One of two gbpC gene homologues is involved in dextran-dependent aggregation of Streptococcus sobrinus.

INTRODUCTION: Streptococcus sobrinus exhibits marked dextran-dependent aggregation mediated by glucan-binding proteins (GBPs). In contrast to Streptococcus mutans, in which the gbpC gene responsible for dextran-dependent aggregation of this organism has been characterized, genes encoding the S. sobrinus GBPs have not yet been identified. METHODS: Recently, we identified the gbpC gene homologue from Streptococcus macacae using polymerase chain reaction primers based on the conserved regions of the gbpC sequence exhibiting intraspecies variations. This method was applied to amplify a S. sobrinus homologue. RESULTS: Unexpectedly, two gbpC gene homologues were identified in S. sobrinus strain 100-4. One homologue, named gbpC, was more similar to the S. mutans gbpC gene than the other and was approximately half the molecular size of its homologue with similar regions interrupted by several non-similar stretches. However, the dextran-binding activity of the protein expressed from gbpC in Escherichia coli was not detected in contrast to the other homologue, a protein designated as Dbl, expressing this activity. The gbpC gene was shown to be intact on the chromosome of strain OMZ176, which does not exhibit dextran-dependent aggregation, while the dbl gene of this strain contained a single adenine nucleotide insertion at approximately one-third the distance from the 5'-end. The insertion mutation in the dbl gene resulted in translation of a premature protein missing its LPXTG sequence signature sequence of the wall-anchored proteins. CONCLUSION: These results suggest that the dbl gene is very likely responsible for S. sobrinus dextran-dependent aggregation.

Streptococcus mutans strains harboring collagen-binding adhesin.

A previously unidentified 120-kDa protein was detected in Streptococcus mutans strain Z1 and was involved in the cold-agglutination of the strain. We have identified the gene, designated cnm, as being involved in the agglutination of strain Z1 following random mutagenesis. The amino acid sequence of the deduced Cnm protein exhibited high similarity to those of collagen-binding adhesins from staphylococci and other organisms. To confirm whether the protein is involved in collagen-binding, we cloned a cnm gene fragment, overexpressed it in E.coli, and prepared crude extracts. The extracts containing recombinant protein exhibited binding to immobilized collagen and laminin but not to fibronectin. Compared with the parental strain Z1, the cold-agglutination-negative mutant 05A02 exhibited reduced binding to collagen and laminin but retained that to fibronectin. This gene was detected in some strains of S. mutans. Therefore, the cnm gene encoded a new strain-specific member of the collagen-binding adhesin family.

An autopsy case of pseudosarcoma of the common bile duct.

An autopsy case of pseudosarcoma in the common bile duct is reported. An 82-year-old Japanese male complaining of jaundice was admitted to our hospital; he was examined by abdominal ultrasonography (US), revealing biliary calculus, dilatation of the common bile duct, and choledocholithiasis, considered to be the possible cause of the obstructive jaundice. Endoscopic retrograde biliary drainage (ERBD) and cholangioscopy were performed concurrently, revealing a vaguely whitish tumor near the papilla of Vater. Two months later, the patient died from complications of the liver, infection, and disseminated intravascular coagulation (DIC). An autopsy study revealed tumor cells with extreme pleomorphic changes, growing diffusely, very like sarcoma. Further examination revealed epithelioid arrangements in the metastatic lymph node. Twelve kinds of immunohistochemical examination showed a positive reaction, reflecting the presence of an epithelioid cytoskeleton. Of 28 cases of true and pseudosarcoma of the biliary system reported in the Japanese literature, only 1 case was reported, in 1990, to involve the common bile duct. We therefore report the present case of pseudosarcoma of the common bile duct.

Effects of probucol and pravastatin on plasma lipids, activities of postheparin lipoprotein lipase, and lecithin cholesterol acyltransferase and apo A-I containing lipoproteins with and without apo A-II in patients with moderate hypercholesterolemia.

