Presence of bacteria in porcine follicular fluid and their ability to generate an inhibitor of follicle-stimulating hormone binding to receptor

Follicular fluid obtained from porcine ovaries collected at slaughter and distributed by the National Institutes of Health was contaminated by bacteria which appeared to be of intestinal origin. This follicular fluid showed increased follicle-stimulating hormone binding inhibition (FSH-BI) activity following incubation under conditions which facilitated bacterial growth. No such increase in FSH-BI activity was observed following incubation of follicular fluid from which bacteria were removed by repeated filtration. Our data suggest that bacteria found in the follicular fluid were capable of generating a substance with FSH-BI activity. This substance has an apparent molecular weight greater than 6000, based on membrane diafiltration studies. The possible presence of bacteria in follicular fluid and their ability to generate a substance which interferes with FSH binding to receptor should be considered in studies on factors in follicular fluid that are considered to regulate ovarian function or development.     

Characterization of a substance P-Gly12 amidating enzyme in human cerebrospinal fluid

Enzyme activity capable of converting the glycine-extended substance P precursor, substance P-Gly12, into substance P was purified from human cerebrospinal fluid. The conversion reaction was monitored by radioimmunoassay measurement of substance P formation. The chemical identity of the product was verified by reversed-phase HPLC. The enzyme reaction was stimulated by Cu(II) ion and ascorbic acid and inhibited by the presence of diethyldithiocarbamate. By HPLC molecular sieving, the major enzyme activity appeared as a protein of 26,000 molecular weight.     

An approximation method for the determination of diffusion and permeability coefficients in nerve trunks

A recently introduced approximation method is applied in order to obtain an expression for the amount of a substance remaining within a nerve at any time, the nerve having been soaked for a long time in a solution containing the substance until the time zero when it is bathed in the same solution but without the substance. The case of a uniform nerve without a sheath leads to substantially the same results as previously obtained by A. V. Hill (1928) for this case. A solution is given for the case of a nerve without sheath but having fibers which are permeable. In this case it is shown how an effective diffusion coefficient for the interstitial fluid can be obtained, as well as the effective inward and outward fiber permeabilities. A solution is given for the case of a nerve with a sheath in which the substance considered does not penetrate the fibers, and it is shown how the effective diffusion coefficients of the sheath and the interstitial fluid can be obtained.     

Production of Nucleic Acid Related Substances by Fermentative Processes

It was found that a guanine and adenine-requiring mutant of Micrococcus glutamicus accumulated the ultraviolet-absorbing substance in the culture fluid. A KY9978 strain, which accumulated the largest amount of the substance, we selected from the guanine auxotrophs derived from the guanineadenine doubleless mutant. The substance was isolated in a crystalline form from the culture fluid by the use of ion exchange resins, Diaion SA 21A and SK No. 1, and identified chemically and enzymatically as 5′-xanthylic acid, an intermediate from 5′-inosinic acid to 5′-guanylic acid on the purine nucleotide biosynthesis.     

Potential error in ventriculocisternal perfusion method for determination of cerebrospinal fluid formation rate in cats

The cerebrospinal fluid (CSF) formation rate (Vf) has been extensively studied by the ventriculocisternal perfusion, a method still regarded as the most precise one. This method as well as the equation for the calculation of the CSF formation rate (Vf) was established by Heisey et al on indicator dilution in perfusate. They assumed that the dilution of the indicator substance in perfusion is a consequence of newly formed CSF i.e. a higher CSF formation rate would result in a higher degree of dilution of the indicator substance. Therefore, such method is indirect and any mistake in the interpretation of the degree of indicator dilution would lead to questionable and often contradictory results regarding CSF formation rates. According to Heisey's equation, Vf shoud not depend on the rate of ventriculocisternal perfusion. However it has been shown that Vf is perfusion dependt value, and also that during perfusion the indicator substance is partially absorbed into surrounding tissue. It is possible that obtained Vf dependence on perfusion rate was caused by observed absorption of indicator substances. For that reason, in anaesthetised cats ventriculocisternal perfusion was performed at higher (252.0 microL/min) and at lower perfusion rate (65.5 microL/min) and Vf was calculated at both experimental and corrected (just for absorbed amount) values of indicator substance. Since (inspite of the correction) the difference of 12.4 microL/min between lower (15.0 microL/min) and higher perfusion rate (27.4 microL/min) was obtained, it is obvious that ventriculocisternal perfusion method cannot be considered reliable for measuring CSF formation rate.     

Purification and Characterization of the Antioxidative Substance Produced by Aspergillus sojae K

An antioxidative substance produced by Aspergillus sojae K is absolutely soluble in water and strongly inhibits autoxidation of Na-ascorbate. The substance, produced extracellularly in the culture ftuid by the mold, was purified by ion exchange chromatography, gel filtration, and HPLC. The substance was purified 34-fold with an activity recovery of 38% from culture fluid. Purity of the substance was confirmed with a single peak through HPLC using an analytical column as well as a single spot on TLC. The purified substance consists of equimolar aspartic acid and glycine, indicating that the substance is a peptide. From mass spectral analysis the molecular weight was 710, but the precise sequence of the amino acids is not clear. The substance is stable at 70°C, but about 80% of the activity was lost at 80°C after 60 min. Besides, the substance is completely stable at pH 3–14. This substance efficiently suppressed the oxidation of fish oil.     

