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E Meezan

Publications and source records attributed to E Meezan.

At least 73 records · Page 4Linked to original sources
PubMed1981

The addition of SDS to the Bradford dye-binding protein assay, a modification with increased sensitivity to collagen.

The standard Bradford protein assay is insensitive to collagen. But if a small, sub-threshold amount of SDS is added to the sample, the response to collagen is increased by at least an order of magnitude, while, on average, the sensitivity for non-collagens is decreased by approximately a factor of 2. As a result, comparable color formation is achieved with both collagen and non-collagens. The addition of protein to a sub-threshold amount of SDS results in the formation of a green color measurable as an increase in absorbance at 700 nm, in contrast to the blue color measured at 595 nm in the standard assay. Depending upon the source, the threshold level for SDS varies from 30 to 50 microgram. The response to protein is linear up to approximately 40 microgram of protein per ml of reagent.

Collagen↗
PubMed1981

A charcoal cartridge for the removal of anionic detergent and electrophoresis stains.

Instructions are given for the construction of a charcoal-containing cartridge that allows the rapid recirculation through charcoal of any fluid in which the cartridge is submerged; recirculatory flow is achieved by magnetic stirring of the fluid by a stirring bar placed under the cartridge. The device is assembled from nylon mesh and conical sections cut from polypropylene beakers. The device can be used to accelerate the destaining of electrophoresis gels and to remove SDS (sodium lauryl sulfate) from SDS gels. The removal of SDS prior to staining is essential for the staining of SDS gels with Coomassie Brilliant Blue G-250.

Charcoal↗
PubMed1981

Human diabetic perineurial cell basement membrane thickening.

The perineurium of peripheral nerves is a cellular sleeve which surrounds and isolates the endoneurium with its axons and Schwann cells from the epineurium. Basement membrane covers the inner and outer surfaces of perineurial cells. This study concerns ultrastructural thickness measurements of human sural nerve perineurial cell basement membranes. Twenty-four patients with diabetes mellitus had a mean and standard deviation thickness of 174.1 +/- 76.2 nm., which was significantly greater than that found in 19 nondiabetics (67.1 +/- 12.8 nm., pooled variance two-tailed t-test, p less than 0.001). For 13 of the diabetics on insulin, the values were 208.4 +/- 78.1 nm. compared to those not on insulin (133.5 +/- 51.8 nm., p = 0.01). The p values when comparing male versus female sex and source of tissue from autopsy versus biopsy indicated no significant differences. This report documents that in diabetes mellitus, in addition to the well known basement membrane thickening of the microvasculature, there is also thickening of the basement membrane covering a nonvascular cell of nerve, the perineurial cell, and this this thickening is exaggerated in diabetics treated with insulin. We suggest that perineurial cell basement membrane thickness quantitation may be useful in distinguishing diabetic from nondiabetic distal symmetrial neuropathy.

Adult↗
PubMed1980

Metachromatic staining with Coomassie Brilliant Blue R-250 of the proline-rich calf thymus histone, H1.

In this report, we show that calf thymus histone 1 stains metachromatically with Coomassie Brilliant Blue R-250. Histone 1 gel bands appear red instead of the more familiar blue color characteristic of the vast majority of proteins. The red color and the spectral properties of histone 1 bands are qualitatively similar to those of collagen and procollagen bands which, as previously shown by others, also stain metachromatically (Micko, S. and Schlaepfer, W.W. (1978) Anal. Biochem. 88, 566-572 and McCormick, P.J., Chandrasekhar, S. and Millis, A.J.T. (1979) Anal. Biochem. 97, 359-366). In contrast to histone 1, histones 2A, 3 and 4 stain blue; histone 2B also stains predominantly blue, but with a faint red tint. Both red and blue bands display an absorption maximum in the vicinity of 560 nm, but red bands display an additional maximum in the vicinity of 530 nm. There are quantitative differences between the red bands; the 530 nm peak is more prominent in the spectrum of the collagen band than in the spectrum of the histone 1 band. The spectra of the blue bands are all very similar except that the spectrum of the histone 2B band is shifted slightly toward lower wavelengths. To account for the spectral differences between protein bands, we propose a model in which closely-spaced proline residues are responsible for the chromotropic effect. Localized concentrations of proline residues are present in both collagens and histone 1 and a small cluster is also present in histone 2B.

Animals↗
PubMed1980

A simple procedure for the purification of bovine fibrin from clotted blood by the use of detergents.

A rapid and simple procedure for the preparation, directly from clotted whole blood, of bovine fibrin free of cell membrane contaminants is described. The procedure exploits the ability of detergents to solubilize and extract cell membrane proteins from homogenized clot fragments. The non-denaturing detergents, TX-100 and DOC, used in sequence, or the denaturing detergent, SDS, used alone, proved equally effective. The resulting fibrin has the solubility properties and the subunit composition on SDS gels of highly cross-linked fibrin.

Animals↗
PubMed1980

Specific insulin receptors in rat renal glomeruli.

