Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.     

Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans.

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.     

Binding of heparin to basic fibroblast growth factor induces a conformational change.

The binding of heparin to basic fibroblast growth factor (bFGF) induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum. The observed spectral changes suggest that the conformational change is highly localized most likely in the beta-turn regions of the bFGF molecule. Heparan sulfate, a component of the endothelial extracellular matrix, was also observed to bind to bFGF and induce a similar conformational change to that observed for heparin. Further, sucrose octasulfate, a compound which mimics the effects of heparin biologically, was also observed to induce this same conformational change. This spectroscopically observable change has allowed us to probe the functional determinants necessary for heparin to bind the bFGF and to induce the observed conformational change. We have determined the effects of binding of various monomeric and polymeric, sulfated and nonsulfated glycosaminoglycans and carbohydrate compounds. The results indicate that the binding of heparin involves highly specific interactions. Further, heparin was observed to greatly increase the thermal stability of bFGF, raising the Tm by 25 degrees C. Sucrose octasulfate was also able to enhance the thermal stability of bFGF, but not to the same extent as heparin.     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Larval host plant affects fitness consequences of egg size variation in the seed beetle Stator limbatus

Egg size variation often has large effects on the fitness of progeny in insects. However, many studies have been unable to detect an advantage of developing from large eggs, suggesting that egg size variation has implications for offspring performance only under adverse conditions, such as during larval competition, periods of starvation, desiccation, or when larvae feed on low-quality resources. We test this hypothesis by examining the consequences of egg size variation for survivorship and development of a seed-feeding insect, Stator limbatus, on both a low-quality (Cercidium floridum) and a high-quality (Acacia greggii) host plant. Our results are consistent with the hypothesis. S. limbatus larval performance was affected by egg size only when developing on the poor-quality host (C. floridum); larvae from large eggs survived better on C. floridum than those from small eggs, while there was no evidence of an effect of egg size on progeny development time, body weight, or survivorship when larvae developed on A. greggii. These results indicate intense selection for large eggs within C. floridum-associated populations, but not in A. greggii-associated populations, so that egg size is predicted to vary among populations associated with different hosts. Our results also support this hypothesis; females from a C. floridum-associated population (Scottsdale) laid larger eggs than females from an A. greggii-associated population (Black Canyon City).     

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions

Abstract A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli , Hafnia alvei , a strain of Citrobacter freundii , and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:H6, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii , EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii , EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eaeA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.     

Characterization of Mhc genes in a multigenerational family of ring-necked pheasants

Little is known about the major histocompatibility (Mhc) genes of birds in different taxonomic groups or about how Mhc genes may be organized in avian species divergent by evolution or habitat. Yet it seems likely that much might be learned from birds about the evolution, organization, and function of this intricate complex of polymorphic genes. In this study a close relative of the chicken, the ring-necked pheasant (Phasianus colchicus), was examined for the presence and organization of Mhc B-G genes. The patterns of restriction fragments revealed by chicken B-G probes in Southern hybridizations and the patterns of pheasant erythrocyte polypeptides revealed in immunoblots by antisera raised against chicken B-G polypeptides provide genetic, molecular, and biochemical data confirming earlier serological evidence for the presence of B-G genes in the pheasant, and hence, the presence of a family of B-G genes in at least a second species of birds. The high polymorphism exhibited by the pheasant B-G gene family allowed genetic differences among individuals within the small experimental population in this study to be detected easily by restriction fragment patterns. Further evidence was found for the organization of the pheasant Mhc class I and class II genes into genetically independent clusters. Whether these gene clusters are fully comparable to the B and Rfp-Y systems in the chicken or whether yet another organization of Mhc genes has been encountered in the pheasant remains to be determined.     

Acceleration of cheese ripening

The characteristic aroma, flavour and texture of cheese develop during ripening of the cheese curd through the action of numerous enzymes derived from the cheese milk, the coagulant, starter and non-starter bacteria. Ripening is a slow and consequently an expensive process that is not fully predictable or controllable. Consequently, there are economic and possibly technological incentives to accelerate ripening. The principal methods by which this may be achieved are: an elevated ripening temperature, modified starters, exogenous enzymes and cheese slurries. The advantages, limitations, technical feasibility and commercial potential of these methods are discussed and compared.