Possible role of peptides derived from otosclerotic bone in the mechanism of sensorineural hearing loss.

Otosclerotic stapes footplates, superstructures and temporal cortical bones were extracted with 0.25 M guanidine X HCl 0.5 M EDTA (pH 7.4) solution. The extracted non-collagenous peptides/proteins were separated chromatographically on a Sephadex G-25 microcolumn. The peptide composition of the bone samples were compared by capillary analytical isotachophoresis (ITP) in the molecular mass range 0.3-5 kD. The otosclerotic stapes footplate contained 13 ITP subfractions, while the stapes superstructures and cortical bone contained only 9 and 10, respectively. An otosclerosis-specific ITP subfraction was also detected in the stapes footplate, but not in the stapes superstructure or cortical bone. This subfraction was previously demonstrated in the otosclerotic perilymph as well. Four ITP subfractions occurred common in the otosclerotic stapes footplate, the superstructure and the cortical bone. Two of these common subfractions were not found in the cortical bone peptide extract, but all of them revealed higher than normal levels in the otosclerotic perilymph.

Non-collagen proteins of stapedial bone matrix in perilymph of otosclerotic patients.

Non-collagen proteins extracted from the otosclerotic stapes footplate, superstructure and temporal cortical bone were compared with the protein patterns of normal and otosclerotic perilymph by analytical isotachophoresis (ITP). Normal and otosclerotic perilymph yielded basically identical isotachophoretic subfractions. Thirteen subfractions were detected moving as anions at pH 9.6 in the normal perilymph, versus 16 subfractions of similar character in the otosclerotic perilymph. Of these 16 protein subfractions, one (which was not detected in the normal perilymph) could be found in the ITP-gram of the stapes footplate non-collagen proteins, but not of the stapes superstructure or temporal cortical bone. Our results support the concept that protein can enter the inner ear fluid spaces from the otosclerotic bone. The biological significance of these proteins has not yet been clarified.

Peptides of the otosclerotic perilymph examined by analytical isotachophoresis.

Perilymphs of normal and otosclerotic origin were separated chromatographically on a Sephadex G-25 microcolumn. Peptide composition of the perilymphs was compared by capillary analytical isotachophoresis in the molecular mass range 0.3-5 kD. Otosclerotic perilymph samples contain a heterogeneous, UV-absorbing peptide subfraction which is not detected in the normal perilymph. Normal and otosclerotic perilymph, furthermore, contain four common subfractions detected in twice the normal concentration in the otosclerotic perilymph. These ITP subfractions are degraded during acid hydrolysis (6 M HCI). On the contrary, otosclerosis is a deficient state compared with the normal, as the number of peptides or oligoglycopeptides is twice as high in normal as in otosclerotic perilymph, beside the otosclerosis specific peptides.

The role of basic amino acids of the seminal plasma in fertility.

The role of basic amino acids in male infertility is discussed. The changes in the arginine, ornithine and total amino acid concentrations of the seminal plasma in various types of pathospermia are commented upon in the light of published evidence and of the authors' own observations. A recent procedure (CV-technique) introduced by the authors into the routine investigation scheme of infertility is reported. The method detects the arginine-deficient type of pathospermia, thus contributing to the possibility of its selective correction.

Non diffusible toxic polypeptides in uraemic sera: a new group of uraemic toxins.

The composition and toxicity of polypeptides prepared from normal and uraemic sera by the combination of Dowex and Sephadex column chromatography have been studied. Significant changes in composition associated with an increased toxicity and decreased diffusibility of peptides (Mwt, 1800--2500 D) isolated from uraemic sera were detected. Due to their moderate of high toxicity as well as their poor or non-diffusibility, these uraemic polypeptides are proposed to be regarded as a new group of uraemic toxins.

Urea as a selective inhibitor of argininosuccinate lyase.

The effect of urea on various ornithine cycle enzymes has been investigated. It was demonstrated that argininosuccinate lyase was the only ornithine cycle enzyme inhibited by urea in a competitive manner. Based on the data presented the possible role of urea in maintaining a physiological range of intracellular and extracellular urea concentration by controlling hepatic ureogenesis was discussed.

Many hitherto unknown peptides are principal constituents of uremic "middle molecules".

The aim of the present investigation was to collect information on the molecular composition of uremic "middle molecules," the 500-5000 molecular-mass serum constituents assumed to be involved in the molecular etiology of uremic intoxication. For this purpose, three fractions of serum containing such molecules were separated by cation-exchange column chromatography. These fractions absorb at 240 nm and are characteristically increased in chronic uremia. By one-dimensional paper chromatography of these fractions it was demonstrated that each could be resolved into at least seven (altogether, 21) ninhydrin-positive subfractions. These, like the original fractions, yielded amino acids on acid hydrolysis. The qualitative amino acid composition of the subfractions differed substantially, both from each other and from that of 31 known peptides with which they were compared. We conclude that hitherto unknown peptides with only limited diffusibility through hemodialysis membranes constitute the main bulk of uremic middle molecules.

Partial purification from bovine pulmonary tissue of a protein capable of inhibiting in vivo DNA synthesis in mouse pulmonary cells.

This paper reports on partial purification and characterization of a natural (endogenous) factor capable of inhibiting in vivo DNA synthesis in mouse pneumocytes in a tissue-specific manner. By using a combination of ultrafiltration and various chromatographic techniques, the active agent has been partially purified from aqueous extracts of both bovine and rat pulmonary tissue. The factor responsible for the observed effect was found to be a heat labile compound, most likely a protein of approx. 40 000 molecular weight. Its chemical and physicochemical properties determined so far, and also the manner of its biological action implies that this lung tissue derived agent might be an endogenous proliferation inhibitor with a chalone-like character operating in the pulmonary epithelial cells.

