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The objectives of the present study were to evaluate the basal concentrations of heat shock proteins (HSP) between Bos indicusand Bos tauruscattle and to determine if HSP basal concentrations change as an animal matures. A total of 40 cattle were used in a 2 × 2 factorial design to evaluate the effects of genotype and age (heifers and mature cows) on basal concentrations of heat shock protein 27 (HSP27), α B-crystallin (Cryab), and heat shock protein 70 (HSP70). Each experimental group of 10 animals was sampled on a separate day over a period of 4 wk during July 2014. A muscle sample was collected from the longissimus thoracis (LT) and concentrations of HSP were quantified using ELISA. There were no significant differences in HSP concentration for the interaction between age and genotype or for age alone. Bos indicuscattle had greater (P< 0.05) basal concentrations of HSP27, Cryab, and HSP70 in the LT than B. tauruscattle. The results of this study show that basal in vivo HSP concentrations differ between B. indicusand B. tauruscattle. However, further studies are needed to investigate the relationship between HSP concentrations and meat tenderness with respect to genotypes to see if HSP concentrations account for at least some variability in tenderness differences.

The physiological and metabolic effects of prophylactic treatment with osmolytes were investigated using twenty-four 2.5-yr-old Bos indicussteers. Animals were allocated to 1 of 4 treatment groups: 1) control, feed and water deprived for 48 h (n = 6); 2) transported, transported for 48 h (n = 6); 3) glycerol, dosed with glycerol (2 g/kg of BW) and then transported for 48 h (n = 6); and 4) betaine, dosed with betaine (0.25 g/kg of BW) then transported for 48 h (n = 6). Body water, electrolytes, blood pH and gases, plasma lactate, glucose, albumin, total protein, anion gap, strong ion difference, total weak acids, and BW were determined at the conclusion of 24 and 48 h of transportation. The glycerol group had greater body water volumes than the control (P= 0.05) and transported (P= 0.02) groups. The glycerol, transported, and betaine groups had lower (P= 0.02) plasma Mg concentrations than the control group at 24 h, whereas the glycerol group maintained lower (P= 0.04) plasma concentrations of Ca than the control group. The betaine group had lower (P= 0.04) hematocrit than the control group at 24 and 48 h. Plasma bicarbonate and pCO2were 13 and 17% greater (P= 0.01 and 0.04, respectively) in the glycerol group at 24 h compared with control and transported groups. However, the ratio of [HCO3]/[CO2] in the glycerol group did not differ from the other groups and thereby maintained pH. The glycerol group maintained a 30% greater (P< 0.001) plasma concentration of glucose than the control group, and 14% greater (P= 0.05) than the transported and betaine groups. In contrast, betaine had little effect on increasing blood glucose compared with glycerol. Glycerol-linked hyperhydration at 24 h may not only help to conserve water loss during long distance transportation, but the increased blood glucose may have an important protein-sparing effect due, in part, to greater insulin concentrations inhibiting the breakdown of muscle proteins, thus, countering the amino-acid mobilizing effect of cortisol after 24 h. Therefore, the osmolyte glycerol shows promise as a prophylactic treatment for attenuating the effects of long distance transportation by maintaining body water, decreasing the energy deficit, and preserving health and muscle quality.

The objective was to compare testis characteristics of Zebu bulls treated with the GnRH agonist, deslorelin, at different times and for different durations during their development. An additional objective was to determine the usefulness of a stain for the transcription factor GATA-binding protein 4 (GATA-4) as a specific marker for Sertoli cell nuclei in cattle. Bulls (54) were allocated to nine groups (n = 6) and received s.c. deslorelin implants as follows: G1 = from birth to 3 mo of age; G2 = from 3 to 6 mo; G3 = from 6 to 9 mo; G4 = from 9 to 12 mo; G5 = from birth to 15 mo; G6 = from 3 to 15 mo; G7 = from 6 to 15 mo; G8 = from 12 to 15 mo; and G9 (control) = no implant. Bulls were castrated at 19 mo of age. Paraffin sections (10 μm) were subjected to quantitative morphometry and GATA-4 immunohistochemistry. At castration, all bulls in the control group (6/6) had attained puberty (scrotal circumference ≥ 28 cm), whereas a smaller proportion (P< 0.05) had reached puberty in G2 (2/5) and G6 (1/6). Bulls in G2 and G6 also had a lesser (P< 0.05) testis weight compared with the control group. Total volume of seminiferous epithelium and total daily sperm production in G2 and G6 were only half that observed in the control group. Spermatids were observed in less than 50% of seminiferous tubules in G2, G6, and G7 compared with 82% in the control group (P< 0.05). Staining for GATA-4 was specific for and abundant in the Sertoli cell nucleus in both pre- and postpubertal bulls, and no other cell nucleus inside the seminiferous tubule was positive for GATA-4. Total number of Sertoli cells was not affected by treatment (P= 0.45), but nuclear volume was smaller in G2 and G6 (P< 0.05) compared with the control group. In conclusion, treatment of Zebu bulls with deslorelin had no apparent beneficial effect on testis development and delayed puberty when treatment was initiated at 3 mo of age. Staining for GATA-4 was a useful method for identifying and quantifying Sertoli cell nuclei in both pre- and postpubertal bulls.

