Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin–resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64?μg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.     

Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage

A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H. Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied. Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage. Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment. When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA. The sum of the fragment lengths equaled the length of the mRNA. Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA. With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.     

Galactosyltransferase and sialyltransferase are located in different subcellular compartments in HeLa cells

Galactosyl- and sialyltransferase have been localized by double immunofluorescence labeling in HeLa cells. Galactosyltransferase was found in a compact juxtanuclear structure previously shown to represent the Golgi apparatus (J. Roth and E.G. Berger (1982) J. Cell Biol. 93, 223-9), whereas sialyltransferase was localized to vesicles spread over the whole cytoplasm. These findings indicate different compartments for both transferases and support a model of subcompartmentation of glycosylation steps along the secretory pathway.     

Platinum toxicity and gene expression in Xenopus embryos: analysis by FETAX and differential display

Since the level of platinum in the environment is destined to increase, because of its use in vehicle catalytic converters, the toxicity of platinum needs further investigation. In this study, the frog embryo teratogenesis assay-Xenopus (FETAX) was used to compare the embryotoxicity and teratogenicity of two common platinum species, (NH4)2PtCl4 and (NH4)2PtCl6. The uptake rates of the two platinum species were studied, and also their effects on the expression of genes encoding metallothionein and heat-shock protein 70, which are known to be induced by several stress factors. In addition, the differential display technique was used to search for genes that were specifically induced by platinum. A gene for the type I collagen alpha-chain and a novel gene were identified.     

Altered profile of secondary metabolites in the root exudates of Arabidopsis ATP-binding cassette transporter mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.     

Biosensors and molecular imaging

The increase in the understanding of the physical and functional properties of the biological material, from the cellular level down to single molecules, owes its success to the development of suitable high-sensitivity platforms to image the biomaterial and analyze its response to specific stimuli. Imaging has indeed reached molecular capabilities, thanks to optical or magnetic markers [1], to the atomic force microscopy (AFM) in surface reconstruction [2], and is nearing success in three-dimensional (3-D) reconstruction thanks to X-ray holography [3].     

Foaming of proteins: New prospects for enzyme purification processes

Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the “White Biotechnologies” for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique.     

Substrate recognition drives the evolution of serine proteases

A method is introduced to identify amino acid residues that dictate the functional diversity acquired during evolution in a protein family. Using over 80 enzymes of the chymotrypsin family, we demonstrate that the general organization of the phylogenetic tree and its functional branch points are fully accounted for by a limited number of residues that cluster around the active site of the protein and define the contact region with the P1-P4 residues of substrate.     

Porous polymers for fixed bed adsorption of aroma compounds in fermentation processes

Summary Deionized water as well as simulated and genuine fermentation media, which contained varying concentrations of benzaldehyde and 4-decanolide, were used to investigate the applicability of selected styrene-divinylbenzene resins for low pressure downflow adsorption of aroma compounds in a fixed bed. The effects of flow rate and matrix on the respective breakthrough curve slopes were examined using the LUB-and the MTZ-model.