The proteins and the formation of gallstones.

Cholesterol supersaturation of bile requires assistance to form gallstones. Proteins have been proposed as candidates either to facilitate or hinder the formation of stones. It is assumed that the identity of these stone proteins should be revealed in order to assess their role in the process. We have used electrodialysis of crushed stones to extract the proteins followed by 2D electrophoresis and N-terminal amino acid sequencing to characterize them. The discovery of bacterial proteins in some stones adds evidence to the importance of an inflammatory process and the deconjugation of bilirubin in mixed gallstone formation.

The content and distribution of glycosaminoglycans in the ejaculates of normal and vasectomized men.

The glycosaminoglycans (GAGs) of sperm and seminal plasma of normal men and seminal plasma of vasectomized individuals have been identified and quantified by two dimensional (2D) electrophoresis. The sperm contains predominantly CSC and HS as well as significant amounts of DS which achieves a high level in the sperm of the youngest man, while HA and LSC are either undetectable or present in small quantities. In normal seminal plasma, characteristically, DS is essentially lacking whereas CSC is the major GAG and HA and LSC account for relatively high percentages. Interestingly, in the ejaculates of vasectomized men the DS content is relatively prominent and the HA concentration varies widely. The oversulfated chondroitin sulfates CSD/CSE were detected in 7 of the 37 specimens. Their presence in a normal human body fluid is reported for the first time and the previous observation of the youthful DS/CSC switch is expanded to this study.

Urinary stone proteins: an update.

The discovery of an organic component in kidney stones dates back to 1684. More than 150 years elapsed before the incrustation of this organic component, which is now called the matrix, was proposed as the mechanism of stone formation. The composition of the matrix remained largely unknown until the development of electron microscopy and the advances in biochemistry combined in the 1950's to usher in the modern era of renal stone matrix investigation. Composed mainly of selectively incorporated proteins generally characterized by high glutamic and aspartic acid content and the frequent occurrence of gamma-carboxyglutamic acid, the matrix displays a variable and complex composition and shares a few proteins in many stones. The embryonic stone may first appear in the renal tubules where it can acquire the blood and cell membrane proteins recently found by analysis of stone protein extracts. The combination of supersaturation, an appropriate environment, the availability of calcium binding proteins which may be abnormal, and the incorporation of proteins extracted from leukocytes and cell wall membranes may induce stone formation.

Sequencing of proteins extracted from stones.

Proteins from urinary tract and gallbladder stones were extracted and characterized to determine the composition of the matrix and possibly unravel the role of the organic phase in stone formation. Proteins from crushed stones were extracted by electrodialysis and concentrated in the Amicon centricon cartridge or by lyophilization after dialysis against distilled water. Aliquots were first analyzed by isoelectric focusing in gel and if suitable subjected to two-dimensional (2D) electrophoresis. The most promising spots were harvested and the N-terminal amino acids sequenced, thus providing maximum information with minimum expenditure of material. The 2D separations and amino acid sequences of several protein extracts demonstrated similarities and differences in composition and achieved the identification or demonstration of previously and recently detected polypeptides.

A cationic protein from a urate-calcium oxalate stone: isolation and purification of a shared protein.

A protein extracted from a urate-calcium oxalate stone by electrodialysis is also excreted in the urine which served as the source material for its purification by FPLC after separation on an ACA44 column. It has an amino acid composition appropriate for a cationic protein. One peptide obtained by cyanogen bromide cleavage has significant (approximately 60%) homology with CD59 protein (protectin). Both proteins have wide distribution, the unknown having been found in bile, cholesterol gallstones, and the wall of the aorta. However, the two proteins appear to be immunologically different.

The glycosaminoglycan composition of the lung with acute and chronic pathology and in senescence.

The content and distribution of GAGs in the anatomic structures of pathological (pneumoconiosis, pneumonia, pulmonary embolism) and senescent lungs have been measured. The total GAG content of the lung structures, except central bronchi is generally lower than normal in the pathological lungs. The GAG distribution in the pleura (DS predominant), central bronchi (C6S predominant), arteries, veins and 'total lung' is similar to the corresponding normal distribution. The other notable observations are: the concentration of HA in peripheral bronchi and alveoli is increased possibly in response to the high local concentration of coal dust; an age related GAG switch from DS in the arteries of the young to C6S in the arteries of the mature lung is confirmed; the arterial GAG content generally increases with age up to age 103 in the male; the arteries of a female smoker display the mature male pattern of GAG composition. The data suggest that gender, smoking and age, more than acute pathology, determine the GAG composition of the anatomic structures of the lung.