This study investigated the effects of excess cortisol on physiological mechanisms that resist dehydration in Bos indicussteers (n = 31, 2 yr of age, 193 ± 21.47 kg mean BW) during a 90-h period. Steers were assigned randomly to one of four groups: 1) no water/no cortisol (n = 8), 2) water/no cortisol (n = 8), 3) no water/cortisol (n = 8), and 4) water/cortisol (n = 7). Animals allocated to cortisol treatment groups were given 0.1 mg•kg BW−1•h−1of hydrocortisone suspended in isotonic saline for the duration of the study. Total body water, osmolality, hematocrit, urine output, feed and water intake, and plasma concentrations of arginine vasopressin (AVP), angiotensin II (AII), electrolytes, total protein, and albumin were determined at 24-h intervals for 90 h. In the presence of excess plasma cortisol, total body water was maintained in the presence of a water deprivation insult for 90 h, whereas hydration indices, such as total plasma protein and albumin, did not change, supporting the body water data. However, plasma osmolality increased for the water-deprived groups from 24 h (P= 0.008). Hematocrit did not reflect dehydration in any group. Water deprivation induced an increase in endogenous plasma cortisol concentrations after 60 h of the study (P= 0.028). Plasma concentrations of AVP increased with water deprivation (P= 0.006). Excess cortisol decreased the plasma concentration of AVP at 72 h only (P= 0.027) and suppressed plasma concentrations of AII at 24 and 72 h (P< 0.001 and P= 0.036, respectively). Animals treated with excess cortisol maintained urinary output for 48 h before decreasing at 72 h (P= 0.057), although there was no effect on water or feed intake. Water deprivation increased plasma sodium concentrations (P< 0.05) until 72 h, whereas potassium decreased under the influence of excess plasma cortisol (P= 0.001) at 24 h. Water deprivation increased plasma chloride concentration at 72 and 90 h (P= 0.051 and P= 0.026, respectively). Plasma phosphorus decreased at 24 h (P= 0.001) and remained at lesser concentrations for the duration of the study (P= 0.05). These results highlight the complexity of endocrine interactions associated with water balance in Bos indicussteers. We accept our hypothesis that, in the presence of excess cortisol, the renin-angiotensin-aldosterone axis is suppressed; however, homeostasis is achieved through other physiological systems.

Caffeic acid phenethyl ester (CAPE) is an anti-inflammatory component of propolis (honeybee resin). CAPE is reportedly a specific inhibitor of nuclear factor-kappaB (NF-kappaB). The aims of our study were 1) to evaluate the effect of CAPE on cytokine production, NF-kappaB, and apoptosis in two cell lines; 2) to assess the effect of CAPE on NF-kappaB in rats with peptidoglycan-polysaccharide (PG-PS)-induced colitis; and 3) to evaluate the efficacy of CAPE against this colitis. In vitro experiments used rat macrophage (NR8383) and colonic epithelial cell (SW620) lines. NF-kappaB was evaluated by electrophoretic mobility shift assay. Cytokines and apoptosis were measured by enzyme-linked immunosorbent assay. Colitis was induced by intramural injections of PG-PS into the distal colon. CAPE (30 mg/kg) or vehicle was administered once daily to rats by intraperitoneal injection, for 1 week. Various macroscopic and biochemical indices were measured on day 21. CAPE (30 microg/ml) significantly inhibited NF-kappaB and TNF-alpha production in the macrophage cell line. In macrophages, CAPE significantly increased DNA fragmentation. CAPE exhibited generally similar effects in the colonic epithelial cell line. CAPE treatment reduced the mean level of colonic NF-kappaB in rats. CAPE also induced a significant reduction in gross colonic injury. Moreover, colonic cytokine levels (TNF-alpha and IL-1beta) were significantly reduced in CAPE-treated rats. In summary, CAPE inhibits NF-kappaB, causes a reduction of pro-inflammatory cytokine production, and induces apoptosis in macrophages. These mechanisms likely contributed to the attenuation of PG-PS-induced colitis by CAPE.

