Chronic food restriction decreases secretion of LH as a result of inhibitory influences on secretion of LHRH. We have previously reported that neuropeptide-Y (NPY) may directly or indirectly mediate this inhibitory effect on LHRH secretion. In the study reported here, we tested the hypothesis that long-term food restriction suppresses tonic release of LH as a result of 1) an increase in biosynthetic activity of NPY neurons in the arcuate nucleus of the hypothalamus, 2) an increase in activity of neurons that secrete beta-endorphin, and 3) a decrease in biosynthesis of LHRH. To test predictions of the hypothesis, we compared levels of mRNA encoding NPY, proopiomelanocortin (POMC; the precursor peptide of beta-endorphin), and LHRH, as well as tonic secretion of LH in food-restricted and well-nourished ewe lambs. Ten ewe lambs were ovariectomized at 18 wk of age and randomly assigned to receive either 100% nutritional requirements (FED; n = 5), or 30% requirements (R; n = 5) between 18 and 25 wk of age. At 25 wk of age, blood samples were taken every 10 min for 6 h and assayed for LH. The tonic release of LH in R lambs was less than that of FED lambs. Hypothalami were collected 4 days after blood sampling and sectioned at 12 microns for use in in situ hybridization. Radiolabeled molecular probes specific for mRNAs encoding NPY, POMC, or LHRH were hybridized to hypothalamic tissue sections. Levels of NPY mRNA were 88% greater in R vs. FED lambs (p < 0.01), whereas levels of POMC mRNA were 52% lower in R vs. FED lambs (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
We examined temporal effects of estradiol (E2) and progesterone on cellular levels of LHRH messenger RNA (mRNA) in several brain regions. Female rats were ovariectomized and implanted with SILASTIC brand capsules of oil or E2 1 week later (day 0). Progesterone capsules were implanted on the morning of day 2. Using in situ hybridization histochemistry, we found that on day 2, E2 induced a complex temporal pattern of changes in LHRH mRNA levels. Levels in ovariectomized E2-treated animals were lower than control values in the morning, then increased before the LH surge and declined again as the surge waned. The magnitude of these changes was greatest in neurons of the rostral preoptic area/organum vasculosum of the lamina terminalis, but a similar pattern was detected in neurons of the medial preoptic area. No changes were seen in neurons of the diagonal band of Broca. Our finding that the effect of E2 on LHRH mRNA levels depends on the time of day and brain region examined largely reconciles discrepancies among previous studies. Progesterone triggered events that advanced the onset of and augmented the LH surge, but had no effect on LHRH mRNA levels. These findings support the hypothesis that the mechanism by which E2 induces region-specific changes in LHRH mRNA levels before the surge is separate from the progesterone-amplified mechanism that induces LHRH release.
To test the hypothesis that changes in LHRH mRNA levels are tightly linked to changes in LHRH secretion, intact male rats were infused with NMDA, a substance which increases LH release by a hypothalamic action. NMDA significantly elevated cellular levels of LHRH mRNA at 15 and 60 min. Similar changes in LH levels were induced by NMDA. These effects of NMDA on LHRH mRNA levels were not due to a generalized excitatory effect in the brain because levels of mRNAs encoding proopiomelanocortin (POMC) and tyrosine hydroxylase (TH) in the arcuate-periarcuate region were not altered by NMDA and levels of TH mRNA were decreased significantly at 15 and 60 min in neurons of the zona incerta. These data strongly support the hypothesis that changes in cellular levels of LHRH mRNA are tightly linked temporally to changes in LHRH secretion.
Beta-endorphin is thought to be an important inhibitor of LHRH neuronal activity and also to play a role in conveying information about changes in steroid levels to LHRH neurons. We have previously shown that the mRNA encoding the precursor of beta-endorphin, proopiomelanocortin (POMC), fluctuates during the estrous cycle with the most dramatic changes occurring on proestrus. POMC mRNA levels decline before the onset of LH surge release but then dramatically rise and remain elevated during the surge. In the present studies we tested the hypothesis that the decline in POMC mRNA levels immediately before the proestrus LH surge is mediated by estrogen and the rise during the surge by progesterone. To test this hypothesis, we compared changes in POMC mRNA levels between ovariectomized (OVX) and OVX estrogen (E2)-treated rats and between OVX E2-treated rats with and without progesterone. Animals were examined at hourly intervals after the administration of progesterone, then at every 4 h during the LH surge. Using in situ hybridization histochemistry, we found that E2 decreased POMC mRNA levels in OVX rats before the onset of the LH surge and further suppressed levels during the surge. Compared to animals treated with E2 alone, progesterone advanced the time at which both the LH surge began and the time at which POMC mRNA levels declined. After a transient decline, POMC mRNA levels rose in these progesterone-treated animals and remained elevated throughout the period of the LH surge. These results support the hypothesis that progesterone times the LH surge and limits its appearance to one day be exerting a biphasic effect on the activity of beta-endorphinergic neurons of the arcuate nucleus.
Invasive methicillin-resistant (MRSA) treated with vancomycin (VAN) is associated with reduced VAN susceptibility and treatment failure. VAN combination therapy is one strategy to improve response, but comprehensive assessments of combinations to prevent resistance are limited. This study identifies optimal combinations to prevent the emergence of VAN-intermediate (VISA). Two standard MRSA and two heterogeneous VISA (hVISA) strains were exposed for 28 days to VAN alone, VAN with cefazolin (CFZ), fosfomycin, gentamicin, meropenem, rifampin, piperacillin-tazobactam (TZP), or trimethoprim-sulfamethoxazole. In addition to VAN susceptibility testing, cell wall thickness (CWT), carotenoid content, and membrane fluidity were determined for Mu3. VAN plus any β-lactam limited the VAN MIC increase to 1 to 4 mg/liter throughout the 28-day exposure, with CFZ and TZP being the most effective agents (VAN MIC = 1 to 2 mg/liter). Similar MIC trends occurred with the lipo-/glycopeptide agents daptomycin and telavancin, where β-lactam combinations with VAN prevented MIC increases to these agents as well. Combinations with non-β-lactams were ineffective in preventing VAN MIC increases with VAN MICs of 4 to 16 mg/liter emerging during weeks 2 to 4 of treatment. VAN plus β-lactam decreased CWT significantly, whereas VAN plus other antibiotics significantly increased the CWT. No correlation was observed between carotenoid content or membrane fluidity and antibiotic exposure. Only the combination exposures of VAN plus β-lactam suppress the development of VISA. Rational selection of VAN plus β-lactam should be further explored as a long-term combination treatment of MRSA infections due to their ability to suppress VAN resistance.
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