Reduced ability of naloxone to stimulate LH and testosterone release in aging male rats; possible relation to increase in hypothalamic met5-enkephalin

Blood luteinizing hormone (LH) and testosterone levels are lower in old than in young male rats. The specific opiate antagonist, naloxone, previously shown to increase serum LH in mature male rats, exhibited relatively little ability to raise serum LH and testosterone levels in old (18–20 mo) as compared to young (4–5 mo) male rats. The brain opiate, met⁵-enkephalin, which depresses LH, was found to be significantly higher in the hypothalamus of old than of young male rats. These observations suggest that hypothalamic opiates may be partially responsible for the lower serum LH and testosterone levels in old male rats, and for reduced release of these hormones in response to naloxone administration.     

Changes in adenosine 3',5'-monophosphate induced by parathyroid hormone and theophylline in the developing rat renal cortex

To test the biochemical responsiveness of developing rat renal cortex to parathyroid hormone (PTH), intracellular concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were measured. Renal cortical slices from 10-day-, 20-day-, and 12-week-old animals contained higher concentrations of cyclic AMP when incubated in the presence of theophylline than in its absence. In the absence of theophylline, tissue from all three age groups responded to PTH with dose-dependent increases in cyclic AMP. In the presence of theophylline the response of tissue from 10-day-old animals was greater than that of 12-week-old animals.It is suggested that the differential effect of theophylline with respect to age may be the result of higher turnover rates of cyclic AMP in the young animals.     

Male sexual behavior in deer mice (Peromyscus maniculatus) following castration and hormone replacement

Sexually experienced male deer mice (Peromyscus maniculatus bairdi) were castrated and tested for male sexual behavior. In the weeks following castration male sexual behavior decreased. Ejaculation disappeared first, followed by intromission and, finally, mounting. Castrated males failing to copulate were assigned to one of four treatment groups: 200 μg testosterone propionate (TP); 200 μg dihydrotestosterone propionate (DHTP); 2 μg estradiol benzoate (EB); or sesame oil (OIL). TP and DHTP were equally effective in restoring the complete male sexual behavior pattern. In contrast, EB was effective in stimulating mounting and minimally effective in stimulating intromissions (vaginal penetration), but did not stimulate ejaculatory responses. These data indicate that in deer mice testosterone may mediate male sexual behavior through reduction to dihydrotestosterone rather than through aromatization to estradiol.     

Pubertally Initiated High-Fat Diet Promotes Mammary Tumorigenesis in Obesity-Prone FVB Mice Similarly to Obesity-Resistant BALB/c Mice

Premenopausal breast cancer is associated with increased animal fat consumption among normal-weight but not overweight women. Our previous findings in obesity-resistant BALB/c mice showed that a diet high in saturated animal fat (HFD) promotes mammary tumorigenesis in both DMBA carcinogenesis and Trp53-null transplant models. Having made these observations in BALB/c mice, which have very modest HFD weight gain, we determined the effects of HFD in FVB mice, which gain significant weight on HFD. Three-week-old FVB mice fed a low-fat diet or HFD were subjected to 7,12-dimethylbenz[a]anthracene-induced carcinogenesis. Like BALB/c mice, HFD promoted mammary tumorigenesis. Development of tumors largely occurred prior to mice becoming obese, indicating the role of animal-derived HFD rather than resulting obesity in tumor promotion. Also similar to BALB/c mice, early-occurring adenosquamous mammary tumors were abundant among HFD-fed FVB mice. Tumors from HFD mice also had increased intra-tumor M2 macrophages. Prior to tumor development, HFD accelerated normal mammary gland development and increased mammary M2 macrophages, similarly to BALB/c mice. The promotional effects of puberty-initiated HFD on carcinogen-induced mammary cancer are thus largely weight gain-independent. Like BALB/c mice, HFD promoted adenosquamous tumors, suggesting a role for early age HFD in promoting this subtype of triple negative mammary cancer. M2 macrophage recruitment was common to both mouse strains. We speculate that a similar effect of HFD on immune function may contribute to epidemiological findings of increased breast cancer risk in young, premenopausal, normal-weight women who consume a diet high in saturated animal fat.     