Endothelin-1 is an endothelium-derived factor which alters tone and proliferation of vascular smooth muscle and has been implicated in the development of atherosclerosis. Estrogen modulates production of and contractile responses to endothelin-1. Since atherosclerosis is less in estrogen-replete women compared to men, experiments were designed to determine whether or not there were gender-associated differences in proliferative responses to endothelin-1 and effect of estrogen status on those responses. Proliferation of smooth muscle cells derived from coronary arteries of sexually mature, gondally intact male and female and oophorect-omized female pigs was determined by thymidine incorporation in the absence and presence of endothelin-1 with and without 17β-estradiol. Endothelin-1 (10-9 M to 10-7 M) significantly inhibited proliferation only in coronary smooth muscle cells from intact female pigs. Addition of β-estradiol inhibited proliferation of cells from intact females but there was not a synergistic effect with endothelin-1. Gender associated inhibition of smooth muscle proliferation by endothelin-1 may contribute, in part, to cardioprotection noted in estrogen-replete states.

RP 73870, the racemic potassium salt of (([N-(methoxy-3-phenyl)-N-(N-methyl-N-phenyl-carbamoylmethyl)- carbamoylmethyl]-3-ureido)-3-phenyl)-2-ethylsulfonate-(RS) is a potent, reversible antagonist of both gastrin and cholecystokinin-B receptors in guinea pig and rat tissues. This compound is a potent inhibitor of pentagastrin-stimulated gastric acid secretion in the perfused rat stomach. RP 73870 also inhibits basal gastric acid secretion in the rat, although at doses higher than that required for inhibition of pentagastrin-stimulated gastric acid secretion. RP 73870 is a potent inhibitor of aspirin-induced gastric damage in the rat. In the prevention of aspirin-induced gastric damage, RP 73870, given p.o., was 10-fold less potent than when given i.v. RP 73870 was as potent as a H2 receptor antagonist or proton pump inhibitor in the prevention of cysteamine-induced duodenal ulcers in the rat. Relative to other gastrin/cholecystokinin-B antagonists, RP 73870 demonstrates greater affinity to gastrin binding sites, and possesses a unique spectrum of in vivo biological activities appropriate for an anti-ulcer indication.

We have compared two methods for collecting earthworm leukocytes (coelomocytes) with respect to cell yield, viability and behaviour in immunoassays. Non-invasive extrusion was more efficient than puncturing the coelomic cavity. Extrusion does not produce trauma to earthworms maintained under long term laboratory conditions. Neither technique modified immune assays, as determined by erythrocyte and secretory rosette formation, and phagocytosis, since all 3 were functionally equivalent. After an initial extrusion, sequential leukocyte collections by extrusion are possible at intervals of 6 weeks without affecting total and differential cell counts and rosette formation.

The transfer of recombinant genes to donor organs may allow for novel therapeutic approaches to the challenges of acute and chronic rejection. Adenoviral vectors are capable of efficient gene transfer, but use of these vectors during donor organ preservation may be less efficient due to the low temperature. This study was designed to examine the effect of temperature on the efficiency of adenovirus-mediated gene transfer.

Abstract: Phytoplankton diversity, primary productivity and community metabolism were measured for 1 year in a 0.94 ha pond located in north-central Texas. Gross primary production ranged from 4.5 to 46.8 kcal m−2 day−1 ( $$\bar X$$ =22.0 kcal m−2 day−1) and community metabolism ranged from 7.3 to 32.4 kcal m−2 day−1 ( $$\bar X$$ =14.8 kcal m−2 day−1). Average production/respiration ratio (1.5) showed that the pond was principally autotrophic. Photosynthetic efficiency (gross primary production/0.5 total solar radiation) ranged from 0.32 to 2.8 with a mean of 1.2. Phytoplankton diversity based on numbers and biomass fluctuated greatly. Highest gross primary productivity occurred during Cyanophyta blooms in late summer-early fall when species diversity was minimal. Water temperature and turbidity, which governed light penetration, were the principal determinants of primary production.

Preferred deep body temperatures of adult bass were determined in the laboratory using a horizontal temperature gradient and telemetry. The method used allowed continuous monitoring of body temperatures of adult bass in the gradient. The preferred temperature of adults (range = 27–32°C) was similar to preferenda reported earlier for sub-adults.

