Oligothiophene isothiocyanates as a new class of fluorescent markers for biopolymers.

The regioselective synthesis of fluorescent oligothiophene isothiocyanates is described. The isothiocyanates were reacted with bovine serum albumin (BSA) following standard procedures and the optical properties of the oligothiophene-BSA conjugates were analyzed as a function of oligomer concentration, time, and irradiation power. The oligothiophene-BSA conjugates were chemically very stable and their photoluminescence characteristics persisted unaltered for several months. Photoluminescence data relative to the conjugate of an oligothiophene-S,S-dioxide isothiocyanate with monoclonal anti-CD8 antibody are reported. No fluorescence quenching was observed following the binding of the isothiocyanate to the antibody and the conjugate displayed high chemical stability and photostability.

Different recognition by clostripain of myelin basic protein in the lipid-free and lipid-bound forms.

Different proteolytic enzymes were tested for their ability to degrade the myelin basic protein of the central nervous system, purified in two different forms, the lipid-free form and the lipid-bound form. As shown by SDS gel electrophoresis only clostripain, a thiol protease, was able to distinguish between the two MBPs since it degraded MBP only in the lipid-free form. The failure to degrade lipid-bound MBP by clostripain could not be ascribed to the presence of lipids, since the other proteolytic enzymes tested degraded both MBPs independently from lipids giving fragments with different size. These results may be related to different conformations of MBPs possibly relevant for the study of myelin structure and antigenic properties of the protein.

[Mental and physical stress of auxiliary nurses: analysis of management]. / Desgaste físico e mental de auxiliares de enfermagem: uma análise sob o enfoque gerencial.

Seeking a better understanding of job stress, auxiliary nurses were evaluated in General Hospitais Belo Horizonte. The emotional stress which accompanies auxiliary nurses caring for the sick and easing physical and mental suffering effects their own mental state and physical health. Elements that cause suffering and diseases can be attenuated or intensified depending on labor relations. However, organizations have not been concerned about working conditions. This paper suggests some protective safety and health measures that can improve labor relations and consequently reduce the suffering of working in hospitals.

Identification of water-soluble proteases in myelin preparations.

Sodium chloride extracts obtained from purified bovine brain myelin were found to contain proteolytic activity capable of degrading isolated myelin basic protein as assessed by SDS gel electrophoresis. Using gels copolymerized with gelatin as substrate, two bands at about 54 and 117-125 KDa, respectively, were detected. Activity corresponding to the 54 KDa band was inhibited by zinc. Data presented in this article suggest that proteolytic activity can be released from the myelin sheath in water-soluble form and recognize MBP as substrate.

Purification of lipid-associated basic protein from guinea pig spinal-cord myelin.

Myelin basic protein (MBP) was purified from guinea pig spinal-cord in a native-like form retaining the binding to all the myelin lipids. Since the guinea pig MBP was found to be much more labile than the corresponding MBP from bovine brain, the original procedure based on the use of hydroxyapatite and detergents was slightly modified as reported here. The product of this purification, lipid-bound MBP, may represent an alternative to lipid-free MBP for the induction, the study and the treatment of experimental allergic encephalomyelitis.

Zinc as an inhibitor of myelin basic protein proteolytic breakdown in the central nervous system.

It is known that central nervous system myelin contains proteolytic enzymes which degrade the myelin basic protein (MBP). We have found that zinc acetate is able to inhibit MBP cleavage at concentrations of 1 mM or higher. Furthermore, the Zn-inhibitable MBP-degrading activity was found to be water-soluble and able to recognize MBP also if this protein was protected by its lipidic environment in the native-like, lipid-bound form. Data presented here suggest a Zn-dependent metallo-protease which recognize MBP as a substrate probably even in the myelin sheath, when the protein is not yet released from the membrane.

Mucin glycoprotein content of human pigment gallstones.

Mucin glycoproteins, a secretory product of the gallbladder, are thought to contribute to the matrix or nucleus of gallstones. Human black pigment stones originate in the gallbladder and have as their major constituent calcium bilirubinate, as well as inorganic salts and small amounts of cholesterol. The object of this study was to estimate the amount of glycoprotein in black pigment stones and to isolate gallbladder mucin from dissolved stones. Black pigment stones containing 18 to 65% calcium bilirubinate were first dissolved in 12.5 mM EDTA/0.1 N NaOH and decolorized, then subjected to glycoprotein assay. The mean glycoprotein content of eight stones was 12.4%. In separate experiments, pigment stones were partially dissolved by brief exposure to EDTA/NaOH to minimize glycoprotein breakdown, and the glycoproteins isolated by gel filtration and ultracentrifugation. Pigment stones contained two glycoprotein fractions on Sepharose 4B; a high molecular weight mucin glycoprotein in the void volume and a lower molecular fraction in the included volume. Mucin was further purified by density gradient ultracentrifugation in cesium chloride. Three separate mucin fractions had an average buoyant density of 1.48 gm per ml which is typical for these glycoproteins. Bile pigment was associated with high molecular weight mucin even after extensive dialysis, gel filtration, and density gradient ultracentrifugation. The identity of mucin was further established by beta-elimination of glycoproteins in alkaline borohydride which yielded galactosaminitol from cleavage of O-glycosidic bonds. Our results indicate that mucin glycoproteins are present in significant concentrations in human black pigment stones and can be purified from stones solubilized in EDTA/NaOH. The association of bile pigment with gallbladder mucin, even after extensive purification, is consistent with the hypothesis that mucin contributes to the matrix of pigment gallstones.

