We have recently shown that the ability of some gram-negative bacteria to dissolve poorly soluble calcium phosphates (Mps ؉ phenotype) is the result of periplasmic oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase (GDH), a component of the direct oxidation pathway. Escherichia coli K-12 derivatives synthesize apo-GDH but not the cofactor pyrroloquinoline-quinone (PQQ) essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, these strains do not produce gluconic acid and are Mps ؊ . Evidence is presented to show that expression of a single 396-base Pseudomonas cepacia open reading frame (designated gabY) in E. coli JM109 (a K-12 derivative) was sufficient to induce the Mps ؉ phenotype and production of gluconic acid. We present the nucleotide sequence of this open reading frame which coded for a protein (GabY) with a deduced M r of 14,235. Coupled transcription-translation of a plasmid (pSLY4 or pGAB1) carrying gabY resulted in production of a protein with an M r of 14,750. Disruption of the open reading frame of gabY via site-directed mutagenesis changed the phenotype to Mps ؊ and eliminated gluconic acid production. The deduced amino acid sequence of gabY has no apparent homology with those of previously cloned direct oxidation pathway genes but does share regions highly homologous with the histidine permease system membrane-bound protein HisQ as well as other proteins in this family. In the presence of 1 M exogenous PQQ, both JM109(pSLY4) and JM109(pGAB1) produced 10 times as much gluconic acid as was seen with either the plasmid or exogenous PQQ alone. The presence of pGAB1 was also sufficient to cause production of gluconic acid in E. coli HB101 (a K-12-B hybrid). In AG121, an apoGDH ؊ , Tn5 mutant of HB101, the presence of pGAB1 did not cause the production of gluconic acid.

The emergence of drug resistant variants of the influenza virus has led to a need to identify novel and effective antiviral agents. As an alternative to synthetic drugs, the consolidation of empirical knowledge with ethnopharmacological evidence of medicinal plants offers a novel platform for the development of antiviral drugs. The aim of this study was to identify plant extracts with proven activity against the influenza virus. Extracts of fifty medicinal plants, originating from the tropical rainforests of Borneo used as herbal medicines by traditional healers to treat flu-like symptoms, were tested against the H1N1 and H3N1 subtypes of the virus. In the initial phase, in vitro micro-inhibition assays along with cytotoxicity screening were performed on MDCK cells. Most plant extracts were found to be minimally cytotoxic, indicating that the compounds linked to an ethnomedical framework were relatively innocuous, and eleven crude extracts exhibited viral inhibition against both the strains. All extracts inhibited the enzymatic activity of viral neuraminidase and four extracts were also shown to act through the hemagglutination inhibition (HI) pathway. Moreover, the samples that acted through both HI and neuraminidase inhibition (NI) evidenced more than 90% reduction in virus adsorption and penetration, thereby indicating potent action in the early stages of viral replication. Concurrent studies involving Receptor Destroying Enzyme treatments of HI extracts indicated the presence of sialic acid-like component(s) that could be responsible for hemagglutination inhibition. The manifestation of both modes of viral inhibition in a single extract suggests that there may be a synergistic effect implicating more than one active component. Overall, our results provide substantive support for the use of Borneo traditional plants as promising sources of novel anti-influenza drug candidates. Furthermore, the pathways involving inhibition of hemagglutination could be a solution to the global occurrence of viral strains resistant to neuraminidase drugs.

The majority of cases of the autosomal recessive disorder Nijmegen breakage syndrome (NBS) are associated with null mutations in the NBS1 gene, which encodes a 95 kDa protein, nibrin. Cell lines established from NBS patients fail to express nibrin and display hypersensitivity to ionizing radiation and dysregulation of the nuclear localization of two key proteins involved in DNA repair, Mre11 and Rad50. Conclusive proof that mutations in the NBS1 gene are responsible for NBS requires that re-expression of normal nibrin in NBS cells complements these phenotypes. In the current study, retroviral expression vectors containing a normal copy of the NBS1 gene or a mutated form derived from a NBS patient were introduced into a well- characterized NBS cell line. Introduction of a normal copy of the NBS1 gene, but not the mutant form, resulted in robust expression of nibrin that displayed correct nuclear localization. Expression of nibrin also restored the ability of nibrin, Mre11 and Rad50 to complex and to redistribute within the nucleus in response to ionizing radiation. Radiation sensitivity of NBS cells expressing wild-type nibrin was restored to normal levels. Hence, introduction of the NBS1 gene can correct the phenotypes observed in NBS cells.

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