Regulatory response to washout of amniotic fluid in sheep

To test the hypothesis that a substance present in the amniotic fluid could serve as a regulator of amniotic fluid volume, we drained and discarded amniotic fluid while replacing it with lactated Ringer solution that was isotonic to amniotic fluid. Seven ewes with singleton fetuses at 119 +/- 1 days of gestation (mean +/- SE) were instrumented with multiple indwelling catheters in the pedal artery, pedal vein, and amniotic cavity. During the exchange periods, an average of 3,019 +/- 171 ml/day of lactated Ringer solution was infused into the amniotic cavity while an equal amount of amniotic fluid was pumped out and discarded. During the control period, amniotic fluid composition and volume were not altered. Exchange and control periods started with the same amniotic fluid volume, lasted 3 or 4 days, and were randomized with regard to order. Amniotic fluid volume measured by vacuum drainage was 556 +/- 98 ml at the end of the control period and 986 +/- 209 ml (P = 0.03) at the end of the exchange period. Fetal arterial blood gases, hemodynamic parameters and the osmolality gradient between fetal plasma and amniotic fluid were not altered by the exchange process. A linear relationship between the control amniotic fluid volume and the volume at the end of the exchange period (P = 0.003) suggests that the animals with larger control volumes responded to isovolumic dilution with a larger volume increase. We conclude that amniotic fluid may contain a substance that regulates amniotic volume.     

Melanotropin-potentiating factor isolated from filtrate of uremic patients suffering from melanosis and carbohydrate intolerance

Summary Following the characterization of a hexadecapeptide,-endorphin, melanotropin-potentiating factor (MPF) was isolated from the filtrate of uremic patients suffering from melanosis and carbohydrate intolerance. The structure of MPF has been determined on the bases of chemical and physicochemical examinations which included HPLC, Edman sequence analysis combined analysis of the amino acid composition and FAB-MS analysis. An accumulation of MPF might be a cause of melanosis in uremic patients.Abbreviations Et₃N triethylamine - TFA trifluoroacetic acid - HF hydrogen fluoride - CH₃CN acetonitrile - DCC dicyclohexyl-carbidimide - HOBT N-hydroxybenzotriazole - OBzl benzyl ester - Boc tert-butoxycarbonyl - Z bebzyloxycarbonyl - EtOH ethanol - ECUM extracorporeal ultrafiltration method - FAB-MS fast atom bombardment spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - AcOH acetic acid - AP-M aminopeptidase-M - PTH phenylthiohydantoin     

Effect of gut regulatory peptides on intestinal luminal fluid in the rat

The effect of intravenous gastrointestinal peptide hormone administration on net fluid transport in the small intestine was assessed in the rat. An increased fluid content was observed during vasoactive intestinal peptide, gastric] inhibitory peptide, and neurotensin infusions, and a decreased content with somatostatin, substance P and pancreatic polypeptide, by comparison with the control series. Motilin had no significant effect on luminal fluid volume. These results suggest that several of the gastrointestinal regulatory peptides may have an influence on the processes of fluid absorption and secretion by the small intestine.     

Chemical characterization of angiotensin I in porcine follicular fluid

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.     

Substance P as a factor enhancing resistance of the surfactant lung system to chronic immobilization stress

In chronic experiments on non-line white rats the influence of system (intraperitoneal) and central (into the lateral brain ventricle) administration of substance P on lung surfactant system was studied in stressed and non-stressed animals. A single injection of substance P limited pulmonary surfactant activity disorders in immobilisation stress. Stress-induced increase of phospholipids level in broncho-alveolar lavage fluid remained the same in intracerebroventricular administration and was partly reduced in intraperitoneal one. In intact rats, a single injection of peptide was accompanied by alveolar phospholipids accumulation. In rabbits, multiple intracerebroventricular injections of substance P enhanced the opposite effect.     

The effect on gastrointestinal motility after ingestion of raw bile of grass carp (Ctenopharyngodon idellus) in the conscious rats

The influence of raw grass carp bile on gastrointestinal motility was studied in conscious rats. It was found that the toxic bile prolonged the gastric emptying time, increased a marker substance traversed along the small intestine and accelerated the transit time along the colon. These findings might be explained by a very efficient protective response of the digestive system, and/or poisoning effect of the invading toxic substance in rats. Accumulation of fluid was found in the stomach, 20 min after ingestion of 0.3 ml of the toxic bile. Besides, ingestion of the raw bile also increased the urea nitrogen in blood, which may result from a loss of fluid volume through the gastrointestinal tract and (or) a direct action of the bile on the kidneys.     