Rapid, concurrent analysis of dopamine, 5-hydroxytryptamine, their precursors and metabolites utilizing high performance liquid chromatography with electrochemical detection: analysis of brain tissue and cerebrospinal fluid

Assays capable of measuring picomole quantities of dopamine (DA), 5-hydroxytryptamine (5-HT), several of their precursors and metabolites concurrently within 25 minutes were developed utilizing high performance liquid chromatography with electrochemical detection (LCEC). Several parameters of the LCEC were altered in order to separate the compounds while maintaining a short assay time. The final LCEC systems demonstrated biological utility in that the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the 5-HT metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were detected in rat cerebrospinal fluid; in addition to these compounds, DA and 5-HT were measurable in the striatum, hypothalamus and median eminence of the rat brain. Pargyline decreased the concentrations of DOPAC, HVA and 5-HIAA and increased the 5-HT concentration in all three brain regions, and increased the DA concentration in the striatum. Probenecid increased all three acid metabolite concentrations in the hypothalamus and median eminence, while only the HVA and 5-HIAA concentrations were increased in the striatum. The DA and 5-HT concentrations were unaltered. The LCEC methods described in this paper should be useful in elucidating the mechanisms and roles of 5-HT and DA neurons in experimental paradigms of biological interest.     

Acitivity measurements as a tool to characterize the microbial composition of methanogenic environments

A rapid and reliable method to assess the potential specific activity of methagenic sludge is presented. The method is based on the gas chromatographic analysis for methane in the headspace of closed vials. Gas is sampled with a pressure lock syringe, which allows quantification independent of the pressure prevailing in the vials.The influence of various parameters as substrates, pH, NaCl and NH₄ Cl concentrations on the activity of methaogenic sludge was investigated with this method. Data on the methanogenic activities on different substrate are discussed in terms of different physiological groups present in the sludge.     

The suppressive effect of a supernate from concanavalin A-activated human lymphocytes: effects of concanavalin A-activated lymphocytes and their supernates on cytotoxic and mixed lymphocyte reactions.

The effects of Concanavalin A-treated human peripheral blood lymphocytes and their supernatants were evaluated on the MLC reaction and on the generation of cytotoxic lymphocytes assessed by cell-mediated lympholysis (CML). Experiments were performed with both allogeneic and xenogenic sensitization. It was found that Con A-activated cells suppressed the MLC and CML reactions in allogeneic and xenogeneic systems. On the other hand, the SIRS-like supernate was able to suppress the MLC reaction and blastogenesis, but had no suppressive effect on the generation of cytotoxic lymphocytes. We found no difference in the magnitude of suppression, whether or not Con A-activated lymphocytes were syngeneic to the responder cells. This finding suggests that there is no requirement for allogeneic restriction in the interaction between suppressor and suppressed cells, and demonstrates a soluble human suppressor substance capable of suppressing some cell-mediated reactions.     

6-Aminohexyl glycopyranosides as ligands for the preparation of affinity adsorbents for the purification of carbohydrate-binding proteins

Syntheses are described of 6-aminohexyl and 2-aminoethyl glycopyranosides of the monosaccharides commonly found in glycoproteins and glycolipids. The ligands have been coupled to agarose, and the adsorbents so formed have been used in the isolation of the lectin from wheat-germ extracts. Various procedures for the formation of glycopyranosides from ¹⁴C-labelled per-O-acetylglycosyl halides and 6-(trifluoroacetamido)hexanol have been examined. Silver carbonate with a trace of iodine is the best catalyst found thus far for glycosidation of simple sugars, and mercuric cyanide for that of 2-acetamido-2-deoxyaldoses. With other catalysts, higher proportions of ortho esters are obtained. The rearrangement of an ortho ester by p-toluenesulfonic acid gives the glycoside, a partially deacetylated glycoside, and a substantial proportion of the aglycon acetate. A simple procedure for isolation of the deacetylated glycosides by use of chromatography on Dowex-50 (pyridinium form) is described.     

