Linked pyridinyl-thiadiazoles: Design and synthesis as potential candidate for treatment of XDR and MDR tuberculosis.

Multi-drug resistant (MDR) and extremely drug resistant (XDR) Mycobacterium tuberculosis strains have turned tuberculosis (TB) as "on the verge of eradication" to "most life threatening" disease. Furthermore, synergy with HIV and other immunosuppressive disease have strengthened its prevalence. This research reports small molecule anti-infectives which are specifically potent against several strains and isolates of TB. The hit compound 7f has also proved to be active against almost 25 clinical isolates comparable to marketed anti-TB agents.

Structural proteins of two different plaque-size phenotypes of fowlpox virus.

Structural polypeptides of two plaque-purified variant isolates of fowlpox virus differing in plaque morphology and size were examined by Coomassie blue-staining and immunoblot analysis of purified virions. A total of 30 structural polypeptides were observed, ranging in molecular weight from 14,100 to 122,600. A late polypeptide of 36,400 molecular weight was quite prominent in the small-plaque clone but absent in the large-plaque clone. Two other polypeptides, of 33,700 and 34,800 molecular weight, were present in virions from large-plaque virus and cell lysates of both clones but were absent in the small-plaque virions. These differences were observed whether the viruses were grown in chorioallantoic membrane or in chicken embryo fibroblast cultures. No difference was observed between the growth curves of the two virus clones. Differences observed in the polypeptides of the two viruses may be due to changes in the less conserved regions of viral DNA and may be used for differentiation of virus isolates.

Inhibition of prostate cancer-cell proliferation by Essiac.

OBJECTIVE: To assess the ability of Essiac tea extracts (Essiac Canada International, Ottawa, Canada) to modulate cancer cell proliferation and immune responsiveness. DESIGN: A noncancerous transformed cell line was compared to a cancerous cell line and spleen cells that had been isolated from mice to examine proliferation responses mediated by the addition of an Essiac preparation. RESULTS: We found in vitro evidence of decreased proliferation of both noncancerous transformed (CHO) and cancerous prostate cell line (LNCaP) when Essiac was present in the culture media. A dose response for inhibition was demonstrated by a linear regression performed on the data for both the CHO and LNCaP cells. The percent inhibition of the LNCaP cells was higher than the percent inhibition of the CHO cells suggesting that Essiac may have a more selective effect on cancer cells than transformed cells. In addition, the effects of Essiac were examined in an immune T-lymphocyte proliferation assay. At low doses of Essiac, augmentation of proliferation of these T cells was demonstrated, but at higher doses Essiac was inhibitory to T-cell proliferation. The same doses of Essiac that stimulated spleen cells were inhibitory for LNCaP cell proliferation. CONCLUSIONS: Essiac preparations may be able to inhibit tumor cell growth while enhancing immune response to antigenic stimulation. This may be especially valuable in immune-suppressed individuals.

Is an activator protein-2-like transcription factor involved in regulating gene expression during nitrogen limitation in fungi?

The upstream sequences of all published lignin peroxidase and manganese peroxidase genomic clones from Phanerochaete chrysosporium were analyzed. This analysis revealed the presence of putative activator protein-2 (AP-2) recognition sequences in 11 of 15 lignin peroxidase genes. The lignin peroxidase clone GLG6 and the manganese peroxidase gene (mnp-1) have two copies of putative AP-2 sequence in the upstream region. Interestingly, the lignin peroxidase gene VLG4 of another white rot fungus, Trametes versicolor, and the nit-2 gene of Neurospora crassa also contain putative AP-2-binding sequences. Since all of these genes are regulated by nutrient nitrogen, I hypothesize that an AP-2-like transcription factor may be involved in inducing gene expression during nitrogen limitation in fungi.

Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions.