An intra-articular method for assessing topical application to a synovial joint

Intra-articular injection of drugs is increasingly used in human medicines. We report a method for the direct administration of a test substance to the synovial fluid of the canine stifle joint. This method caused little distress or pathology, making it suitable for pre-clinical assessment of new drugs in dogs and other species.     

Purification and structural determination of an inhibitor of starfish oocyte maturation from a Bacillus species.

Inhibitors of bacterial origins of starfish oocyte maturation were sought to obtain biologically active substances which act on either hormonal signal transduction or cell cycle regulation. An oocyte maturation-inhibiting substance found in culture fluid of a Bacillus species was purified to homogeneity. This substance possessed the nature of a detergent and specifically inhibited 1-methyladenine-induced oocyte maturation (50% inhibitory concentration, 3.3 microM) but not dithiothreitol-induced maturation. Its total structure was established to be the lactone of 3-hydroxy-13-methyltetradecanoyl-Glu-Leu-Leu-Val-Asp-Leu -Leu through COOH of the carboxy-terminal Leu. This structure is identical to surfactin, although although the configuration of the substance's amino acid residues has not yet been determined. Surfactin was shown to be identical with this substance in its inhibitory effect on starfish oocyte maturation as well as its chromatographic and electrophoretic properties. Therefore, it was concluded that the oocyte maturation-inhibiting substance produced by a Bacillus species is surfactin.     

Role of substance P in hypersensitivity reactions induced by paclitaxel, an anticancer agent

The role of substance P in adverse pulmonary reactions induced by an anticancer agent paclitaxel was investigated in rats and humans who undertook post-operative chemotherapy for ovarian cancer. In rats, paclitaxel caused a marked plasma extravasation and edema in lungs with a concomitant decrease in arterial partial oxygen pressure, which were reversed by an NK1 antagonist LY303870. Substance P level in rat plasma and bronchoalveolar lavage fluid increased after paclitaxel injection. In 13 patients, plasma level of substance P but not histamine significantly (P < 0.05) increased during paclitaxel infusion. Therefore, substance P rather than histamine may be involved in paclitaxel hypersensitivity.     

Cytochemical differentiation of nucleic acids with a Schiff-methylene blue sequence.

A method is described whereby a Feulgen type of hydrolysis of deoxyribonucleic acid is carried out on paraffin sections of routinely fixed tissues by controlled exposure of the sections to Bouin's fluid. Subsequent staining with Schiff reagent followed by methylene blue distinguishes red-to purple-stained deoxyribonucleic acid from blue-stained ribonucleic acid. This Schiff-methylene blue sequence visualizes ribonucleic acid in nucleoli and the chromidial substance of various normal and neoplastic cells and provides an assessment of their protein synthetic activity. The method has proved valuable in demonstrating normal immunocytes and immunoglobulin-forming tumor cells in pathologic specimens.     

A homodimer of the beta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone

A 24,000 Dalton protein with follicle stimulating hormone (FSH)-releasing activity, named activin, has been characterized previously from porcine follicular fluid as a heterodimer composed of the beta-subunits of inhibins A and B linked by disulfide bond(s) [Ling et al. (1986) Nature, in press]. In this paper we report the isolation of another 24,000 Dalton protein with FSH-releasing activity from porcine follicular fluid, using successive steps of heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and four steps of reversed-phase HPLC, followed by preparative sodium dodecyl-sulfate-polyacrylamide gel electrophoresis chromatography. Based on the molecular weight of the isolated molecule and its deduced NH2-terminal sequence, we propose that this second FSH-releasing substance present in porcine follicular fluid is a homodimeric protein composed of two beta-subunits of inhibin A joined together by disulfide bond(s). The name homo-activin-A is proposed for this substance.     

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 ml-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and L-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5-120 microM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E2 and prostaglandin F2 alpha to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100 degrees C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.     

Parasympathetic NANC-secretion of saliva in the mink, and effects of substance P and VIP.

In both parotid and submandibular glands a parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked secretion of saliva was demonstrated. Saliva evoked by exogenous substance P was poor in protein, while saliva evoked by VIP was protein-rich. In a subthreshold dose for fluid secretion VIP released protein and potentiated the responses elicited by substance P, particularly regarding the output of protein. The two neuropeptides may contribute to the parasympathetic NANC secretion of saliva in the mink. Further, agonists responsible for the secretory NANC response are also likely to contribute to the secretory response of the glands to parasympathetic stimulation in the absence of autonomic receptor blockade in this species.     

Antibiotic activity of marine gram-negative bacteria]

An antimicrobial substance was isolated from the culture fluid of Vibrio fischeri belonging to ++gram-negative sea bacteria. The substance was shown to be highly active against ++gram-positive cocci and less active against some other ++gram-positive bacteria and fungi. The active substance was defined as a low molecular weight compound which appeared to be thermostable and had positive ninhydrin reaction. The antibiotic formed when the culture was grown in the media containing sea water and protein hydrolysates or yeast extract during the exponential phase. The maximum antibiotic activity was observed during the stationary phase of culture development.