Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases

We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp. strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei. These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains.     

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

Extractive separation of penicillin G by facilitated transport via carrier supported liquid membranes

The facilitated transport of penicillin G (Pen G), through a supported liquid membrane with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, were studied. The distribution coefficient was obtained from a batch extraction experiment. The effects of flow rate, carrier concentration, initial concentration of Pen G, and the pH of feed and stripping phases on the transport rate of Pen G through the supported liquid membrane were also investigated. The results are in agreement with theoretical predictions, and it is demonstrated that the transport of Pen G through the supported liquid membrane is controlled simultaneously by mass transfer across both aqueous and liquid membranes. (c) 1993 John Wiley & Sons, Inc.     

Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.     

Similar structures in gamma-carboxymuconolactone decarboxylase and beta-ketoadipate succinyl coenzyme A transferase.

gamma-Carboxymuconolactone decarboxylase (EC 4.1.1.44) and beta-ketoadipate succinyl coenzyme A transferase (EC 2.8.3.6) mediate different steps in the beta-ketoadipate pathway. Antisera prepared against the Pseudomonas putida transferase cross-reacted immunologically with the decarboxylase from the same organism. The transferase is formed by association of two nonidentical protein subunits. The NH2-terminal amino acid sequences of the two nonidentical transferase subunits resembled each other and also were similar to the NH2-terminal amino acid sequence of the decarboxylase.     

Loss of transferrin receptors following induced differentiation of HL-60 promyelocytic leukemia cells

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.     

Clonotypic analysis of anti-acetylcholine receptor antibodies from experimental autoimmune myasthenia gravis-sensitive Lewis rats and experimental autoimmune myasthenia gravis-resistant Wistar Furth rats

A single immunization of Lewis rats with purified acetylcholine receptor (AChR) emulsified in adjuvant typically stimulates the production of oligoclonal AChR-reactive antibodies (as demonstrated by IEF) dominated by the IgG2a subclass, of moderate but clonotypically heterogeneous relative Ag-binding avidity, and capable of inducing symptoms of experimental autoimmune myasthenia gravis. Although similar immunization of Wistar Furth rats produces AChR-reactive antibodies with similar characteristics of clonotypic heterogeneity, avidity, and isotype expression, no detectable signs of AChR-dependent muscle impairment is observed. This contrasts the ability to induce impaired AChR function upon the passive transfer of pre-formed Lewis anti-AChR antibodies into naive Wistar Furth rats, suggesting that disease resistance in this model is not conferred at the level of the AChR itself. Moreover, if more aggressive immunization protocols are used (i.e., multiple injections of AChR), a transient breakthrough of AChR-dependent muscle dysfunction can be induced directly in the Wistar Furth strain indicating that the potential for the production of disease-causing antibodies does exist in the Wistar Furth repertoire. IEF analysis of Wistar Furth anti-AChR antibodies has revealed that hyperimmunization results in modified antibody clonotype expression that might explain changing expression of disease symptoms; however, explanations for the apparent "resistance" of Wistar Furth rats to disease induction are likely to be complex.     

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.     

Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells

Tyrosine phosphorylation of membrane-associated proteins is involved at two distinct sites of contact between cells and the extracellular matrix: adhesion plaques (cell adhesion and de-adhesion) and invadopodia (invasion into the extracellular matrix). Adhesion plaques from chicken embryonic fibroblasts or from cells transformed by Rous sarcoma virus contain low levels of tyrosine-phosphorylated proteins (YPPs) which were below the level of detection in 0.5-microns thin, frozen sections. In contrast, intense localization of YPPs was observed at invadopodia of transformed cells at sites of degradation and invasion into the fibronectin-coated gelatin substratum, but not in membrane extensions free of contact with the extracellular matrix. Local extracellular matrix degradation and formation of invadopodia were blocked by genistein, an inhibitor of tyrosine-specific kinases, but cells remained attached to the substratum and retained their free-membrane extensions. Invadopodia reduced or lost YPP labeling after treatment of the cells with genistein, but adhesion plaques retained YPP labeling. The plasma membrane contact fractions of normal and transformed cells have been isolated form cells grown on gelatin cross-linked substratum using a novel fractionation scheme, and analyzed by immunoblotting. Four major YPPs (150, 130, 81, and 77 kD) characterize invadopodial membranes in contact with the matrix, and are probably responsible for the intense YPP labeling associated with invadopodia extending into sites of matrix degradation. YPP150 may be an invadopodal-specific YPP since it is approximately 3.6-fold enriched in the invasive contact fraction relative to the cell body fraction and is not observed in normal contacts. YPP130 is enriched in transformed cell contacts but may also be present in normal contacts. The two major YPPs of normal contacts (130 and 71 kD) are much lower in abundance than the major tyrosine-phosphorylated bands associated with invadopodial membranes, and likely represent major adhesion plaque YPPs. YPP150, paxillin, and tensin appear to be enriched in the cell contact fractions containing adhesion plaques and invadopodia relative to the cell body fraction, but are also present in the soluble supernate fraction. However, vinculin, talin, and alpha-actinin that are localized at invadopodia, are equally concentrated in cell bodies and cell contacts as is the membrane-adhesion receptor beta 1 integrin. Thus, tyrosine phosphorylation of the membrane-bound proteins may contribute to the cytoskeletal and plasma membrane events leading to the formation and function of invadopodia that contact and proteolytically degrade the extracellular matrix; we have identified several candidate YPPs that may participate in the regulation of these processes.