In order to delineate the roles of lignin and manganese peroxidases in the degradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium, the biodegradation of phenanthrene (chosen as a model for polycyclic aromatic hydrocarbons) was investigated. The disappearance of phenanthrene from the extracellular medium and mycelia was determined by using gas chromatography. The disappearance of phenanthrene from cultures of wild-type strains BKM-F1767 (ATCC 24725) and ME446 (ATCC 34541) under ligninolytic (low-nitrogen) as well as nonligninolytic (high-nitrogen) conditions was observed. The study was extended to two homokaryotic (basidiospore-derived) isolates of strain ME446. Both homokaryotic isolates, ME446-B19 (which produces lignin and manganese peroxidases only in low-nitrogen medium) and ME446-B5 (which totally lacks lignin and manganese peroxidase activities), caused the disappearance of phenanthrene when grown in low- as well as high-nitrogen media. Moreover, lignin and manganese peroxidase activities were not detected in any of the cultures incubated in the presence of phenanthrene. Additionally, the mineralization of phenanthrene was observed even under nonligninolytic conditions. The results collectively indicate that lignin and manganese peroxidases are not essential for the degradation of phenanthrene by P. chrysosporium. The observation that phenanthrene degradation occurs under nonligninolytic conditions suggests that the potential of P. chrysosporium for degradation of certain environmental pollutants is not limited to nutrient starvation conditions.

Structural analysis of unstable intermediate and stable forms of recombinant fowlpox virus.

The stability and structure of the products of recombination in a fowlpox virus (FPV) system using the thymidine kinase (TK) gene as the insertion site were examined. A 4.6 kb chimeric DNA fragment from the pUV1 expression vector, containing the bacterial lacZ gene and the vaccinia virus P7.5 promoter, was ligated into the XbaI site of the FPV TK gene. The resulting vector, pFTKlacZb, was transfected into chicken embryo fibroblast cultures infected with FPV at an m.o.i. of 0.1. Recombinants were screened for the expression of beta-galactosidase. Five recombinants were isolated and plaque-purified to 80 to 90% for expression of beta-glucosidase. Serial cell culture passage of the recombinants led to the gradual reappearance of the non-recombinant parental phenotype. Southern hybridization analysis of EcoRI fragments from all five recombinants indicated that a single cross-over homologous recombination had occurred between either the 5' or the 3' end fragments of the TK gene, generating unstable intermediate recombinants incorporating the entire pFTKlacZb vector. Secondary intermolecular or intramolecular recombination of intergenic repetitive sequences within the intermediate recombinants appears to have resulted in frequent regeneration of the parental genotype and an infrequent generation of more stable recombinants. A method was developed to select stable recombinants by passage of the intermediate recombinants in chicken embryo fibroblast cultures treated with 5-bromo-2'-deoxyuridine.

Transformation of Neurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA.

We have conducted a detailed study of 108 qa-2+ Neurospora transformants which were obtained by use of circular plasmid DNAs and various linear DNAs. Parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which the qa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass the qa-2 gene through a cross. The non-transmissible class comprises the majority of transformants and may identify those which harbor autonomously replicating plasmids. Evidence is presented which suggests that a 1.2 kB BamHI-BglII qa-2+ DNA fragment might possess an ars sequence. Transformation with linear plasmid DNAs and DNA fragments carrying the qa-2 gene resulted in a demonstrable increase in transformation frequency beyond that achieved with circular plasmid DNAs, but did not permit precise targeting to the resident locus. Southern analysis showed that linked transformants have only the normal resident qa-2 band whereas the unlinked transformants always possess the resident band plus at least one additional band. Multiple integration events appear to be common and include cases where only a portion of the transforming DNA has been integrated.

Alternative methods for production and staining of Phanerochaete chrysosporium basidiospores.

Homokaryotic isolates of Phanerochaete chrysosporium are generally obtained by stimulating the production of basidiospores. The most commonly used method requires a special incubator that is maintained at 28 degrees C with continuous illumination. Here we report an alternate method which permits the production of basidiopores with common laboratory incubators and requires no special illumination conditions. This alternate method gives reproducible results and yields basidiospores that are not contaminated with conidia. We also report a detailed optimized method for staining basidiospores for visualizing nuclei.

A new, rapid and efficient transformation procedure for Neurospora.