In this study, plasma HDL fractions were separated by ultracentrifugation and apo A-I containing lipoproteins (A-I Lp) were then isolated using anti-apo A-I immunoaffinity chromatography. The A-I Lp were further separated into two fractions with the use of anti-apo A-II immunoaffinity chromatography. One fraction, Lp A-I, contained apo A-I without apo A-II, while the other, Lp A-I/A-II, contained both apo A-I and apo A-II. These techniques were applied to investigate the changes in HDL apoprotein composition in hypercholesterolemic subjects treated with either probucol or pravastatin. Treatment with probucol (500 mg/day) or pravastatin (10 mg/day) reduced mean plasma total cholesterol concentrations by 24% (p < 0.01) and 16% (p < 0.05), respectively. Both drugs caused some reduction in lipoprotein lipase activity, but neither had any influence on the activity of hepatic triglyceride lipase or lecithin cholesterol acyltransferase. Their effects on HDL-cholesterol levels and apoprotein composition differed markedly. Probucol significantly decreased the HDL-cholesterol concentration, the plasma apo A-I/apo A-II ratio, and the number of large particles of diameter greater than 10.4 nm. When the ratios of Lp A-I and Lp A-I/A-II for the probucol-treated subjects were compared with those in the normolipidemic controls, and with the ratios before and after administration of probucol, a remarkable decrease in the level of Lp A-I was apparent. It is presumed that the decrease in HLD-cholesterol by prolonged probucol administration reflects the decrease of Lp A-I more than the decrease of Lp A-I/A-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al.

Human plasma in vitro inhibits the growth of coagulase negative staphylococci, S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity against S. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated with S. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation. To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition. These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.

[Separation of high density lipoprotein (HDL) using anti-apo A-I, apo A-II immuno-affinity chromatography to evaluate the effects of probucol].

An assessment has been made regarding the changes of the particles of lipoprotein A-I without A-II (Lp A-I) and lipoprotein A-I with A-II (LpA-I/A-II) which correspond to HDL subfraction isolated by the use of anti-apo A-I and A-II antibody affinity columns in order to quantitatively and qualitatively investigate the change of HDL caused by administration of probucol and pravastatin which are therapeutic drugs for hypercholesterolemia. Probucol caused significant decreases of HDL-cholesterol, plasma apo A-I/apo A-II ratio and particles larger in diameter than 10.4 nm. Comparing Lp A-I and A-I/A-II ratios with those in normolipidemic controls and the ratios before and after administration of probucol, the decrease of LpA-I ratio was found to be remarkable after prolonged administration of probucol, and it was presumed that the decrease of HDL cholesterol by prolonged administration reflects the decrease of LpA-I particles more than the decrease of LpA-I/A-II. On the other hand, no significant change was seen in HDL cholesterol, plasma apo A-I/apo A-II ratio or HDL particle size in the pravastatin group. It is considered essential to observe HDL from the aspect of apoprotein, which plays an important role in the metabolism of lipoprotein, in the assessment of the anti-atherogenic activity of HDL cholesterol in future. In other words, it is necessary to analyze the change of HDL from the aspect of Lp A-I and Lp A-I/AII and investigate their respective metabolisms and roles.

Ischemia-reperfusion arrhythmias and lipids: effect of human high- and low-density lipoproteins on reperfusion arrhythmias.

The effect of high- and low-density lipoproteins separated from human serum on the postischemic reperfusion arrhythmias was investigated. The hearts were perfused by working heart mode with Krebs Henseleit bicarbonate buffer containing arachidonic acid (1 microgram/ml) for 5 minutes. Whole heart ischemia was induced by the use of a one-way ball valve, and hearts were perfused for 15 minutes followed by 20 minutes of reperfusion. Physiologic concentrations of high- and low-density lipoproteins were constantly infused through the atrial route during ischemic perfusion. Coronary effluent was collected via pulmonary artery cannulation for subsequent radioimmunoassay of thromboxane B2 and 6-keto-prostaglandin F1 alpha, the major stable metabolites of thromboxane A2 and prostacyclin, respectively. The incidence of ventricular arrhythmias during reperfusion was 6/6 (100%), 1/6 (17%), and 6/6 (100%) in control, high-density lipoprotein and low-density lipoprotein infusion groups, respectively. There was no significant difference in coronary flow among the three groups throughout the perfusion. Both thromboxane B2 and 6-keto-prostaglandin F1 alpha increased significantly during ischemia compared with preischemic values in all groups of hearts. However, the ratio of these two parameters varied in control and low-density lipoprotein infusion groups during ischemia, while there was no significant change in the high-density lipoprotein infusion group. These results provide the possibility that arachidonate metabolites may be involved in the regulation of ischemia-reperfusion arrhythmias and that high-density lipoprotein that was infused during ischemia markedly inhibits the incidence of ischemia-reperfusion-induced ventricular arrhythmias, due in part at least, to stabilizing the arachidonate metabolites during ischemic perfusion.