This study examined the effects of live mass on ovarian follicular dynamics in heifers before and after the onset of nutritional anoestrus. The effects of decreased live mass on secretion of progesterone, LH and FSH were also investigated. Ovarian characteristics were monitored in 16 postpubertal Brahman heifers using daily transrectal ultrasonography. After two normal oestrous cycles, 11 heifers (treatment group) were allocated to a restricted diet, until the cessation of their oestrous cycles, that resulted in a decrease in live mass of 0.8 kg day−1. Heifers were then fed an ad libitumdiet, resulting in increasing live mass until resumption of oestrous cycles. Five heifers (control group) were fed an unrestricted diet throughout the experimental period. The concentrations of progesterone in plasma were monitored twice a week and the concentrations of LH and FSH were determined on day 6 after ovulation, before initiation of dietary restriction, after a 17% decrease in live mass and after the onset of anoestrus in the treatment group. Onset of anoestrus was preceded by linear decreases in the size of ovarian follicles and corpora lutea, and in the persistence of the first dominant follicles of oestrous cycles. These changes were proportional to the decrease in live mass (P< 0.001). The frequency of pulses of LH or mean concentration of FSH on day 6 of the oestrous cycle were not changed after a 17% decrease in live mass, and did not increase following the onset of anoestrus, although concentrations of progesterone were < 0.2 ng ml−1, suggesting that release of LH was suppressed at this time. Increasing live mass following anoestrus resulted in a linear increase in dominant follicle size and persistence, which were correlated with changing mass (P< 0.001); ovulation resumed when dominant follicles were 105 ± 4% of the size of initial ovulatory follicles.

Changes in pulsatile secretion of LH, FSH, oestradiol and progesterone were related to the growth phase, early plateau phase and regression phase of the first ovarian dominant follicle of the oestrous cycle in Bos indicusheifers. Relationships between these hormones during the three phases were also investigated. Accurate measurements of episodic ovarian steroid secretion were obtained by catheterizing the caudal vena cava via the lateral saphenous vein; the tip of the catheter was positioned just cranial to the ovarian vein using transrectal ultrasonography. Pulsatile secretion of oestradiol was increased only during the growth phase of the dominant follicle and was associated with high frequency release of LH pulses. However, mean concentrations of oestradiol were reduced when the dominant follicle attained its maximum diameter. Between the growth and plateau phases, as the amount of progesterone released increased and oestradiol released decreased, LH pulse frequency and mean concentration of LH decreased. Pulses of LH released were followed within 15 min by increases in mean concentrations of oestradiol (P< 0.001); however, there was no apparent relationship between LH and progesterone release (P= 0.19). Although there was little evidence of pulsatile release of FSH, mean concentrations of FSH were increased by 0.2 ng ml−1(P= 0.04) during the plateau phase, which was on average 2.1 days before the day of emergence of the second dominant follicle of the oestrous cycle. This increase in FSH, in conjunction with the decrease in secretion of oestradiol, may be an indication of the loss of functional dominance by the first dominant follicle of the oestrous cycle.

Previously we showed that mononuclear cells from about half of human T- lymphotropic virus (HTLV)-seropositive persons exhibit spontaneous proliferation in vitro. We sought to determine if proliferation was associated with other immunologic changes characteristic of HTLV infection. The parameters assessed were (1) percentages of lymphocytes expressing CD4 and/or CD25 (interleukin-2 receptor), (2) serum levels of soluble CD25, (3) serostatus for other viruses, (4) anti-HTLV antibody levels, and (5) HTLV type determined by polymerase chain reaction or serologic reactivity with type-specific peptides. The proliferation+ HTLV (PROL+) group, proliferation HTLV (PROL-) group, and control group showed similar percentages of CD4+, CD25+, and CD4+CD25+ lymphocytes; serum levels of soluble CD25 were also similar. Antibodies to cytomegalovirus, hepatitis B core, and hepatitis C were present in similar proportions of PROL+ and PROL+ groups. However, a significant association was found between spontaneous proliferation and anti-HTLV antibody levels; sera from 67% of PROL+ persons, but only 18% of PROL- persons, required dilution to yield absorbance values within the linear range of the anti-HTLV antibody assay. In the PROL+ group, persons whose sera required the most dilution had proliferative responses significantly higher than those whose sera required no dilution. The PROL+ and PROL groups were similar with regard to the relative distribution of HTLV-I and HTLV-II infection. These findings indicate that HTLV-related spontaneous lymphocyte proliferation is related to levels of circulating anti-HTLV antibodies, and characterizes both HTLV-I and HTLV-II infection.