Molecular weight distribution, diffusibility and comparability of middle molecular fractions prepared from normal and uremic sera by different fractionation procedures.

Sephadex G-25 (SG-25) chromatography of normal and uremic (U) sera yielded three (A, B, C) optically active (280 nm) fractions. Only fraction B disclosed a substantial increase in uremia that could not be counteracted by routine hemodialysis. Although middle molecules (MM) might be included in all SG-25 fractions (B greater than C greater than A), none of them was exclusively composed of MM. Solutes in U fraction B and C were found to be (a) heat stable; (b) soluble in ethanol; (c) diffusible through a Visking membrane, and (d) adsorbed by activated charcoal. Solutes in U fraction B and C and those in a toxic ethanol U serum extract could be partially or entirely recovered in the same Dowex fractions which were obtained by Dowex chromatography of native U sera. Methodical pitfalls leading to misinterpretation of molecular weight data obtained by SG chromatography and to erroneous classification of MM are also thoroughly discussed in this paper.

Possible origin of amniotic fluid constituents affecting thromboplastic activity.

The clotting-accelerating activity of amniotic fluid (AF) has been known for long, but a clinical importance has only recently been attributed to this phenomenon. In order to obtain some informations on the origin of AF component(s) responsible for this action, the effects on the coagulation process of extracts prepared from the placenta, fetal membranes and fetal excretes were investigated. Normal saline extracts from the placenta, the amniotic and chorionic membranes, and those from the mucus aspirated from the upper respiratory tract of the fetuses were shown to accelerate, whereas meconium extract and untreated fetal urine were found to inhibit blood coagulation. Results presented in this paper indicate that AF components affecting the thromboplastic system in one way or other may be of both fetal and extrafetal gestational tissue origin, and that the quality of the actual effect exerted on the clotting system by AF depends on the relative amount of AF constituents oppositely affecting the hemostatic system.

Possible origin of amniotic fluid constituents influencing fibrinolytic activity.

An attempt was made to clarify the origin of the amniotic fluid constituents affecting fibrinolysis. For this purpose, saline extracts were prepared from various extra-fetal gestational tissues (EGT) as well as from various fetal excretory products (FEP) and their effect on an euglobinolytic system was studied in vitro. It was found that extracts from FEPs stimulated, whereas those from EGTs inhibited fibrinolysis. It is concluded that the fibrinolysis inhibiting amniotic fluid constituents originate from the placenta and the fetal membranes, whereas the fibrinolysis stimulating ones from various sources of fetal origin.

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro.

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.

Proliferation related peptides in quiescent and regenerating rat liver.

Aqueous extracts of resting (C) and 30 h regenerating (P) rat livers were partially purified by a combination of ion exchange-, Sephadex- and paper chromatographic techniques. It was demonstrated that a limited number of the semipurified chromatographic fractions inhibited or stimulated both DNA synthesis and cell proliferation, the effect being partially determined by C or P liver origin of the fractions. It was also shown that biological activities were mediated by hepatic peptides of middle molecular size in the semipurified fractions. It is suggested that regulation of hepatocyte proliferation might be under a dual control operating through an interplay of stimulatory and inhibitory peptides, the final outcome determined by their interaction.

Biochemical characterization of substances with myometrium contraction inhibitory action, originated from ovaries of pregnant sows.

In order to understand life itself, we need biotechnology to purify biologically active substances as well. One of these processes results peptides with myometrium contraction inhibitory effect, originated from the ovaries of pregnant sows, but different from relaxin. After collection and acidic-acetone extraction of the ovaries (product generation, primary purification) the substance with myometrium contraction inhibitory effect was further purified on Sephadex G-25 (fine), CM-52 and again Sephadex G-25 (fine) gels (secondary purification) without loosing the bioactivity of the substance (quality control). Product analysis by analytical high performance liquid chromatography and capillary isotachophoresis were performed at this stage of the experiments. The analysis mentioned above resulted in 2-15 different subfractions in the studied extract. This shows that the biologically active ovarian extract needs further purification before the planned final amino-acid analysis is performed.

Effect of rehydration of rat liver tissue after water deprivation.

Male albino rats were deprived of water for 6 days, then they were allowed to drink tap water ad libitim. The structure of the liver was examined by light and electron microscopy, and the protein and dry matter contents, oxygen consumption and glucose-6-phosphatase activity of the liver were determined after rehydration. At 10 minutes, the mitochondria showed signs of division and a peculiar transformation of the cristae. At 60 minutes, the membranes of the rough endoplasmic reticulum were found to have proliferated. At 12 hours, the smooth-surfaced membranes showed hypertrophy and the bile canaliculi were distended. At 24 hours all rehydration induced organelle alterations were declining. The biochemical findings agreed well with the fine structural changes and both were indicative of an enchanced functional capacity of liver cells during rehydration.

Relationship between lysosomal damage, fatty infiltration and hepatocellular necrosis in the course of acute liver injury induced by carbon tetrachloride in the rat.

In the course of liver injury induced by CCl4 in rats the change of the endoplasmic reticulum takes 5 hours and that of the lysosomal membrane, 18 hours to develop. The latter change precedes hepatocellular necrosis. Elevation of plasma free fatty acids and fatty infiltration of the liver can be observed at 3 hours after CCl4 administration. The maximum of fatty infiltration, hepatocellular necrosis and the highest degree of lysosomal damage develop at the same time. Since CCl4 is eliminated in a few hours, it must initiate a cellular process which then leads to lysosomal membrane damage and hepatocellular necrosis.