Profiling of urinary medium-sized peptides in normal and uremic urine by high-performance liquid chromatography

This report describes the profiling of medium-sized peptides in both normal and uremic urine by ion-pair reversed-phase high-performance liquid chromatography using an acetonitrile—heptafluorobutyric acid solvent system as eluent. Several medium-sized peptide peaks could be detected in both normal and uremic urine at low picomole level by using post-column fluorescence derivatization with fluorescamine. Contrary to expectation, uremic urine contained slightly larger amounts of medium-sized peptides compared with normal urine.     

Interleukin 1 activity in normal human urine

Human leukocyte dialysates contain components capable of amplifying cutaneous delayed-type hypersensitivity (DTH) reactions. In the present study, two such amplifiers, both less than 3500 m.w., were partially purified from human leukocyte dialysates by gel filtration on Sephadex G-10 followed by high pressure reverse-phase liquid chromatography. These amplifiers of DTH were examined for their effects on production of the migration inhibitory lymphokines leukocyte migration inhibition factor (LIF) and macrophage migration inhibition factor (MIF). The amplifiers were found to increase LIF and MIF production by antigen- or alloantigen-stimulated human peripheral blood lymphocytes in a dose-dependent fashion. Further analysis demonstrated that although antigen-stimulated T4 and T8 cell subpopulations could produce LIF activity under the assay conditions employed, amplification of lymphokine production by modulator was only observed with the T4 subset.     

Variation of metabolites in normal human urine

Urine is often sampled from patients participating in clinical and metabolomic studies. Biological homeostasis occurs in humans, but little is known about the variability of metabolites found in urine. It is important to define the inter- and intra-individual metabolite variance within a normal population before scientific or clinical conclusions are made regarding different pathophysiologies. This study investigates the variability of selected urine metabolites in a group of 60 healthy men and women over a period of 30 days. To monitor individual variation, 6 women from the normal population were randomly selected and followed for 30 days. To determine the influence of extraneous environmental factors urine was collected from 25 guinea pigs with similar genetics, diet, and living environment. For both studies, 24 metabolites were identified and quantified using high-resolution ¹H nuclear magnetic resonance spectroscopy (NMR). The data demonstrated large inter and intra-individual variation in metabolite concentrations in both normal human and control animal populations. A defined normal baseline is essential before any conclusions may be drawn regarding changes in urine metabolite concentrations.     

The heterogeneity of arylsulphatase-A and arylsulphatase-B in normal human urine and urine from cancer patients

Arylsulphatase-A and arylsulphatase-B heterogeneity in normal and cancer patient urine was investigated using high resolution agarose isoelectricfocusing. Normal urine contained up to nine forms of arylsulphatase-A activity with isoelectric points from 4.45 to 5.43 and at least 5 forms of arylsulphatase-B between 8.58 and 9.15 along with a broad zone of activity between pH 6.5 and 7.6. Although cancer patients had significantly higher levels of arylsulphatase-A and arylsulphatase-B activity, their pattern of activity was essentially the same as for the normals with only minor quantitative differences in some peaks.     

Tight junctions are sensitive to peptides eliminated in the urine

We prepare an extract of dog urine (DLU) that, when applied to monolayers of MDCK cells (epithelial, derived from a normal dog), enhances the transepithelial electrical resistance (TER) in a dose-dependent manner. This increase is not reflected in variations of the linear amount of TJ nor in changes of the pattern of junctional strands as observed in freeze fracture replicas, nor in the distribution of claudin 1 (a membrane protein of the TJ) nor ZO-1 (a TJ-associated protein). A preliminary characterization of the active component of DLU indicates that it weighs 30-50 kDa, bears a net negative electric charge, and is destroyed by type I protease but not by 10-min boiling. DLUs prepared from human, dog, rabbit and cat are effective on MDCK cells. However, dog DLU increases TER in MDCK (dog) as well as LLCPK1 (pig) monolayers, but not in other epithelial cell lines such as LLCRK1 (rabbit), PTK2 (kangaroo) and MA-104 (monkey), nor in the endothelial cell line CPA47 (cow). Given that in its transit from the glomerulus to the urinary bladder the filtrate increases its concentration by more than two orders of magnitude, the substance(s) we report may act at increasingly higher concentrations in each segment, and afford a potential clue to the progressive increase of TER across the walls of the nephron from the proximal to the collecting duct.     