Energy flow (A = R + P; assimilation = respiration + production) and life-history/density-biomass cycles were followed for 1 year in a mayfly (Brachycercussp.) population and two chironomid (Procladiussp. and Tendipes decorus) populations in a 0.94 ha pond located in north-central Texas. Temperature (5, 10, 15, 20, 25 and 30°C) effects on R and patterns of metabolic compensation/acclimatization were determined for the mayfly and chironomids. Chironomid and mayfly populations dominated the nine benthic macroinvertebrates identified in the pond, with greatest abundances in winter and winter-spring respectively. Annual energy flow was 51 kcal m−2(R = 40 kcal, P = 11 kcal or 1.9 g) in Brachycercussp., 23 kcal m−2(R = 5 kcal, P = 18 kcal or 2.4 g) in Procladiussp. and 74 kcal m−2(R = 17 kcal, P = 57 kcal or 6.0 g) in T. decorus. Each species metabolically compensated partially to temperatures en countered during their life histories.

In the neurological mutant mouse weaver (wv/wv), the majority of males are infertile due to hypospermatogenesis. Heterozygous weaver mice (wv/+) cease mating successfully when males reach an average age of 3.5 months. The contents of epididymal fluid were scored for the number of sperm and sperm motility in wv/wv, wv/+ and controls. Testes were examined in mice of the three genotypes at various ages using light and electron microscopy. In wv/+ males, sperm counts were significantly lower than in controls and were significantly higher than in wv/wv. The seminiferous epithelium in weaver mice appears depleted soon after puberty and a wide range of degenerative changes was identified in both germ cells and Sertoli cells. Analogous cellular aberrations were detected in heterozygous males, but they appeared at an older age and were not as severe as in wv/wv. We hypothesize that in weaver homo- and heterozygosity the damage of Sertoli cells may induce degeneration of germinal cells and particularly affect the most advanced spermatogenic cells.

The objective of this study was to determine the effects of inducing pituitary desensitization by treatment with an LHRH agonist (deslorelin) on reproductive hormone secretion and ovarian follicular status in heifer calves, before and during stimulation with FSH. The recovery and in vitro development of oocytes was also investigated. Brahman (Bos indicus) calves, 6 mo old, received either no treatment from Day 0 to Day 8 and injections of FSH on Days 9, 10, and 11 (controls, n = 10), or bioimplants of deslorelin on Day 0 and injections of FSH on Days 9, 10, and 11 (deslorelin calves, n = 10). Ovarian follicular characteristics were determined on Days -2, 0, and 8 by ultrasonography; follicle sizes (2–4 mm, 5–7 mm, 8–9 mm, or ≥ 10 mm) were recorded. Ovaries were removed surgically on Day 12, surface follicle numbers and sizes were recorded, and oocytes were aspirated, graded (A-grade, B-grade, denuded, atretic), and prepared for in vitro fertilization and culture. Blood samples were taken throughout the experiment to monitor plasma concentrations of LH, estradiol-17 β (estradiol) and progesterone. Treatment with deslorelin desensitized the pituitary in heifer calves and altered patterns of LH and estradiol secretion. There were no apparent consistent effects of deslorelin treatment on follicle numbers and growth. A higher number of combined A-grade and B-grade oocytes were obtained from heifers treated with deslorelin, which, in turn, resulted in twice the number of blastocysts. Treatment with an LHRH agonist provides a model for studying the hormonal requirements for follicle growth and in vivo oocyte maturation in heifer calves.

Quazolast, a mast cell stabilizer, was evaluated for efficacy against acid independent (alcohol, HCl), or dependent (aspirin, indomethacin) gastric damage in rats. Its gastroprotective profile was compared to that of ranitidine. In addition, the antisecretory and gastic ulcer (acetic acid induced) healing capabilities of these agents were examined. Quazolast, in direct contrast to ranitidine, protected the rat gastric mucosa from acid-independent, but not acid-dependent gastric damage. Quazolast lacked antisecretory activity in rats; however, it did heal acetic acid induced gastric ulcers in this species. On day 15 after acetic acid injection, quazolast significantly healed such ulcers, while ranitidine did not. Although the exact mechanisms of gastroprotection and ulcer healing action for quazolast remain to be determined, it may be an effective agent for the treatment of gastric ulcers.

