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The public availability of numerous microbial genomes is enabling the analysis of bacterial biology in great detail and with an unprecedented, organism-wide and taxon-wide, broad scope. Streptococcus pneumoniae is one of the most important bacterial pathogens throughout the world. We present here sequences and functional annotations for 2.1-Mbp of pneumococcal DNA, covering more than 90% of the total estimated size of the genome. The sequenced strain is a clinical isolate resistant to macrolides and tetracycline. It carries a type 19F capsular locus, but multilocus sequence typing for several conserved genetic loci suggests that the strain sequenced belongs to a pneumococcal lineage that most often expresses a serotype 15 capsular polysaccharide. A total of 2,046 putative open reading frames (ORFs) longer than 100 amino acids were identified (average of 1,009 bp per ORF), including all described two-component systems and aminoacyl tRNA synthetases. Comparisons to other complete, or nearly complete, bacterial genomes were made and are presented in a graphical form for all the predicted proteins.

Background/aims: The risk for hepatitis C virus infection in kidney transplant recipients has been reduced by the introduction of accurate diagnostic tests. Little is known, however, of the current risk factors and the molecular genetics of hepatitis C virus infection in Italy., Methods: We studied 101 Italian kidney allograft recipients, transplanted between 1975 and 1995, in Italy or abroad. Sera were assayed for biochemistry, presence of HBsAg, anti-hepatitis C virus antibodies, hepatitis C virus-RNA (by reverse transcription nested PCR) and hepatitis C virus genotyping., Results: HBsAg was found in 4 sera and anti-Hepatitis C Virus antibodies in 33 (33%). The duration of pre-transplant dialysis was longer in anti-hepatitis C virus positive than in anti-hepatitis C virus negative patients (5.9 +/- 4.3 vs 2.8 +/- 1.9 years, p = 0.0004). Anti-hepatitis C virus seropositivity was more frequent among patients grafted before than after 1990 (50% vs 27%, p = 0.04) and varied depending on the country of transplantation (25% in Italy; 56% in other European countries; and 40% in non-European developing countries). Twenty-seven sera were hepatitis C virus-RNA positive, including 5 without anti-hepatitis C virus antibodies. Hepatitis C virus genotype 1b was found in 13 (48%) patients, the remainder being infected with genotypes 1a (6 cases), 2a, 2c, 3a and 4. Genotype 1b was largely predominant among patients grafted in Europe but never found in those transplanted in developing countries. All but one patient without a sustained antibody response were infected by non-1b genotypes. Hepatitis C virus-RNA seropositivity was associated (p = 0.03) with a higher dose of prednisone (p = 0.03) and a lower dose of cyclosporine (p = 0.05) used as immunosuppressants., Conclusions: Current risk factors for hepatitis C virus infection in Italian kidney graft recipients include the duration of haemodialysis, transplantation in developing countries and the level of post-transplant immunosuppression. The pattern of hepatitis C virus genotypes is changing from predominantly 1b to non-1b genotypes and the latter infection often occurs without a sustained antibody response. Few patients develop clinical liver disease.

Insulin binding and insulin receptor down-regulation characteristics were evaluated, as well as cholesterol and phospholipid levels in erythrocytes from 22 patients with liver cirrhosis. These parameters were correlated with clinical characteristics and with scores related to severity of the liver injury. Nine healthy subjects were studied as a control group. It was observed that insulin binding was generally greater in patients than in controls because of an increase in surface insulin receptor numbers, rather than from a change in receptor affinities. In cirrhotic patients, the erythrocyte insulin receptors did not undergo the normal down-regulation process but, in most cases, increased in response to insulin incubation. The alterations in insulin processing characteristics were more frequent in patients with alcoholic cirrhosis and more severe liver impairment and correlated with the changes in the lipid composition of erythrocyte membranes. In particular, an increase in the cholesterol to phospholipid molar ratio and a decrease in polyunsaturated fatty acid content of phospholipids in erythrocyte membranes of cirrhotic patients was associated with impairments in insulin receptor processing. Similar changes in insulin receptor processing were observed when the molar ratio of cholesterol to phospholipid in normal erythrocytes was modified in vitro by incubation with cholesterol-rich liposomes.

