The GM2-1 ganglioside islet autoantigen in insulin-dependent diabetes mellitus is expressed in secretory granules and is not beta-cell specific

The pancreatic islet monosialo-ganglioside (GM2-1), an autoantigen in insulin-dependent diabetes mellitus (IDDM) recently shown to be the target of autoantibodies associated with diabetes development in relatives of IDDM patients, is islet specific within the pancreas, and its expression is metabolically regulatable. In the present study we sought to establish 1) whether GM2-1 is beta-cell specific, and 2) its intracellular localization. To this end, we analyzed the pattern of ganglioside expression in highly purified beta- and non-beta-cells isolated from rat islets. In addition, ganglioside levels were determined in subcellular fractions of a rat beta-cell line (INS). No qualitative or quantitative difference was found in the pattern of ganglioside expression between beta and non-beta rat islet cells, with GM3, GM2-1, and GD3 gangliosides expressed in both cell populations. Within INS cells, GM2-1 ganglioside was expressed in the fraction containing secretory granules and, to a lesser extent, in plasma membranes; GM3 was expressed in secretory granules, whereas GD3 was found only in plasma membranes. These data indicate that the GM2-1 autoantigen is not beta-cell specific within the islets, in accordance with the observation that this molecule is a target of islet cell autoantibodies that bind to the whole pancreatic islet. Interestingly, this autoantigen is present in secretory granules similarly to other autoantigens in IDDM (insulin, carboxypeptidase H, 38-kDa protein, etc.), suggesting that the autoimmunity to the components of this organelle may be central to the pathogenesis of the disease.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

Plasma albumin induces calcium waves in rat cortical astrocytes

At the end of the 19th century, ectopic pregnancy became a surgical procedure. A century later, one third of ectopic pregnancies are treated medically. In the meantime, early detection of ectopic pregnancy became possible due to sensitive serum hCG and progesterone combined assays with transvaginal sonography and a knowledge of risk factors. Consequently, a nonsurgical approach appears to be an attractive alternative to surgery. Expectant management is recommended with a plateau or decreasing hCG and an initial level < or = 1.000 mIU/ml in asymptomatic women. Medical treatment by local or parenteral methotrexate is recommended in patients with clear evidence of an unruptured pregnancy in based on initial hCG and progesterone level, size of hemoperitoneum, ultrasound diameter of hematosalpinx and absence of clinical pain. Laparoscopy remains the gold standard but in prospective randomized trials between medical treatment and laparoscopy, in selected patients, the non-surgical approach appears to be equivalent with a similar reproductive performance.     

Matrix metalloproteinase activity in equine synovial fluid: influence of age, osteoarthritis, and osteochondrosis

OBJECTIVE: To investigate the influence of age, osteoarthritis (OA), and osteochondrosis (OC) on the matrix metalloproteinase (MMP) activity in the synovial fluid (SF) of equine joints. METHODS: SF was collected from normal and osteoarthritic metacarpophalangeal joints (normal: 14 adult, 28 juvenile; OA: 22 adult). And from normal and osteochondrotic tarsocrural joints (5 months: 11 normal, 8 OC; 11 months: 7 normal, 6 OC). Subsequently, overall MMP activity was measured. RESULTS: The level of active MMPs was almost twofold higher in SF from juvenile horses (age up to 11 months) than in SF from mature animals (4-30 years; p < 0.001). In juvenile horses MMP activity was higher in 5 month old foals than in 11 month old foals (p < 0.01). In adult horses MMP activity was independent of age. In OA joints the activity was nearly twice as high as in normal joints (p < 0.001). In OC joints MMP activity was not significantly different from normal, age matched, control joints. CONCLUSIONS: MMP activity in SF from normal adult joints is not related to age. In juvenile joints MMP activity is significantly higher than activity in joints from adult animals. It is hypothesised that the gradual decrease in MMP activity with increasing age reflects the declining metabolic activity resulting from ceasing growth and the accompanying decrease in cartilage remodelling. The increased MMP activity in osteoarthritis joints most likely reflects matrix destruction. In osteochondrosis MMP mediated matrix degradation appears not to be different from normal joints.     