Inhibition of estrogen-activated sexual behavior by androgens

Experiments to determine the potential of androgen to inhibit estrogen-activated female sexual behavior in rats were conducted. Treatment with either testosterone propionate (0.8 or 1.6 mg/day) or dihydrotestosterone propionate (0.2, 0.4, or 0.8 mg/day) significantly reduced the incidence of lordosis in ovariectomized females receiving estradiol benzoate (1 microgram/day). A similar suppression of estrogen-activated lordosis by testosterone was observed in castrated male rats. Flutamide, an androgen-receptor blocker, prevented the inhibition of lordosis by testosterone in females, indicating that the interaction of testosterone or a metabolite with an androgen receptor may be an important feature of this inhibition. Furthermore, the ability of dihydrotestosterone to inhibit lordosis at lower doses than testosterone suggests that the conversion of testosterone to dihydrotestosterone may also be necessary. These experiments demonstrate the potential of testosterone to inhibit the occurrence of female sexual behavior in rats, in contrast to its established facilitative effect on this behavior.     

Regulation of B cell tolerance by murine gangliosides

The potential role of gangliosides as modulators of the triggering of neonatal primary B lymphocytes at the single precursor cell level was evaluated. Tolerance was induced in splenic fragment cultures containing an excess of carrier-primed T cells. Gangliosides at low concentrations (20 ng/culture) abrogated the tolerogenic effect of haptens presented on carriers not recognized by environmental T cells. The permanent arrest of immature B-cell responsiveness resulting from tolerogen treatment was eliminated by the presence of gangliosides during tolerogen treatment. The active moiety in the glycolipid preparation which protected B cells during tolerogen treatment was separated by ion-exchange chromatography and demonstrated to be in the disialoganglioside fraction.     

Interaction of gangliosides with B cells in splenic fragment cultures

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.     

Intestinal protein dynamics in the stonefly, Pteronarcys californica

Characteristics of the intestinal proteins of a detritus-eating aquatic insect, Pteronarcys californica were assessed to determine their possible role as a nutrient source during metamorphosis. Total soluble intestinal protein was significantly correlated to larval weitht, averaging 3.9% of dry weight for both males and females. Proteinase activity in the gut was separated into several bands by electrophoresis. Electrophoresis patterns of total intestinal proteins of P. californica larvae of differing instar, sex, and diet were remarkably similar. Larvae incubated with tritium-labelled amino acids produced labelled intestinal proteins. When early instar larvae were removed from label, there was no significant loss of tritium from the intestine after 2 months. Penultimate instars labelled in the same manner however, showed depuration of tritium from the gut according to first order kinetics. This loss was correlated to the 6–8 wk non-feeding stage of metamorphosis, just prior to adult emergence. These results indicate that proteins are accumulated in the intestinal tract of immature P. californica, then are reabsorbed from the gut during metamorphosis.     

Further evidence for masculinization of female rats by males located caudally in utero

The morphology and behavior of female rodents is partially masculinized as a result of residence near males in the same uterine horn (Clemens effect). Two hypothetical mechanisms have been proposed to account for this effect. In the first hypothesis (“contiguity”) androgens secreted by males in utero are proposed to diffuse across the amniotic membrane, reaching adjacent fetuses. In the second hypothesis (“caudal male”) androgens are transported via the cervical-to-ovarian blood flow and may diffuse directly between closely apposed uterine veins and arteries. This study was designed to test directly which of these mechanisms appears more influential in masculinizing the morphology of female rats. Pregnant Sprague-Dawley rats were decapitated early on Day 22 of gestation and pups were Caesarean delivered. Their anogenital distance and body weight were recorded, location in utero coded by means of footpad tatooing, and each litter fostered to a maternal female. Measurements were taken again when the animals were weaned. Statistical analysis revealed that the presence of one or more males caudal to a female in the uterine horn has a more critical influence on that female's morphology than contiguity per se. Such a mechanism may result in partial masculinization of dimorphic behaviors later in life.     