Radiolucent gallstones are common in young adults with cystic fibrosis. In the mid-1970s, it was suggested that gallstones are made of cholesterol, but this hypothesis has never been tested. Several recent studies have shown that the detection of cholesterol monohydrate crystals in bile has high sensitivity and specificity for the diagnosis of cholesterol gallstones. We therefore used this approach to study 17 young adults with cystic fibrosis, 10 of whom had radiolucent gallstones. The two groups of patients were comparable in age and gender (all patients but one were male). Duodenal bile was obtained after gallbladder contraction with intravenous cerulein; it was used for lipid and protein chemistry studies and for polarizing microscopy. The latter was performed both in whole bile and in the postultracentrifugation (100,000 g) sediment. Bile cholesterol saturation did not significantly differ between patients with (1.21 +/- 0.28) or without gallstones (0.99 +/- 0.54). Slight cholesterol supersaturation was found in 7 of 10 gallstone and three of seven nongallstone patients. At no time were cholesterol crystals detected in either the group, even after bile ultracentrifugation. Two more cystic fibrosis patients with gallstones died of severe bronchopneumopathy, and small pigment gallstones were obtained at autopsy. At stone analysis, cholesterol content was 44% and 28% of dry weight, respectively. Infrared spectroscopy of stone powder was compatible with the presence of calcium bilirubinate and proteins as major components. We conclude that radiolucent gallstones of cystic fibrosis are not of the conventional cholesterol type.

Lysophosphatidylcholine is a major metabolic product in the plasma and cellular turnover of phospholipids, with well-known membrane-toxic and proinflammatory properties. Because the liver plays a key role in plasma lysophosphatidylcholine removal and biotransformation and because virtually nothing is known of these processes in a diseased organ, the hepatobiliary metabolism of lysophosphatidylcholine was investigated in rats with carbon tetrachloride-induced liver cirrhosis. Twelve adult male Wistar rats with histologically confirmed cirrhosis and 8 control animals were fitted with jugular and biliary catheters and allowed to recover. The animals were kept under constant IV infusion of taurocholate (1 mumol/min). Two microcuries of sn-1[14C]palmitoyl-lysophosphatidylcholine was administered as a single bolus. The fate of the injected radioactivity, including removal from plasma, uptake, and subcellular location in the liver and molecular and aggregative forms, was studied by combined chromatographic and radiochemical methods. Major findings were (a) that lysophosphatidylcholine has a prolonged permanence in plasma of cirrhotic rats, due both to decreased hepatic clearance and to depressed conversion into phosphatidylcholine; (b) that the rate of lysophosphatidylcholine acylation is much slower in the cirrhotic than in the normal liver, both at the microsomal and at the cytosolic level; (c) that cytosolic lysophosphatidylcholine in the cirrhotic liver, but not in the normal liver, is predominantly non-protein bound; (d) that the strict molecular selectivity of lysophosphatidylcholine acylation observed in controls is partially lost in cirrhosis; and (e) that a consistent fraction of lysophosphatidylcholine is converted into triacylglycerols in cirrhotics but not in controls. These findings show a profound derangment of lysophosphatidylcholine handling and processing in the cirrhotic liver, which is of potential pathogenetic significance.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Liver cirrhosis is associated with a high prevalence of pigmentary cholelithiasis. The major compound of pigment gallstones is unconjugated bilirubin (UCB) in the form of calcium bilirubinate salts or a black pigment polymer. Most of UCB in bile derives from enzymic or non-enzymic hydrolysis of mono- or diconjugated bilirubin. Changes in the relative ratios between these two bilirubin species have been associated with pigment gallstones. It has also been shown that UCB solubilization in bile depends on its interaction with bile salts. In order to clarify the factors predisposing cirrhotic patients to pigment stone formation, we measured UCB, monoconjugated bilirubin (MCB) and diconjugated bilirubin (DCB) in duodenal bile of 15 patients with cirrhosis, ten patients with chronic active hepatitis (CAH) and ten normal subjects, we also analyzed their relationships with lipids. In cirrhotic patients, the MCB concentration in bile was significantly (p less than 0.05) higher than in normal subjects and was correlated with the severity of the disease. Bile salts and lecithin concentrations were significantly lower in cirrhosis (p less than 0.005 vs. CAH or normals). Cirrhotic patients have a bile salts/UCB molar ratio which is one third that of CAH patients or normal subjects (p less than 0.01). No differences were found between CAH patients and controls in each of the parameters tested. In conclusion, we propose that the very low BS/UCB molar ratio and the very high biliary content in MCB represent two independent physico-chemical factors predisposing cirrhotic patients to pigmentary cholelithiasis.