Recognition of analogous and homologous protein folds--assessment of prediction success and associated alignment accuracy using empirical substitution matrices

Fold recognition methods aim to use the information in the known proteinstructures (the targets) to identify that the sequence of a protein ofunknown structure (the probe) will adopt a known fold. This paperhighlights that the structural similarities sought by these methods can bedivided into two types: remote homologues and analogues. Homologues are theresult of divergent evolution and often share a common function. We defineremote homologues as those that are not easily detectable by sequencecomparison methods alone. Analogues do not have a common ancestor andgenerally do not have a common function. Several sets of empirical matricesfor residue substitution, secondary structure conservation and residueaccessibility conservation have previously been derived from aligned pairsof remote homologues and analogues (Russell et al., J. Mol. Biol., 1997,269, 423-439). Here a method for fold recognition, FOLDFIT, is introducedthat uses these matrices to match the sequences, secondary structures andresidue accessibilities of the probe and target. The approach is evaluatedon distinct datasets of analogous and remotely homologous folds. Theaccuracy of FOLDFIT with the different matrices on the two datasets iscontrasted to results from another fold recognition method (THREADER) andto searches using mutation matrices in the absence of any structuralinformation. FOLDFIT identifies at top rank 12 out of 18 remotelyhomologous folds and five out of nine analogous folds. The averagealignment accuracies for residue and secondary structure equivalencing aremuch higher for homologous folds (residue approximately 42%, secondarystructure approximately 78%) than for analogues folds (approximately 12%,approximately 47%). Sequence searches alone can be successful for severalhomologues in the testing sets but nearly always fail for the analogues.These results suggest that the recognition of analogous and remotelyhomologous folds should be assessed separately. This study has implicationsfor the development and comparative evaluation of fold recognitionalgorithms.     

氯化聚合物废物的热裂解方法

发明综述了在流动气体和固体流化床存在下,含氯的废聚合物在反应器中的热裂解,粒状流化物质在３５０－６００℃温度范围内裂解成低烃气相混合物,使其氧的质量分数低于５＊１０＾－５。在本生产工艺中,在４００－６００℃的温度范围内,裂解产物在流化床形式,并通过一个或多个防护床,该防护床由氧化钙或可引发氧化钙的物质组成。通过这种方法,产品中氯的质量分数低于５＊１０＾－５。,     

Candidate genes for nonsyndromic cleft lip and palate and maternal cigarette smoking and alcohol consumption: evaluation of genotype-environment interactions from a population-based case-control study of orofacial clefts

Previous studies suggest that the relationship between genes and nonsyndromic cleft lip +/- cleft palate (CLP) or cleft palate only (CP) may be modified by the environment. Using data from a population-based case-control study, we examined allelic variants for three genes, i.e., transforming growth factor alpha (TGFA), transforming growth factor beta 3 (TGFB3), and Msh (Drosophila) homeobox homolog 1 (MSX1), and their interactions with two exposures during pregnancy (maternal cigarette smoking and alcohol consumption) as risk factors for CLP and CP. For each cleft phenotype, risk estimates associated with most allelic variants tended to be near unity. Risk estimates for maternal smoking (> or = 10 cigarettes/day) were significantly elevated for CP and were most elevated among infants with allelic variants at the TGFB3 or MSX1 sites. By comparison, risk estimates for maternal alcohol consumption (> or = 4 drinks/month) were significantly elevated for CLP and were most elevated among infants with allelic variants at the MSX1 site. Our results suggest that development of CLP and CP may be influenced independently by maternal exposures but more significantly by interaction of such exposures and specific allelic variants.     

Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: role in mitogenesis

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extra-cellular signals are transduced to intracellular signaling pathways. The mitogen-activated protein kinase (MAPK) cascade has been identified as a key factor in transducing numerous mitogenic stimuli. MAPK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinoma (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model of HCC we investigated changes in Gq/G11-protein expression in tumorigenic tissue versus adjacent, non-neoplastic liver. In addition we addressed the role of Gq/G11-proteins in the regulation of MAPK-linked mitogenesis by using rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture. Western blot analysis showed significant increases in Gqalpha and G11alpha expression in tumorigenic liver versus normal liver specimens, an effect that was augmented in cultured H4IIE cells versus isolated cultured hepatocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activity was significantly increased in HCC versus normal liver. A specific PLC inhibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated DNA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, serum-stimulated MAPK activity was significantly enhanced in H4IIE versus cultured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenuated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gqalpha-PLC specific uncoupler) and GP2A (Gqalpha specific inhibitor) mirrored the effects of those observed for Et-18-OCH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-stimulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is associated with enhanced Gq/G11-PLC expression/activity as compared with normal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.     

Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI

Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible N-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of B. cepacia K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a luxR homolog that was designated cepR. The flanking DNA region was used to clone the wild-type copy of cepR. Sequence analysis revealed the presence of cepI, a luxI homolog, located 727 bp upstream and divergently transcribed from cepR. A lux box-like sequence was identified upstream of cepI. CepR was 36% identical to Pseudomonas aeruginosa RhlR and 67% identical to SolR of Ralstonia solanacearum. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a cepR mutation restored parental levels of ornibactin and protease but not lipase. An N-acylhomoserine lactone was purified from culture fluids and identified as N-octanoylhomoserine lactone. K56-I2, a cepI mutant, was created and shown not to produce N-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for cepIR in the regulation of extracellular virulence factor production by B. cepacia.