Characterization of fibronectin metabolites in normal rat urine

Fibronectin fragments showing immunoreactivity against an anti-rat plasma fibronectin antibody were detected in normal rat urine. Untreated urine showed immunoreactivity and this reactivity could not be dialyzed. These rat urinary fibronectin-related substances (RUF) were adsorbed on heparin-immobilized gel but hardly at all on gelatin-immobilized gel. They were separated into three fractions by DEAE-Sephadex ion exchange chromatography and each fraction was further purified by heparin-Sepharose affinity chromatography. The partially purified preparations, RUF-1, RUF-2 and RUF-3, were analyzed by the sandwich-type enzyme-linked immunosorbent assay (ELISA) method, and it was found their maximum reactivities were about 30%, 45% and 20% that of the intact plasma fibronectin, respectively. Plasma fibronectin was digested with elastase and then analyzed by the ELISA method to elucidate the patterns shown by the fragments. The fragments showed almost the same patterns on ELISA analysis, and the maximum immunoreactivity decreased in parallel with the degradation. On SDS-polyacrylamide gel electrophoresis, the RUF preparations were found to be composed of multiple forms of molecules, Mr 15,000-100,000, which reacted with the antibody. These results suggest that various forms of fibronectin metabolites are excreted in normal rat urine.     

Determination of proteins in unconcentrated normal human urine

The protein content of repeatedly dialyzed urine is determined by measuring its absorbancy at 210 nm.     

Hormone-containing peptides from normal and goiter human thyroglobulins

A series of low iodine human thyroglobulin samples derived from colloid-rich goiter tissue was examined by HPLC mapping of tryptic digests and compared to normal human thyroglobulin. These samples ranged in iodine content from 2 to 8 gram-atoms of iodine (g.a. I) per mole and were not further iodinated in vitro. Peptides containing the principal hormonogenic sequence were detected using the long wavelength absorbance of the iodotyrosine derivatives at 325 nm. Two such peptides were isolated and sequenced. Their thyroxine content was confirmed by radioimmunoassay. The number of 325-nm-absorbing peaks was significantly lower in the normally iodinated human thyroglobulin than that observed the thyroglobulins of cattle and dog. This suggests a more restricted iodination in the human protein. Sodium dodecyl sulfate gel patterns of the reduced and alkylated proteins showed significant molecular size heterogeneity in all of the samples. Polypeptide fragments ranged in molecular size from approximately 330 to 45 kDa in the goiter derived material and from approximately 330 to 15 kDa in the normal human material. This difference between the proteins is consistent with earlier observations that peptides less than 45 kDa appear concomitantly with hormone formation. These data confirm that the human thyroglobulin molecule is capable of forming at least limited amounts of thyroid hormone at iodine levels as low as 4 g.a. I per mole. The hormone detected in this study was located at residue 5 near the amino terminus of the thyroglobulin molecule.     

5''-Methylthioadenosine in urine from normal subjects and cancer patients

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 mumol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 micron in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 mumol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4 degrees C, and less than 10% Ara-C at 37 degrees C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

Alzheimer beta-amyloid peptides: normal and abnormal localization

Alzheimer's disease (AD) neuropathology is characterized by accumulation of "senile" plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are principally composed of aggregates of up to 42/43 amino acid beta-amyloid (A beta) peptides. The discovery of familial AD (FAD) mutations in the genes for the amyloid precursor protein (APP) and presenilins (PSs), all of which increase A beta42 production, support the view that A beta is centrally involved in the pathogenesis of AD. A beta42 aggregates readily, and is thought to seed the formation of fibrils, which then act as templates for plaque formation. A beta is generated by the sequential intracellular cleavage of APP by beta-secretase to generate the N-terminal end of A beta, and intramembranous cleavage by gamma-secretase to generate the C-terminal end. Cell biological studies have demonstrated that A beta is generated in the ER, Golgi, and endosomal/lysosomal system. A central question involving the role of A beta in AD concerns how A beta causes disease and whether it is extracellular A beta deposition and/or intracellular A beta accumulation that initiates the disease process. The most prevalent view is that SPs are composed of extracellular deposits of secreted A beta and that A beta causes toxicity to surrounding neurons as extracellular SP. The recent emphasis on the intracellular biology of APP and A beta has led some investigators to consider the possibility that intraneuronal A beta may directly cause toxicity. In this review we will outline current knowledge of the localization of both intracellular and extracellular A beta.