A 100-kDa Protein Tyrosine Phosphorylation Is Concurrent with β1 Integrin-Mediated Morphological Differentiation in Neuroblastoma and Small Cell Lung Cancer Cells

IMR32, a neuroblastoma cell line, and CADO LC6, a small cell lung cancer (SCLC) cell line, extended neurite-like processes when cultured on fibronectin (FN)-coated surfaces or cultured in a serum-free medium. Monoclonal antibodies against the integrin β1 subunit inhibited this process formation, suggesting that their morphological change is initiated by β1 integrin-dependent signal transduction to the cell interior. Anti-phosphotyrosine immunoblots demonstrated that the phosphorylation level of a 100-kDa protein, but not 125-kDa focal adhesion kindase, correlated well with the morphological change in both cell lines. This 100-kDa protein phosphorylation did not accompany FN-induced morphological changes in NIH 3T3 fibroblasts or A549 adenocarcinoma cells. These findings suggest that neuroblastoma and SCLC may share β1 integrin-mediated signaling events distinct from nonneuronal cells.     

A second lysine-specific serine protease from Lysobacter sp. strain IB-9374

A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA. The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.     

Cloning and tissue expression of cDNAs from chromosome 5q21–22 which is frequently deleted in advanced lung cancer

Previously, we have reported that the inactivation of putative tumor-suppressor gene(s) on chromosome 5q21–22 may play an important role in the progression of lung cancer. Here, we describe the establishment of a yeast artificial chromosome (YAC) contig that spans 8–10 Mb at the 5q21–22 region. Six cosmid contigs have also been established in this YAC contig. About 35 exon-like fragments have been detected by exon-amplification, direct screening, cross-species hybridization, and searches of a database. Thus far, 14 cDNAs have been isolated, and two of them coincide with known genes, viz., cysteine dioxygenase I and geranylgeranyltransferase I. The other 12 cDNAs are considered to be novel genes. Two of these novel cDNA show partial homology to known genes, viz., semaphorin CD100 and the 28S rRNA gene. In addition, four known genes, including APC (adenomatous polyposis coli), MCC (mutated in colorectal cancer), proto-oncogene tyrosine kinase FER, and genomic imprinted gene U2AF1-RS1, have also been mapped in this contig. This large contig and expression map should prove crucial in the identification of susceptibility gene(s) related to the progression of lung cancer. Received: 27 May 1997 / Accepted: 3 September 1997     

Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis,molecular docking and anticonvulsant studies

Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.     

Prediction of protein motions from amino acid sequence and its application to protein-protein interaction

Background  

Structural flexibility is an important characteristic of proteins because it is often associated with their function. The movement of a polypeptide segment in a protein can be broken down into two types of motions: internal and external ones. The former is deformation of the segment itself, but the latter involves only rotational and translational motions as a rigid body. Normal Model Analysis (NMA) can derive these two motions, but its application remains limited because it necessitates the gathering of complete structural information.     

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.     

Beta-hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates

Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes β-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as β-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that β-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that β-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.     

Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the 'steric gate' of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.