Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene

A wild isolate of Penicillium canescens was subjected to mutagenesis, and 150 chlorate-resistant mutants were isolated and classified in respect of their ability to utilize various nitrogen sources. Strains supposedly deficient in nitrate reductase have been transformed with the nitrate-reductase gene from Aspergillus niger. Transformation probably occurred by non-homologous integration of the transforming vector into the chromosome. Co-transformation with the AMA1 replicating element from A. nidulans enhanced transformation frequency up to 2000-fold, and was shown to result in autonomous maintenance of replicating concatenates, one of which was re-isolated by transformation of E. coli.     

Intergenic HA-NA interactions in influenza A virus: postreassortment substitutions of charged amino acid in the hemagglutinin of different subtypes

In our previous studies influenza A virus reassortants having neuraminidase (NA) gene of A/USSR/90/77 (H1N1) strain and hemagglutinin (HA) genes of H3, H4 and H13 subtypes were shown to produce a low virus yield and to exhibit a strong tendency to virion aggregation. More detailed studies with the use of a H3N1 reassortant and its high-yield non-aggregating variants revealed that NA of A/USSR/90/77 strain is inefficient in the removal of the terminal sialic acid residues from the virion components, and that the inefficiency of NA may be compensated by mutations in HA gene leading to a decrease of the receptor-binding affinity (Kaverin, N.V. , Gambaryan, A.S., Bovin, N.V., Rudneva, I.A., Shilov, A.A., Khodova, O.M., Varich, N.L., Sinitsin, B.V., Makarova, N.L., Kaverin, N.V., 1998. Postreassortment changes in influenza virus hemagglutinin restoring HA-NA functional match, Virology 244, 315-321). The present report describes studies performed with the use of H2N1 and H4N1 reassortants having HA genes of A/Pintail/Primorie/695/76 (H2N3) and A/Duck/Czechoslovakia/56 (H4N6) strains respectively and NA gene of A/USSR/90/77 strain. The low-yield reassortants and their high-yield non-aggregating variants were studied in both direct and competitive binding assays with sialic acid-containing substrates. The non-aggregating variants were shown to have a decreased affinity as compared to the initial reassortants toward high-molecular-weight sialic acid-containing substrates. The sequencing of HA genes revealed that all non-aggregating variants of H2N1 and H4N1 reassortants had amino acid substitutions increasing the negative charge of the HA molecule in the vicinity of the receptor-binding pocket. The results suggest that the influenza virus reassortants containing low-functional NA undergo similar postreassortment changes irrespective of the HA subtype: their receptor-binding activity decreased due to negatively charged amino acid substitutions in the vicinity of the receptor-binding pocket.     

Lineage-specific gene expansions in bacterial and archaeal genomes

Gene duplication is an important mechanistic antecedent to the evolution of new genes and novel biochemical functions. In an attempt to assess the contribution of gene duplication to genome evolution in archaea and bacteria, clusters of related genes that appear to have expanded subsequent to the diversification of the major prokaryotic lineages (lineage-specific expansions) were analyzed. Analysis of 21 completely sequenced prokaryotic genomes shows that lineage-specific expansions comprise a substantial fraction (approximately 5%-33%) of their coding capacities. A positive correlation exists between the fraction of the genes taken up by lineage-specific expansions and the total number of genes in a genome. Consistent with the notion that lineage-specific expansions are made up of relatively recently duplicated genes, >90% of the detected clusters consists of only two to four genes. The more common smaller clusters tend to include genes with higher pairwise similarity (as reflected by average score density) than larger clusters. Regardless of size, cluster members tend to be located more closely on bacterial chromosomes than expected by chance, which could reflect a history of tandem gene duplication. In addition to the small clusters, almost all genomes also contain rare large clusters of size > or =20. Several examples of the potential adaptive significance of these large clusters are explored. The presence or absence of clusters and their related genes was used as the basis for the construction of a similarity graph for completely sequenced prokaryotic genomes. The topology of the resulting graph seems to reflect a combined effect of common ancestry, horizontal transfer, and lineage-specific gene loss.     

Clinical and genetic features of variegate porphyria in a Chinese patient

Acute porphyria is rare in orientals. We describe a Chinese woman with recurrent generalised tonic-clonic seizures and abdominal pain. Genomic DNA studies identified a heterozygous base substitution from guanine to adenine at nucleotide position 503, resulting in substitution of arginine by histidine at position 168 of the protein (R168H). This genetic abnormality is similar to the mutation reported in Caucasians with variegate porphyria. To the best of our knowledge, this is the first report in the English literature a Chinese patient with variegate porphyria with an identifiable mutation. A brief review of porphyria is presented.     

Bladder pheochromocytoma encountered on sonography

Pheochromocytomas of the bladder are rare neoplasms, constituting <0.06% of all vesical tumours. Common presenting features of this tumour include episodes of sweating, hypertension, haematuria and postmicturition syncope. We describe a case of bladder pheochromocytoma in a 66‐year‐old man whose only symptom of macroscopic haematuria was initially assessed with ultrasonography. Clinical presentation highlights the need for a high index of suspicion during sonographic evaluation of bladder neoplasms because such tumours might present without symptoms of adrenergic excess.     

Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells

Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 microM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox, did not prevent the inhibition of GJIC by H2O2, which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione- sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH.      

Quality of life (QOL) assessment of MIP (mitomycin, ifosfamide and cisplatin) chemotherapy in advanced non-small cell lung cancers (NSCLC)

BACKGROUND: Quality of life (QOL) assessment has emerged to measure and quantify the balance between treatment benefit and toxicity, and has a value in predicting response and overall survival in cancer patients. METHODS: From July 1995 to February 1997, 38 symptomatic patients with advanced non-small cell lung cancer (NSCLC) were treated with MIP chemotherapy (mitomycin 6 mg/m2, ifosfamide 3000 mg/m2 and cisplatin 50 mg/m2 on day 1 every 3 weeks). Patients were assessed for QOL including physical well-being, general symptoms and lung cancer-specific symptoms, as well as objective response. RESULTS: The overall response rate was 38.9% (14/36, all were partial response) and the median duration of response was 3.5 months [95% confidence interval (CI) 2.0-4.0]. The median duration of overall survival was 7 months (95% CI 5.9-8.5). The overall improvement of QOL was 58.3% with 21 patients feeling better on treatment. The toxicity of chemotherapy was mild, mainly nausea/vomiting and minimal alopecia. Using multiple clinical predictors of survival (age, histology, stage, performance status), only change of QOL emerged significantly (P = 0.0007). CONCLUSIONS: MIP had an endurable response and low toxicity profile, and provided good QOL. Integral QOL data in our study provided the strong prediction of survival in advanced NSCLC. Further experienced QOL study will provide greatly enhanced outcome data in clinical trials.