The matrix of urinary tract stones: protein composition, antigenicity, and ultrastructure.

We have extracted proteins from urinary tract stones by electrodialysis and have developed antisera to the core and the shell of a renal stone. The protein composition varies between stones but is identical in the core and the shell of the same stone. One stone antigen is present in the urine of normal individuals and stone formers, as well as in cholesterol gallstone extracts. Electron microscopy of the core of a urate-calcium oxalate stone before and after demineralization reveals a fibrillar structure associated with mineral deposits, as well as aggregates of crystals.

Physical chemical studies of calcium oxalate crystallization.

The physical chemical approach to the investigation of the calcium oxalate (CaOx) crystallization and urolith formation is the systematic examination of the various aspects of mineral precipitation and growth in pure solution, in the presence of individual urinary components, and in whole urine media. Recent experimental studies have indicated that while small urinary ions such as citrate, magnesium, and phosphocitrate retard the mineralization rate of CaOx, urinary macromolecules may act either as inhibitors of growth or promoters of nucleation. Some CaOx mineralization inhibitors have also been found to influence the growth mechanism of the phase and its flocculation properties. Therefore, urinary macromolecules that are adsorbed on the mineralizing crystals and incorporated into the developing stone may play a significant role in urolithiasis.

The influence of urinary macromolecules on calcium oxalate monohydrate crystal growth.

The Constant Composition (CC) kinetics method has been used for studying the mineralization of calcium oxalate monohydrate (COM) at sustained supersaturations in the presence of pre-bladder urine and macromolecules isolated from normal urine and kidney and bladder stones. The method is especially sensitive for investigating the inhibitory activities of these urinary macromolecular components (UMMC) and matrix macromolecular components (MMMC) with a coefficient of variation in growth rate of approximately 2%. Significant COM mineral inhibition was observed in a wide molecular weight region of urine components. Urine removed directly from the kidney showed appreciable inhibitory activity towards COM crystallization. Normal urinary proteins and the dissolved precipitate resulting from urine centrifugation were fractionated by gel filtration. The resulting solutions were mostly COM mineralization inhibitors. Electrodialysis was utilized to isolate the MMMC (greater than 7000 d) of renal and bladder calculi. While these solutions inhibited COM crystallization, they were also found to be calcium binders as measured by the calcium electrode.

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta.

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.

Myocardial chondroitin sulfates D and E in a case of acute carbon monoxide poisoning.

The glycosaminoglycans of the myocardium of an individual who died of acute carbon monoxide poisoning were quantified by two-dimensional electrophoresis on cellulose acetate membranes. The total glycosaminoglycan content was found to be approximately twice the normal value. In contrast to the content of each glycosaminoglycan of the normal heart, the level of the chondroitin sulfates of this patient's myocardium was found to be markedly increased whereas that of hyaluronic acid was decreased and dermatan sulfate could not be detected. Further, this tissue contained significant amounts of keratan sulfate and an oversulfated dermatan sulfate, glycosaminoglycans not found in normal myocardium. Of particular interest was the presence of two unusual oversulfated chondroitin sulfates (chondroitin sulfates D and E).

The optimal temperature for preservation of the myocardium during global ischemia.

To determine the myocardial temperature that provides maximal preservation of the heart during global ischemic arrest, five groups of dogs were studied (6 per group). In all animals, the aorta was cross-clamped for 120 minutes. Serial biopsies were done for determination of adenosine triphosphate and creatine phosphate, and study by electron microscopy. Starling curves were derived prior to cardiopulmonary bypass and 60 minutes after bypass. Mitochondrial changes were graded on a scale of 0 to 4. In the control group (Group 1), the aorta was clamped when the rectal temperature reached 25 degrees C (myocardial temperature, 18 degrees to 22 degrees C). In Groups 2, 3, 4, and 5, myocardial temperature was maintained at 6 degrees C, 10 degrees C, 14 degrees C, and 18 degrees C (all +/- 2 degrees C), respectively, by the use of systemic and topical hypothermia and repeated injections of cold cardioplegic solution into the aortic root. All groups showed a depression of left ventricular stroke work index, particularly Group 1 (no survivors), Group 2, and Group 3. The high-energy phosphate stores were well preserved in all groups except Group 1. The mitochondrial ultrastructure showed significant changes in all groups, especially Groups 1 and 5. These data indicate that satisfactory preservation of mitochondrial ultrastructure and high-energy phosphates was achieved at myocardial temperatures lower than 18 degrees C. Extreme hypothermia (Groups 2 and 3) was associated with significant reduction in ventricular function under the experimental conditions employed.

