Effects of a luteinizing hormone-releasing hormone antagonist in late-juvenile female rats: blockade of follicle growth and delay of first ovulation following suppression of gonadotropin concentrations

Subcutaneous injections of an antagonist against luteinizing hormone-releasing hormone (LHRH-A, Org, 30276) were administered to late-juvenile female rats. The effects on timing of vaginal opening and first ovulation on serum gonadotropin concentrations and on follicle growth were studied. The dose of 100 micrograms LHRH-A/100 g body wt, given on Days 28, 31, and 34, did not influence timing of first ovulation. After administration of 500 micrograms LHRH-A/100 g body wt, ovulation was retarded by 4.7 days if injections were given on Days 28 and 31; by 6.7 days if given on Days 28, 31, and 34; and by 11.5 days if given on Days 28, 31, 34, and 37. Serum LH and FSH concentrations 3 days after the first, second, and third injections of 500 micrograms LHRH-A were significantly (p less than 0.01) lower than in saline-treated controls. Ovarian follicle counts showed decreased numbers of (antral) Class 2, 3, and 4 follicles 3 days after injection of 500 micrograms LHRH-A/100 g body wt on Day 28; a significantly higher number of Class 1 follicles and a further decrease in Class 2, 3, and 4 follicles 3 days after the second LHRH-A injection; and total absence of Class 3, 4, and 5 follicles 3 days after the third LHRH-A injection. Six days after the third LHRH-A injection, Class 3 and 4 follicles reappeared in the ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.     

Isolation and partial characterization of rat duodenal-gland (Brunner''s-gland) mucus glycoprotein.

A mucus glycoprotein was isolated from the duodenal glands of the rat and purified by repeated density-gradient centrifugation. The characterized glycoprotein is unique to the mucous cells of the duodenal glands and is not present in parts of the small intestine devoid of these glands. The chemical composition of the purified glycoprotein is characteristic for glycoproteins of the mucin-type. Its protein content is relatively high and amount to 35% by weight. No neuraminic acid and little sulphate (2%) is present. Evidence is presented that the native glycoprotein is built up from subunits held together via disulphide bridges in a non-glycosylated region of the protein core.     

Dietary administration of eicosapentaenoic and linolenic acid increases arterial blood pressure and suppresses vascular prostacyclin synthesis in the rat

The role of the ‘prostacyclin-thromboxane system’ in the regulation of arterial blood pressure was investigated in rats receiving diets which contained different amounts of eixosapentaenoic (EPA) and linolenic acid (LNA). Forty rats were divided into five groups of 8 animals, each group receiving 25 energy (en) % as fat. All diets contained equal amounts of linoleic acid (5 en%) and oleic acid (5 en%). In the control group I, the remaining 15 en% of fat were given as saturated fat. Two groups of animals received cod liver oil as a source for EPA in amounts of 2.5 (group II)_and 5 en% (group III) while the two remaining groups were given diets supplemented with linseed oil as a source for LNA in amounts of 2.5 (group IV) and 5 en% (group V), respectively. After six weeks of feeding period the animals were sacrificed and portions of their isolated aorta incubated in Tris buffer (pH 9.3) for determination of prostacyclin (PGI₂)-like activity. Arterial blood pressure was uncharged in group I animals, but significantly increased in all rats receiving dietary EPA or LNA supplements. This rise is arterial blood pressure was associated with a marked suppression of the appearance of PGI₂-like activity in the incubation buffer while platelet thromboxane release during blood clotting was unchanged. Our results show that dietary adminis- tration of EPA and LNA increases arterial blood pressure in the rat and that this effect is associated with a suppressed generation of vasodilator prostacyclin by vascular tissue.     

Multimodal biomarker discovery for active Onchocerca volvulus infection

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.     

Extreme heat- and pressure-resistant 7-kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single-point amino acid substitution

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance. Proteins 29:381–390, 1997. © 1997 Wiley-Liss, Inc.     

Microfungal Community Changes in Rodent Food Stores over Space and Time

Abstract This paper reports the changes over time in the microfungal communities that inhabit three rodent species' food stores at two climatically different locations. Results reveal that microfungal diversity values calculated from above-ground food stores are highest in the more commonly disturbed portions of the rodent dens. Interactions among food-inhabiting microbes and between the rodents and food-inhabiting microbes also appear to influence the microfungal communities within the rodent dens. For example, our data suggest that transport by animal vectors, and not by air currents, is more effective at dispersing microbial propagules. Furthermore, although fungal communities inhabiting food stores within dens varied in composition and diversity over time, standardized substrates (sorghum seeds) simultaneously placed within the food stores converged in microfungal composition the longer they were left within the dens. We hypothesize that animal vectors, including rodents, make neighboring fungal communities more alike by introducing similar communities of microbes, which in turn initiate a cascade of biological interactions that, over time, result in similar microfungal communities inhabiting newly stored food items. Received: 14 January 1999; Accepted: 2 April 1999     

Regenerative Potential of Blood-Derived Products in 3D Osteoarthritic Chondrocyte Culture System

Intra-articular injection of different types of blood-derived products is gaining popularity and clinical importance in the treatment of degenerative cartilage disorders such as osteoarthritis. The regenerative potential of two types of platelet-rich plasma (PRP), prepared in the presence of EDTA (EPRP) and citrate (CPRP) and an alternative blood product-hyperacute serum (hypACT) was evaluated using a 3D osteoarthritic chondrocyte pellet model by assessing the metabolic cell activity, cartilage-related gene expression and extracellular matrix deposition within the pellets. Chondrocyte viability was determined by XTT assay and it revealed no significant difference in metabolic activity of OA chondrocyte pellets after supplementation with different blood products. Nevertheless, the selection of blood products influenced the cartilage-related genes expression, ECM morphology and the tissue quality of pellets. Both PRP types had a different biological effect depending upon concentration and even though CPRP is widely used in clinics our assessment did not reveal good results in gene expression either tissue quality. HypACT supplementation resulted in superior cartilage-related genes expression together with tissue quality and seemed to be the most stable product since no remarkable changes were observed between the two different concentrations. All in all, for successful regenerative therapy, possible molecular mechanisms induced by blood-derived products should be always carefully investigated and adapted to the specific medical indications.     

Melampolides from Argentinean Acanthospermum australe

The investigation of the ethanol extract of Acanthospermum australe, collected in the province of Misiones, Argentina, yielded eight melampolides (1–8) of the acanthospermal type. Two of them, 8β-hydroxy-9α-(2-methylbutyryloxy)-14-oxo-acanthospermolide (3) and 9α-hydroxy-8β-(2-methylbutyryloxy)-14-oxo-acanthospermolide (7) are new compounds. Two other compounds (4 and 8) have been previously reported, and the NMR data of 4 are corrected. Compounds 1, 2, 5 and 6 have not been previously reported, but are probably artifacts formed during extraction. Compounds 3, 6 and 7 showed slight antibiotic activity against Gram-positive bacteria.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.