A sensitive method to measure physical and chemical carcinogen-induced "unscheduled DNA synthesis" in rapidly dividing eukaryotic cells

A sensitive assay for quantitating ‘unscheduled DNA synthesis’ (repair synthesis) in transformed human amnion (AV₃) cells has been developed. The combined use of hydroxyurea and arginine-deficient culture medium enabled the detection of 10–20 fold increases in ‘unscheduled DNA synthesis’ after treatment with N-acetoxy-2-acetylaminofluorene or ultraviolet light. The technique allows the detection of ‘DNA repair synthesis’ following treatment with extremely low doses of mutagens and carcinogens.     

Alteration in the action of cholinergic and anti cholinergic drugs after chronic haloperidol: indirect evidence for cholinergic hyposensitivity.

Following repeated treatment with haloperidol, spontaneous locomotor activity and the locomotor stimulation produced by amphetamine and the anti-cholinergic, dexetimide was enhanced compared with normal rats. The reduction in locomotor activity produced by the cholinergic agonist, pilocarpine, was retarded following this treatment. These results suggest that a dopaminergic supersensitivity and a cholinergic hyposensitivity develop as a result of chronic neuroleptic treatment.     

Effect of intermittent injections of gonadotrophin releasing hormone at various frequencies on the release of luteinizing hormone and ovulation in dairy cows

Gonadotrophin releasing hormone (GnRH, 5 μg every 4 h) was administered to six dairy cows between days 5 and 10 post-partum and the release of luteinizing hormone (LH) and the onset of ovulation were determined. LH was measured using a specific radioimmunoassay and the occurrence of ovulation was assessed from changes in the concentration of progesterone in milk. Treatment with GnRH resulted in a median time of first ovulation of 17.0 days after calving. This was less (P < 0.05) than that observed for control cows (21.5 days, n = 7). Determinations of plasma LH concentrations over an 8-h period on days 6 and 10 post-partum indicated that there was a tendency for GnRH-treated cows to have higher levels of LH on these days. The 5 μg dose of GnRH did not repeatably induce a release of LH between days 6 and 10. Endogenous pulsatile release of LH did, however, increase in frequency from 3.18 pulses per 8 h on day 6 to 5.18 pulses per 8 h on day 14 post-partum (P < 0.01).In a second experiment groups of 20 cows were treated with either 5 μg GnRH every 4 h or 15 μg GnRH every 12 h from days 5 to 10 post-partum. Seventeen untreated cows served as controls. The median times to first ovulation were 27.0 days for the control cows, 22.5 days for those cows treated with 5 μg GnRH every 4 h and 17.0 days for cows treated with 15 μg every 12 h. The latter treatment significantly advanced the time of first ovulation (P < 0.05) relative to controls. This difference had, however, disappeared by the time of the second and third ovulations. Primiparous cows ovulated later (P < 0.01) than the pluriparous cows in the group treated with 5 μg GnRH every 4 h. This was a major reason for the lack of effect of this treatment. Some treated cows were blood sampled at frequent intervals on day 8 to evaluate the LH responses to GnRH injections. The administration of 5 μg GnRH on day 8 did not elicit a pulse of LH which could be distinguished from endogenous pulsatile secretion at this time. The dose of 15 μg on this day did, however, elicit a more defined pulse on some, but not all, occasions.The injection of a small dose of GnRH twice a day from day 5 to day 10 after calving, therefore, advanced the time of first ovulation in dairy cows by 10 days.