During studies on the effect of bile salt-pool depletion in the bile-fistula rat (adult male Sprague-Dawley), the spontaneous formation of an orange-brown precipitate was noted. The nature of this phenomenon and its relationship to BS and calcium concentration was investigated in depth. Bile from 18 animals was collected in the dark into transparent tubes containing sodium azide, ascorbic acid, and glucaro-1,4-lactone. The tubes were flushed with nitrogen, sealed, and incubated at 37 degrees C. The pigmentary precipitate formed in all the bile salt-depleted (less than 3-5 mM) bile samples (i.e., those collected after 5-7 h of external biliary drainage), but not in bile salt-rich biles. It appeared within 30-240 min after collection, both in bile samples collected at room temperature and at 37 degrees C, initially as a pale flocculation and then slowly sedimenting to form, after centrifugation, a solid, dark-orange pellet. There were no pH changes during incubation, and bile cultures were negative. Under polarizing microscopy, the precipitate appeared amorphous, and there was no evidence of birefringence. High-performance liquid chromatography showed that unconjugated bilirubin was the prevalent pigmentary component, but significant amounts of monoconjugated bilirubin also coprecipitated. Lipid chemistry showed the presence of lecithin (80.1% of total lipids), which was rich in palmitoyl and linoleoyl fatty acids, and of fatty acids (predominantly palmitic and oleic). Infrared spectroscopy and x-ray diffraction showed the presence of calcium bilirubinate and palmitate. In-vivo replenishment of the bile salt pool by intravenous infusion of either taurocholate or taurochenodeoxycholate (1 mumol/min) completely prevented the pigmentary precipitation. In vitro experiments showed inhibition of the precipitate formation by the addition of individual bile salt in concentrations approximating their critical micellar concentration. Precipitate formation was hastened by the addition of calcium chloride (4-12 mM), but only in bile salt-depleted biles. As the composition of the precipitate closely resembles that of human brown-pigment stones and sludge, these findings may provide new insights into an understanding of the pathogenesis of pigment gallstone disease.

The content of polyunsaturated phosphatidylcholines (PCs) is one of the parameters which regulate membrane functions. Polyunsaturated PCs are preferentially synthesized in the liver by the microsomal enzyme phosphatidylethanolamine N-methyltransferase. The activity of this enzyme may be stimulated in vitro in isolated rat hepatocytes by supplementation with dimethylethanolamine (DME), the polar head group of the precursor of PC along this pathway. The aim of this study was to evaluate in vivo the effect of an intravenous infusion of DME in the rat on the hepatic phospholipid composition. Bile fistula rats were intravenously infused for 15 h with sodium taurocholate (1 mumol/kg per min), with or without the addition of 0.3 mg/kg per min of [14C]DME. The concentration per gram of wet liver of individual phospholipid classes, PC molecular species and of total triacylglycerols, as well as the distribution of radioactivity in liver phospholipids, in rat tissues and body fluids were analyzed. A significant (P less than 0.01) enrichment in PC was found in the liver of DME-infused rats with respect to controls. No differences in the other phospholipid classes were found. DME-infused rats showed a significant (P less than 0.01) decrease in the hepatic concentration of triacylglycerols. At HPLC analysis, the enrichment in PC in DME-infused rats was found to be selectively due to three molecular species (i.e., sn-1 stearoyl/sn-2 arachidonoyl, sn-1 stearoyl/sn-2 linoleoyl, sn-1 stearoyl/sn-2 docosahexanoyl molecular species). In agreement with quantitative data, more than 70% of hepatic radioactivity was recovered in polyunsaturated PC species, with the highest specific activity in the sn-1 stearoyl PCs. The specific activity of hepatic PC approximates that of phosphatidyldimethylethanolamine. This finding together with the effective incorporation of DME in PC suggests that this amino base is methylated after its incorporation into phosphatidyldimethylethanolamine, throughout the stimulation of hepatic N-methyltransferase activity. The selective hepatic enrichment with polyunsaturated PC species after DME infusion may offer a new experimental tool for studying hepatic membrane metabolism.