Cysteamine and prostaglandin F2 beta stimulate rat gastric mucin release.

Gastric mucin glycoproteins form an adherent gel over the surface epithelium that is thought to protect the stomach against chemical and physical damage. The purpose of this study was to measure the release of mucin glycoproteins from rat stomach after treatment with cysteamine and prostaglandin F2 beta, two structurally unrelated drugs that have been shown to protect the stomach against the noxious effects of alcohol and other damaging agents. Gastric mucin was separated into soluble (washout) and insoluble (adherent) phases before colorimetric quantitation of total mucin, protein-bound hexose, and sialic acid. Cysteamine produced a dose-dependent increase in release of soluble and gel mucin. Prostaglandin F2 beta caused a dose-dependent release of hexose-containing mucin but had no effect on sialic acid-containing glycoproteins. Sepharose 4B chromatography of both the soluble and adherent mucus revealed that greater than 90% was a high molecular weight glycoprotein fraction. N-Ethylmaleimide, a known inhibitor of cytoprotection by cysteamine, had no effect on mucin secretion. Similarly, indomethacin inhibited mucin secretion by cysteamine but did not significantly influence cytoprotection. Thus the secretion of mucin by cytoprotective agents is unlikely by itself to explain the ability of the stomach to resist chemical or physical damage.

Purification and composition of colonic epithelial mucin.

Colonic mucin was purified from homogenized scrapings of rat colonic epithelial cells using gel filtration and ion-exchange chromatography. High molecular weight water-soluble mucin was separated from low molecular weight proteins by gel exclusion chromatography on Sepharose 4B, and was further separated into two major mucin fractions and several non-mucin fractions on DEAE-cellulose. Fraction IV, the major mucin, was a sulphated glycoprotein with 62% carbohydrate by weight, and high concentrations of serine and threonine. A more acidic mucin, fraction V, had similar composition. Approx. 85% of the sialic acid of fractions IV and V were removed after incubation with Clostridium perfringens neuraminidase. Blood group A but not group H activity was present in fractions III, IV, and V. Ultracentrifugation experiments showed that fraction IV migrated as a single peak, whereas fraction V contained two components. Our study indicates that colonic mucin consists of at least two closely related acidic high molecular weight glycoproteins which can be separated from non-mucin contaminants by ion-exchange chromatography.

Synthesis and secretion of colonic glycoproteins: evidence for shedding in vivo of low molecular weight membrane components.

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [3H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M alpha-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.

Galactosyltransferase in fetal, neonatal, and adult colon: relationship to differentiation.

Microsomal galactosyltransferase activity of fetal rat colon increased fourfold between 18 and 22 days of gestation and then more slowly during neonatal life reaching adult levels after 14 days. The Km for uridinediphosphate- (UDP) galactose, pH optimum, cation, and detergent requirements were identical in fetal and adult galactosyltransferase. Cytidine 5'-diphosphate-choline stimulated the adult but not fetal colonic galactosyltrasferase activity by inhibition of UDP-galactose pyrophosphatase. The increase in colonic galactosyltransferase in late fetal development is correlated with our previous observation that incorporation of [3H]galactose is markedly increased during differentiation of the fetal colon.

Stimulation of colonic glycoprotein synthesis by dibutyryl cyclic AMP and theophylline.

The effects of dibutyryl cyclic AMP (B2cAMP) and theophylline on glyoprotein synthesis in rabbit colon were studied in mucosal organ cultures using [3H]glucosamine as a precursor. Addition of B2cAMP (1 mm) to culture medium caused a significant increase in glycoprotein synthesis after 12 and 24 hr compared with biopsies cultured in control medium. The increase in glycoprotein synthesis was observed only if the cyclic nucleotide was present continuously in the incubation medium for at least 12 hr. The stimulatory effect of B2cAMP on [3H]glucosamine incorporation was blocked by cycloheximide. B2cAMP also stimulated mucosal uptake of glucosamine into the intracellular pool and markedly enhanced specific activity of mucosa galactosyltransferase, an enzyme involved in glycoprotein synthesis. Addition of 5mM theophylline caused a greater than 2-fold increase in cAMP levels, which was also accompanied by an increase in glucosamine uptake and incorporation into mucosal glycoproteins. This study demonstrates that elevation of intracellular cAMP concentration in colon epithelium in vitro is associated with an increase in glycoprotein synthesis. These effects may be mediated in part by (1) increased uptake of glycoprotein precursors such as glucosamine, and (2) increased activity of glycoprotein synthetic enzymes.