The efficacy of various drugs used to treat ulcerative colitis, (sulfasalazine, 5-aminosalicylate, hydrocortisone) was investigated in a model of acetic acid-induced colitis in the rat. Subsequently, we tested the ability of antioxidant/5-lipoxygenase inhibitors (gossypol and nordihydroguiaretic acid [NDGA]) and a cyclooxygenase inhibitor (indomethacin) to attenuate the macroscopic colonic damage and/or neutrophil influx (myeloperoxidase activity [MPO]) associated with this model of colitis. Oral pretreatment with either sulfasalazine, gossypol, or NDGA significantly decreased colonic MPO activity induced by acetic acid. Intrarectal administration of such drugs resulted in an even larger reduction of the colonic inflammation, with gossypol being the most potent compound. Oral or intrarectal administration of corticosteroids (dexamethasone, hydrocortisone) also attenuated the parameters of acetic acid induced colitis. In contrast, pretreatment with indomethacin was ineffective, or when administered daily after colitis induction, indomethacin actually increased colonic neutrophil influx significantly. Our data suggest that both the route of drug administration and dosing regimen employed affect the antiinflammatory potency and/or efficacy of compounds on colitis induced by acetic acid in the rat. Drugs which were effective against this colitis may act by scavenging of oxygen derived free radicals.

The energy metabolism of a mammalian cell line grown in vitro was analyzed by substrate consumption rates and metabolic flux measurements. The data allowed the determination of the relative importance of the pathways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture contributed to the changing catalytic activities of key enzymes, which were determined.A murine B-lymphocyte hybridoma (PQXB1/2) was grown in batch culture to a maximum cell density of 1–2×106cells/mL in 3–4 d. The intracellular protein content showed a maximum value during the exponential growth phase of 0.55 mg/106cells. Glutamine was completely depleted, but glucose only partially depleted to 50% of its original concentration when the cells reached a stationary phase following exponential growth.The specific rates of glutamine and glucose utilization varied during culture and showed maximal values at the midexponential phase of 2.4-nmol/min/106cells and 4.3 nmol/min/106cells, respectively.A high proportion of glucose (96%) was metabolized by glycolysis, but only limited amounts by the pentose phosphate pathway (3.3%) and TCA cycle (0.21%).The maximum catalytic activity of hexolinase approximates to the measured flux of glycolysis and is suggested as a rate-limiting step. In the stationary phase, the hexokinase activity reduced to 11% of its original value and may explain the reduced glucose utilization at this stage.The maximal activities of two TCA cycle enzymes were well above the measured metabolic flux and are unlikely to pose regulatory barriers. However, the activity of pyruvate dehydrogenase was undetectable by spectrophotometric assay and explains the low level of flux of glycolytic metabolites into the TCA cycle.A significant proportion of the glutamine (36%) utilized by the cells was completely oxidized to CO2.The measured rate of glutamine transport into the cells approximated to the metabolic flux and is suggested as a rate-limiting step.Glutamine metabolism is likely to occur via glutaminase and amino transaminase, which have significantly higher activities than glutamate dehydrogenase.The calculated potential ATP production suggests that, overall, glutamine is the major contributor of cellular energy. However, at the midexponential phase, the energy contribution from the catabolism of the two substrates was finely balanced—glutamine (55%) and glucose (45%).

In a previous report, we described antibodies in autoimmune hypoparathyroidism (AHP) that are cytotoxic for cultured bovine parathyroid cells. In the present study, we show that sera from six AHP patients, but not from 26 patients with other autoimmune diseases or from 7 healthy subjects, react with bovine endothelial cells in culture (by flow cytometry and fluorescence microscopy) and in tissue sections (by immunohistology). We found uniformly that the immunoglobulin class reacting is IgM. Adsorption experiments showed that the antigenic determinants reacting with AHP sera were similar on bovine cultured endothelial cell membranes and in tissue sections of bovine parathyroid glands. The AHP sera also reacted with endothelial cells cultured from bovine adrenal medulla and pulmonary artery. Immunoblot analysis showed antibody binding to two major bands of 200 and 130 kDa solubilized from the membrane fraction of bovine parathyroid endothelial cells. Only one AHP serum consistently recognized endothelium-related structures on frozen sections of three different human parathyroid adenomas; two other sera reacted with one adenoma each; and three did not react with human adenomas. This indicates that human material is less suitable than bovine in detecting endothelium-related immune phenomena in AHP sera. The anti-endothelium IgM antibodies appear to be disease-specific but are not organ- or species-specific. The identification of endothelial cells as the target for antibodies in AHP raises the possibility that the endothelium subserves an important local function for endocrine epithelium.

We have shown previously that Ca2+-channel agonists, which open Ca2+ channels, inhibit parathyroid hormone (PTH) secretion from dispersed bovine parathyroid cells, whereas Ca2+-channel antagonists, which close Ca2+ channels, stimulate PTH release. We now have tested the effects of mouse antibodies specific for purified alpha subunits of rat skeletal muscle Ca2+-channel proteins on PTH secretion by bovine parathyroid cells in vitro. Mouse antisera (MC-2, MC-3, MC-4) blocked the secretion of PTH from parathyroid cells incubated with 0.5 mM Ca2+ ions. Affinity-purified MC-4 antibodies inhibited PTH release in a concentration-dependent manner. Incubation of parathyroid cells with pertussis toxin markedly reduced MC-4-dependent inhibition of PTH secretion. Parathyroid cell membrane proteins were fractionated by NaDodSO4/polyacrylamide gel electrophoresis under either reducing or nonreducing conditions and immunoblotted with MC-4 antiserum. Antibodies bound to one major band of protein with Mr approximately equal to 150,000. These results suggest that the antibodies bind to Ca2+-channel alpha subunits and act as agonists that open the channels and inhibit PTH release.