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone.

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.

Enhanced ultrastructural preservation of cartilage proteoglycans in the extended state.

We investigated the preservation of proteoglycan (PG) structure in rat epiphyseal cartilage using N-N-dimethylformamide (DMF) dehydration before embedding. After aldehyde fixation, specimens with and without routine osmium post-fixation were dehydrated in graded DMF and embedded in either Spurr's resin or Lowicryl K4M resin. Standard ethanol dehydration with Spurr or Lowicryl embedding techniques resulted in the formation of condensed PGs, called matrix granules. DMF dehydration before embedding greatly improved the preservation of PG structure and resulted in an extended appearance of PGs closely resembling the fine filamentous network of cartilage tissues processed by rapid freezing and freeze-substitution. However, en bloc staining of aldehyde-fixed specimens with cationic reagents before or during DMF dehydration induced the condensation of PGs and resulted in the formation of matrix granules. These observations demonstrate that DMF, a mild dehydration agent, dramatically improves PG preservation without a harmful effect on aldehyde-fixed PG structure and can be utilized regardless of routine post-fixation.

Immunohistochemical localization of glycosaminoglycans and proteoglycans in predentin and dentin of rat incisors.

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aldehyde fixation of rat incisors using (a) four monoclonal antibodies (1-B-5,9-A-2,3-B-3, and 5-D-4) which recognize epitopes in unsulfated chondroitin (C0-S), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatment of tissue sections with chondroitinase ABC localized the majority of stainable PG (C4-S, KS, DSPG, C0-S, and C6-S) in predentin and, to a lesser extent (C4-S and small DSPG), in the dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a definite difference between PG of predentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules during dentinogenesis and mineralization.

Ultrastructural demonstration of increased sulfated proteoglycans and calcium associated with chondrocyte cytoplasmic processes and matrix vesicles in rat growth plate cartilage.

A monoclonal antibody (3-B-3) to chondroitin 6-sulfated proteoglycan was used with immunoperoxidase electron microscopy to study the relationship of chondrocyte cytoplasmic processes and matrix vesicles in rat epiphyseal growth plate cartilage. Immunoperoxidase staining of the chondrocyte plasmalemma was found at all levels in the growth plate and was most prominent in the hypertrophic zone. The plasmalemma and matrix of the cytoplasmic process often demonstrated stronger reactivity than the remainder of the cell surface. Matrix vesicles showed weak to strong surface or internal reactivity. The majority of them stained very similarly to the cytoplasmic process. X-ray microanalysis of specimens processed by rapid freezing and freeze substitution confirmed that both sulfur and calcium were localized within or in close association with both the cytoplasmic process and the matrix vesicle, suggesting a chemical combination of calcium with sulfated proteoglycans at both sites. These results indicate that there is a selective increase in the concentration of membrane-associated sulfated proteoglycan and calcium in the cell process, from which matrix vesicles may be released into the extracellular matrix.

Effects of gammalinolenic acid on plasma lipoproteins and apolipoproteins.

