two groups of animals was statistically significant only at 30 and 60 min. The role of endotoxin in the pathogenesis of galactosamine liver injury is reviewed and discussed.

Unlike their +/+ and nu/+ littermates, homozygous athymic-nude (nu/nu) mice that had been sensitized with Bacillus Calmette-Guérin (BCG) failed to produce interferon (IFN) in response to purified protein derivative (PPD). Likewise, spleen cells derived from BCG-sensitized nu/nu mice failed to produce IFN when exposed to PPD in vitro. These findings suggest that the T-cell subset that responds to specific antigens (i.e., PPD) is absent in athymic-nude mice.