The kidney is a major site of pathologic complications in diabetes mellitus including those affecting the basement membrane. Although several hypotheses of the etiology of renal diabetic microangiopathy involve effects of insulin on basement membrane metabolism, little is known about the localization or properties of insulin receptors in isolated renal glomeruli. We have incubated isolated glomeruli and tubules with 125I-insulin and examined the binding of the hormone to these purified renal subfractions. Glomeruli and tubules exhibited specific binding of 125I-insulin at 22 degrees C which increased with time and protein concentration. Nonspecific binding accounted for 20-35% of the total binding observed. At a concentration of 1 ng/ml 125I-insulin specific binding reached a plateau within 60-90 min at 22 degrees C. Unlabeled insulin competitively displaced 125I-insulin from its binding sites with 2.8 x 10(-9) M insulin inhibiting half of the initial binding to glomeruli while a 10-fold higher concentration of unlabeled insulin was required to achieve a similar inhibition of tubular binding. Insulin analogues were less potent than insulin itself in inhibiting the binding of 125I-insulin to both glomeruli and tubules in the order of relative potency: insulin greater than despentapeptide insulin greater than proinsulin indicating the specificity of the binding. Scatchard analysis of the competition binding data of 125I-insulin to glomeruli and tubules was consistent with two distinct binding sites and/or negative cooperativity between one class of receptor sites. The binding of 125I-insulin to the glomerular receptor(s) exhibited a higher affinity than that to the tubular receptor(s) which had a greater binding capacity. These two subunits of the nephron, therefore, contain insulin receptors with different binding properties which may serve different functions.

Animals↗
PubMed1980

Rabbit glomerular and tubular basement membranes: morphology and differential solubilization.

Rabbit glomeruli and tubules were obtained by retrograde perfusion of magnetite into the lower aorta as previously described for the isolation of rat glomeruli. In addition, an alternative method of separating rabbit glomerular and tubular basement membranes from a single renal cortex preparation was developed. These preparations, which represent the Triton X-100-insoluble material, were extracted in sequence with deoxycholate, dodecyl sulfate in the presence and absence of 2-mercaptoethanol at 8 degrees C and again in the latter at 100 degrees C. The polypeptides solubilized at each step of this sequence were characterized by electrophoresis. The final extract consisted almost exclusively of red-staining, presumably collagenous bands, while the earlier extracts consisted of blue-staining, presumably noncollagenous bands. No significant differences were found between the glomerular and tubular basement membrane extracts.

Animals↗
PubMed1979

Isolation and characterization of human placental chorionic villar extracellular matrix.

The cell-free extracellular matrix of human placental chorionic villi has been prepared by a procedure employing extraction of the terminal villar fragments with the detergents Triton X-100 and sodium deoxycholate. The isolated human placental extracellular matrix retains an intact, but collapsed, histoarchitecture, as observed by scanning and transmission electron microscopy. It remains intact, in large part because of the presence of continuous sheets of villar basement membranes and associated interstitial collagen fibers and scattered patches of fibrin. The staining charcteristics and chemical composition of the isolated human placental extracellular matrlix are similar to those reported for basement membranes in several tissues and indicate the presence of collagen-like and glycoprotein components in this preparation. Gel electrophoresis of urea-SDS-mercaptoethanol extracts of the matrix showed that it consists of several polypeptide components of various molecuar weights, some of which are associated into high molecular weight complexes by disulfide bonds.

Amino Acids↗
PubMed1979

pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-sepharose.

Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained less than 1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% Ig2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.

Animals↗
PubMed1979

Biosynthesis of basement membrane matrix by isolated rat renal glomeruli.

The incorporation of radioactive precursors into the extracellular basement membrane matrix has been investigated in a purified preparation of isolated rat kidney glomeruli. Using deoxycholate extraction of isolated glomeruli which were incubated with radioactively labeled amino acids and carbohydrates, as an assay system for the measurement of incorporation of basement membrane precursors into intact deoxycholate insoluble basement membrane material, we demonstrated the in vitro biosynthesis of this structure. The assay system minimized the possibility of contamination of the isolated labeled basement membrane with labeled cell membrane fragments which may occur with the standard isolation procedures involving sonication and centrifugation. A linear incorporation of labeled proline, lysine, glycine, glucosamine, and galactose into glomerular basement membrane was shown. Basement membrane synthesis was inhibited by metabolic poisons and protein synthesis inhibitors as well as by inhibitors of collagen synthesis, but not by colchicine, an inhibitor of collagen secretion. Rhe appearance of 14C-hydroxyproline in the basement membrane matrix was negligible during the first 4 hours of incubation and rose only to 1% of the total proline counts thereafter. The results are consistent with the characterization of glomerular basement membrane synthesis and deposition as a two component system comprising a rapidly synthesized and deposited noncollagenous glycoprotein component(s) and a collagenous component which is only deposited after a delay of 4 to 6 hours.

Aminopropionitrile↗
PubMed1979

Effect of paraquat treatment on prolyl hydroxylase activity and collagen synthesis of rat lung and kidney.

Morphological evidence has shown that the herbicide paraquat produces severe lung fibrosis in experimental animals. In order to investigate the biochemical basis for this fibrosis, prolyl hydroxylase activity, total hydroxyproline content and collagen and protein synthesis were estimated in rats (200 gm) which were pretreated with paraquat (25 mg/Kg; I.P.) nine days before sacrifice. Prolyl hydroxylase activity in the treated rats was significantly elevated in the lungs. However, no such increase was seen in the kidney. There was no significant change in the total hydroxyproline content or collagen formation in the treated animals. When paraquat was added to the culture medium and incubated with lung slices, there was a marked decrease in collagen synthesis along with a decrease in protein synthesis. When paraquat was added to lung cells in culture, it was found that lung explants were more sensitive to paraquat toxicity than cells obtained by trypsinization. In explants which were sensitive to paraquat, there was a marked decrease in both protein synthesis and collagen synthesis.

Animals↗