We utilized a recently developed long-term serum-free culture system for bovine parathyroid cells to detect antibodies in seven patients with autoimmune hypoparathyroidism (AHP). Antibodies were tested by indirect immunofluorescence methods and by cytotoxicity utilizing the chromium (51Cr) release technique. Seven AHP sera caused specific lysis [57 +/- 6% release of 51Cr vs. 5 +/- 1% for 56 controls (15 normal subjects and 41 patients with diverse other conditions associated with immune dysfunction)]. The least effect of any of the AHP sera on cell lysis exceeded the greatest effect of any of the control sera. Absorption of AHP sera (two cases) with bovine pituitary, thyroid, liver, or kidney cells did not affect lysis, but absorption with adrenal or parathyroid cells caused a marked decrease in specific lysis. Cytotoxicity determined by 51Cr release increased with antiserum concentration and time of incubation. Cytotoxicity was dependent on complement. Replicating parathyroid cells provide a uniform reproducible detection method for anti-parathyroid antibodies in AHP. The autoantibodies in AHP appear to be specific for tissue (parathyroid and adrenal cortex) but not for species.

Primary cultures of bovine parathyroid cells were developed using Coon's modified Ham's F-12 medium containing low (0.3 mM) concentrations of calcium and supplements of bovine hypothalamic extract, bovine pituitary extract, epidermal growth factor, insulin, transferrin, selenous acid, hydrocortisone, triiodothyronine, retinoic acid, and galactose. These cells were cultured serially on serum-coated dishes for 140 population doublings before signs of senescence were detected. The cells were epithelioid and maintained a high degree of differentiation as evidenced by calcium regulation of both growth and secretion and by prostaglandin E1 stimulation of cAMP formation and hormone release.

L-365,260, a nonpeptide antagonist of gastrin/CCK-B receptors, was evaluated in receptor binding, antisecretory and gastrointestinal damage assays. L-365,260 binds potently and stereo-selectively to gastrin and CCK-B sites in guinea pig tissue. In contrast, L-365,260 binds to the isolated canine parietal cell gastrin receptor weakly, and without stereoselectivity. In the pylorus-ligated rat, low doses of L-365,260, given i.v., attenuated pentagastrin-stimulated acid secretion, whereas higher doses were required to inhibit both histamine-stimulated and basal acid secretion. In an aspirin-induced gastric damage model, L-365,260 was 2.4-fold less potent than the standard histamine H2 antagonist cimetidine in preventing gastric damage when given i.v., and was 8.3-fold less potent than cimetidine when given p.o. Moreover, the ED50 value for L-365,260, given i.v., in prevention of aspirin-induced gastric damage (11.5 mg/kg) agreed well with its ED50 value for inhibition of basal acid secretion (12.6 mg/kg). At doses as great as 100 mg/kg p.o., neither L-365,260 nor cimetidine had an effect on ethanol-induced gastric damage. L-365,260, although p.o. less bioavailable relative to cimetidine in the aspirin gastric damage model, was as potent as cimetidine in the prevention of cysteamine-induced duodenal ulcers in the rat. We conclude that the gastrin/CCK-B receptor antagonist L-365,260, at doses supramaximal for the inhibition of pentagastrin-stimulated secretory responses in vivo, inhibits gastrointestinal damage in models of peptic ulcer disease by an antisecretory mechanism of action.