The distribution of the glycosaminoglycans in the anatomic components of the lung and the changes in concentration of these macromolecules during development and aging.

The glycosaminoglycans of the normal human and bovine lungs and of the major structural components of these organs (pleura, 'alveoli', peripheral and central bronchi, arteries and veins) were investigated. To carry out this study, a micromethod for the separation and quantitative determination of these macromolecules, namely two-dimensional electrophoresis on cellulose acetate plates, was employed. This procedure made it possible to measure the content of each glycosaminoglycan present in the mentioned anatomic components. In the human lung the distribution of the glycosaminoglycans varies considerably from one component to another: dermatan sulfate was the predominant mucopolysaccharide of the pleura, chondroitin 6-sulfate that of the central bronchi, and heparan sulfate and chondroitin sulfate those of the alveoli. Heparin and keratan sulfate were not detected in any of the structural components. Significant changes in the mucopolysaccharide levels were found during maturation and aging. Further age-related changes were noted between 22 and 39 years. In the bovine lung significant changes in the glycosaminoglycan levels were also observed during growth and aging. Heparin appeared in the lung at an age between 1 and 16 months. Similarities and differences in the total contents and compositions of the glycosaminoglycans between the human and bovine lung were noted.

Old age and immunity.

Changes in immunoglobulin production and delayed-type hypersensitivity occur in the elderly, making it difficult for the aging immune system to meet new challenges, such as infectious agents or neoplastic cells. The maintenance and repair of host defenses is thus both a significant scientific and social goal.

Chemical and physicochemical studies on the mineral deposits of the human atherosclerotic aorta.

The mineral deposits of the human atherosclerotic aorta were prepared by a new method characterized by the use of mild conditions. Both large and small mineral deposits were isolated from the atherosclerotic plaque and were shown to possess essentially the same chemical composition. The deposits consisted mainly of calcium apatite (71%), carbonate (9%) and contained a relatively high percentage of protein (15%). X-ray diffraction pattern analysis revealed the presence of microcrystals with an average size of approximately 0.1 micron. Electron probe analysis showed that the surface and interior of the mineral deposit had the same chemical composition. However, scanning electron microscopy revealed that the deposits were heterogeneous and consisted of five different structures: (1) individual and conglomerates of smooth-surfaced apheres consisting of spherical layers; (2) spheres consisting of spindle-like, radially arranged particles; (3) fibres forming networks and bundles which sometimes included spherical particles; (4) irregularly shaped particles with fuzzy surfaces and (5) flat plates with smooth surfaces.

Isolation and characterization of a serine-threonine-rich galactoglycoprotein from normal human plasma.

A serine-threonine-rich galactoglycoprotein was isolated in homogeneous form from pooled normal human plasma. After precipitation of the major portion of the plasma proteins by Cohn's low temperature-low salt-ethanol procedure, the proteins and glycoproteins remaining in the supernatant solution of Cohn Fraction V were concentrated with the aid of DEAE-cellulose. Fractional elution from this ion exchange resin yielded a crude preparation of the galactoglycoprotein which was purified subsequently by gel filtration through Sephadex G-100. Homogeneity of the protein was established by several criteria of purity including disc polyacrylamide gel electrophoresis and COOH-terminal amino acid analysis. The major physicochemical constants of the macromolecule including its molecular weight of 81,000 were measured. The chemical composition of this protein was found to be unusual: the total carbohydrate moiety accounts for 76% of its weight and consists of 23% sialic acid, 23% total neutral sugar, including 20% galactose, 1% fucose, and almost equal amounts of GalNAc and GlcNAc totalling 27%. The polypeptide moiety accounting for 26% of the weight of the glycoprotein distinguishes itself by a high content of serine and threonine.

The primary structure of the asialo-carbohydrate units of the first glycosylation site of human plasma alpha 1-acid glycoprotein.

The elucidation of the structures of the carbohydrate units linked to glycosylation site I of human plasma alpha 1-acid glycoprotein is described. These carbohydrate units can be grouped into compounds with bi- (class A) and triantennary (class B) structures and the triantennary structure with a fucose residue (class BF) (Fig. 1). The structural variability of the carbohydrate units of glycosylation site I and also of glycosylation sites II to V (Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206--5214) accounts largely for the microheterogeneity of alpha 1-acid glycoprotein.