A new, rapid, efficient and reliable method for transforming Neurospora crassa is described. In this procedure, germinated conidia are treated with lithium acetate, then incubated with DNA, followed by exposure to polyethylene glycol and then a brief heat shock, prior to plating on selective medium. Optimal conditions to achieve a high transformation rate are reported. Transformation can be obtained with both circular and linear plasmid DNA and also with genomic DNA. Although the rate is substantially decreased, transformation was also obtained with relatively impure DNA preparations, such as that made via rapid "miniprep" procedures. This transformation technique is simple and reliable and provides a considerable savings in time and materials.

Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.

Transient expression assay for qualitative assessment of gene expression by fowlpox virus.

A transient expression assay for fowlpox virus (FPV) was developed to assess the feasibility of using heterologous promoters in FPV and to qualitatively determine relative promoter strength. A transient expression system for FPV has not been reported, and various methods used for transient expression in vaccinia-virus-infected cells produced negative results when used with FPV. Here a successful method for transient expression of E. coli beta-galactosidase in FPV-infected chick embryo fibroblasts is reported. This transient expression assay has been developed to qualitatively assess promoter recognition and gene expression by FPV. It should also prove useful in the identification of promoters from the FPV genomic library and in testing the accuracy of chimeric promoter-gene constructs.

Increase of chalcone synthase mRNA in pathogen-inoculated soybeans with race-specific resistance is different in leaves and roots.

Soybeans (Glycine max [L.] Merr.) respond to pathogens by producing isoflavonoid-derived phytoalexins. Chalcone synthase (CHS) is the first enzyme of the flavonoid/isoflavonoid biosynthetic pathway. We investigated changes in the steady state levels of CHS mRNA and other specific mRNAs at increasing times after inoculation in two different race-specific interactions, one between leaves and the bacterium Pseudomonas syringae pv glycinea (Psg), and one between roots and the fungus, Phytophthora megasperma f. sp. glycinea (Pmg). The amount of CHS mRNA increases significantly in soybean leaves inoculated with an avirulent race of Psg but not with a virulent race or water. In contrast, the increase in CHS mRNA is similar in roots inoculated with zoospores of either an avirulent or virulent race of Pmg. CHS mRNA increases significantly in pathogen inoculated roots but not in uninoculated controls. Hydroxyproline-rich glycoprotein (HRGP) has been observed by others to increase in wounded or pathogen-inoculated plants. We report here that HRGP mRNA levels are greater in roots inoculated with an avirulent Pmg race than with a virulent race, but inoculation with either race causes a significant increase in HRGP mRNA with respect to controls. Calmodulin or ubiquitin mRNA do not increase in either uninoculated or inoculated roots and leaves. The possibility that race-specific resistance in soybeans is expressed differently in different organs of the plant is discussed.

The cell content and secretion of water-soluble vitamins by several freshwater algae.

Three green algae, Chlamydomonas reinhardii, Chlorella vulgaris and Scenedesmus obliquus, and one blue-green alga, Anabaena cyclindrica, were grown in chemically defined media. All the algae examined contained folates, beta-carotene and vitamins C and E; several of the B-vitamins and vitamin A were found in varying amounts in some but not in all the algae examined. All the green algae secreted significant amounts of folate and biotin and all but Scenedesmus secreted pantothenate into their growth medium; Anabaena secreted folate and pantothenate.

Regulatory interactions between mitochondrial genes: interactions between two mosaic genes.

We have studied the mitochondrial DNA and the phenotypes of strains of Saccharomyces cerevisiae with specific intervening sequences in two mosaic genes: cob (the gene for apocytochrome b) and oxi3 (the gene for subunit I of cytochrome oxidase). The results suggest the following. (i) The presence of an intervening sequence downstream encompassing the intron box7 is sufficient for the regulation of oxi3 by cob (BOX phenotype); two sequences (containing intron loci box3 and box10) upstream in cob and two in oxi3 are dispensable. (ii) Strains without the two sequences upstream still contain the downstream sequence and the competence to specify a functional trans-acting element. Mutational lesions in this segment are phenotypically indistinguishable from box7 mutants, including the accumulation of polypeptides with homologous amino acid sequences. (iii) A catabolite-sensitive BOX phenotype, characteristic of mutants in the first exon, requires the simultaneous presence of an adjacent intervening sequence. A model is presented in which a hypothetical product specified by an intron (locus box7) of the cob gene controls the expression of a second mosaic gene (oxi3).