RG 12915 [4-[N-(1-azabicyclo[2.2.2.]octan-3-(S)-yl)]2-chloro-cis 5a-(S)-9a-(S)-5a,6,7,8,9,9a-hexahydrobenzofurancarboxamide hydrochloride] is a potent and effective agent against cisplatin-induced emesis in the ferret after i.v. or p.o. administration. This agent (p.o.) is also highly protective against cisplatin-induced emesis in the dog, as well as cyclophosphamide/doxorubicin-induced emesis in the ferret. When administered either p.o. or i.v., RG 12915 has a lower ED50 value (0.004 mg/kg) than GR 38032F, BRL 43694 and metoclopramide for attenuating cisplatin-induced emetic episodes in the ferret. It also has a long duration of action against cisplatin-induced emesis in the ferret. In contrast to metoclopramide, RG 12915 lacks significant antidopaminergic activity both in vitro [( 3H]spiroperidol displacement), as well as in vivo (apomorphine-induced emesis). In radioligand binding assays, RG 12915 is a potent and selective displacer of binding of 5-hydroxytryptamine (5-HT)3 binding sites (IC50 value = 0.16 nM), whereas failing to displace binding of ligands for the alpha-1, alpha-2 and beta adrenergic, 5-HT1 or 5-HT2 or cholinergic-muscarinic sites with IC50 values less than 1 microM. At a p.o. dose (1 mg/kg) in which RG 12915 is highly protective against cisplatin-induced emesis in the dog, RG 12915 has no significant gastroprokinetic activity in the same species. In summary, RG 12915 is a potent and p.o. effective agent against cytotoxic drug-induced emesis in animal models. The antiemetic potency of RG 12915 against cisplatin is unrelated to antidopaminergic or gastroprokinetic activity, but may be related to its affinity for 5-HT3 binding sites.

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains unresolved. A recently developed technique for the isolation of avian osteoclasts has been modified to obtain highly purified multinucleated cells from human giant cell tumors. These osteoclast-like cells have been examined for evidence of estrogen receptors (ERs) and responses to 17 beta-estradiol (17 beta-E2). Analysis of giant-cell RNA demonstrated expression of ER mRNA. Furthermore, immunoblot analysis revealed that the giant cells contained a 66-kDa protein that was recognized by a monoclonal antibody specific for the human ER. When isolated multinucleated cells were cultured on slices of bone, there was a dose-dependent decrease in resorption in response to treatment detectable at 10 pM 17 beta-E2. Treatment with 10 nM 17 alpha-estradiol or vehicle (control) did not inhibit resorption. Moreover, the multinucleated cells isolated from these tumors had decreased mRNA levels for cathepsin B, cathepsin D, and tartrate-resistant acid phosphatase (TRAP) as well as secreted cathepsin B and TRAP enzyme activity in response to treatment with 10 nM 17 beta-E2. In contrast to these data, no change in gene expression was detected in mononuclear cells from these tumors in response to 17 beta-E2 treatment. These data support the proposition that human osteoclasts are target cells for estrogen and that estrogen can inhibit bone resorption by human osteoclasts.

The aim of the present study was to develop a treatment protocol for the precise synchronization of oestrus that would avoid the development of persistent dominant ovarian follicles. Bos indicusheifers, in which oestrous cycles had been presynchronized, were allocated randomly, according to the day of their oestrous cycle, to one of five treatment groups. All heifers received a subcutaneous ear implant containing 3 mg of norgestomet for 17 days starting on day 0 and an injection of an analogue of prostaglandin F2αon days 0 and 4. Heifers in group 1 (control group; n= 7) received no other treatment, while heifers in groups 2 (n= 8), 3 (n= 7), 4(n= 7), and 5 (n= 7) received a single progesterone-releasing controlled internal drug release device (CIDR) for 24 h on days 10, 12, 14 and 16, respectively. Treatment with a single CIDR delayed the mean time of ovulation and the day of emergence of the ovulatory follicle in heifers treated on days 14 and 16 compared with control heifers (P<0.05). There was less variation in the interval to ovulation in heifers treated on day 10 compared with other treated heifers (P< 0.05). The variation among heifers in the day of emergence of the ovulatory follicle and the age of the ovulatory follicle at ovulation was less for all groups treated with a CIDR than for the control group (P<0.05). The duration of dominance and variation in the duration of dominance of the ovulatory follicle was less in heifers treated with a CIDR device on days 10 and 16 than for control heifers (P< 0.05). Mean age (days from emergence to ovulation) of the ovulatory follicle did not differ among treatment groups (P> 0.05). Concentrations of LH and oestradiol decreased coincident with increased concentrations of progesterone on the days of CIDR treatment in treated compared with control heifers (P< 0.02) but increased again after removal of the CIDR. A smaller proportion of follicles in the growing phase of follicular development at the time of CIDR treatment become atretic compared with follicles that had reached a plateau phase of follicular growth (14.3% (1/7) versus 90.5% (19/21), respectively; P< 0.001). It was concluded that acute treatment with progesterone can influence the growth pattern of ovarian follicular development. However, the effect varies with the stage of ovarian follicular development. Short term treatment with progesterone 7 days before the end of a 17 day period of norgestomet treatment resulted in precise synchrony of ovulation without the ovulation of a persistent dominant ovarian follicle.