Nineteen hypercholesterolemic patients (10 without and 9 with hypertriglyceridemia) were given evening primrose oil rich in gammalinolenic acid (GLA, 18: 3n - 6), in a placebo controlled cross-over design, over 16 weeks (8 + 8 weeks), with safflower oil as the placebo. During supplementation with evening primrose oil, dihomogammalinolenic acid (20: 3n - 6) increased in plasma lipids and red blood cells, and in subjects without hypertriglyceridemia there was a significant decrease in low density lipoprotein-cholesterol and plasma apolipoprotein B compared with the levels observed during safflower oil administration. Our results confirmed that evening primrose oil is effective in lowering low density lipoprotein in hypercholesterolemic patients.

Specific binding of high density lipoprotein (HDL3) is not related to sterol synthesis in rat intestinal mucosa.

There is good evidence that high density lipoprotein (HDL) is involved in the flux of cholesterol into the cells of some organs and out of the cells of other tissues. Because we have previously found that HDL is bound specifically by mucosal cells of the small intestine, we have examined the possibility that this was associated with regulation of cholesterol flux. We have, therefore, compared the specific binding of 125I-labeled HDL3 with cholesterol synthesis in mucosal cells obtained from rats that had been treated to alter intestinal cholesterol metabolism. The rate of sterol synthesis measured in tissue slices, by the incorporation of [3H]water into sterols, was altered up to fivefold by treatment with cholestyramine (to induce bile salt loss), by surformer treatment (to reduce absorption of cholesterol), and by biliary diversion. Yet the capacity of mucosal cells to bind, internalize, and degrade 125I-labeled HDL3 was unchanged. Cholesterol feeding influenced neither the interaction of 125I-labeled HDL3 with cells nor the rate of sterol synthesis. Furthermore, the interactions of 125I-labeled HDL3 with mucosal cells isolated from the proximal and distal halves of the intestine or between the upper and lower villus cells were similar, despite differences in sterol synthesis. These data suggest that, in rat intestine, the specific binding of HDL is not related to sterol synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a high density lipoprotein binding protein from adrenocortical membranes.

A protein band with an apparent molecular weight of 78,000 daltons has been identified in the solubilised plasma membrane extract of sheep adrenal cortex which binds HDL3 devoid of E apolipoprotein. Following 'Western' blotting, and development of the nitrocellulose strips with appropriate antisera and color reagent, the same band, unlike other cortical membrane proteins or albumin, bound AI and AII apolipoproteins. Human LDL bound weakly to the same band but more strongly to another two proteins of higher molecular weight. These studies confirm the same degree of specificity of HDL3 binding found with cultured adrenal cells and strengthen the suggested existence of a specific HDL receptor.

Characteristics of the binding of high-density lipoprotein3 by intact cells and membrane preparations of rat intestinal mucosa.

We have investigated the binding of high-density lipoprotein (HDL3, d = 1.12-1.21 g/ml), and apolipoprotein E-deficient human and rat HDL, obtained by heparin-Sepharose affinity chromatography, to intact cells and membrane preparations of rat intestinal mucosal cells. Binding of 125I-labeled HDL3 to the basolateral plasma membranes was characterised by a saturable, specific process (Kd = 21 micrograms of HDL3 protein/ml, Bmax = 660 ng HDL3 protein/mg membrane protein) and E-deficient human HDL demonstrated a similar affinity for the binding site. The basolateral plasma membranes isolated from proximal and distal portion of rat small intestine showed similar binding affinities for HDL3, whereas the interaction of HDL with brush-border membranes was characterised by mainly nonspecific and nonsaturable binding. The binding of 125I-labeled HDL3 to basolateral plasma membranes was competitively inhibited by unlabeled HDL3 but less efficiently by unlabeled human LDL. The putative HDL receptor was not dependent on the presence of divalent cations but was markedly influenced by temperature and sensitive to pronase treatment. We have also demonstrated, using whole intestinal mucosal cells, that lysine and arginine-modified HDL3 inhibited binding of normal 125I-labeled HDL3 to the same extent as normal excess HDL3. These data suggest that basolateral plasma membranes of rat intestinal mucosal cells possess a specific receptor for HDL3 which contains mainly apolipoprotein A-I and A-II, and the mechanisms of recognition of HDL3 differ from those involved in binding to the B/E receptor.