Pleiotropic function of intersectin homologue Cin1 in Cryptococcus neoformans

The manifestation of virulence traits in Cryptococcus neoformans is thought to rely on intracellular transport, a process not fully explored in this pathogenic fungus. Through interaction cloning, we identified a multi‐modular protein, Cin1 (cryptococcal intersectin 1), whose domain structure is similar to that of the human endocytic protein ITSN1. Cin1 contains an N‐terminal EH domain, a central coiled‐coil region, a WH2 domain, two SH3 domains and a C‐terminal RhoGEF (DH)‐PH domain. Interestingly, alternative mRNA splicing resulted in two Cin1 isoforms, and Cin1 homologues are also restricted to basidiomycetous fungi. Disruption of the CIN1 gene had a pleiotropic effect on growth, normal cytokinesis, intracellular transports and the production of several virulence factors. Additionally, Cin1 interacts with cryptococcal Cdc42 and Wsp1 (a WASP homologue) proteins in vitro, suggesting a conserved role in the regulation of the actin cytoskeleton. However, deletion of RhoGEF or SH3 and RhoGEF domains did not result in any phenotypic changes, suggesting that functional redundancy exists in proteins containing similar domains or that the activities by other domains are necessary for Cin1 function. Our study presents the first evidence of a multi‐modular protein whose function in intracellular transport underlies the growth, differentiation and virulence of a pathogenic microorganism.     

Genetic relationships between resistances to Fusarium head blight and crown rot in bread wheat (Triticum aestivum L.)

Fusarium head blight (FHB) and crown rot (CR) are two wheat diseases caused by the same Fusarium pathogens. Progress towards CR resistance could benefit from FHB-resistant germplasm if the same genes are involved in resistance to these two different diseases. Two independent studies were conducted to investigate the relationship between host resistances to these two diseases. In the first study 32 genotypes were assessed and no significant correlation between their reactions to FHB and CR was detected. The second study was based on a QTL analysis of a doubled haploid population derived from a variety with resistance to both diseases. Results from this study showed that loci conferring resistance to FHB and CR are located on different chromosomes. Together, these results suggest that, despite a common aetiology, different host genes are involved in the resistance against FHB and CR in wheat. Thus, although it is possible that genes affecting both diseases may exist in other germplasm or under different conditions, separate screening seems to be needed in identifying sources of CR resistance.     

A new carbazole-based phenanthrenyl ruthenium complex as sensitizer for a dye-sensitized solar cell

Three new amphiphilic ruthenium complexes (Ru-1, Ru-2 and Ru-3) based on phenanthrenyl derivatives have been synthesized and used as photosensitizers for dye-sensitized solar cells (DSSCs). The ruthenium complex Ru-1 containing a carbazole group showed especially improved photophysical properties (red-shifted metal-to-ligand charge-transfer transition absorptions and enhanced molar extinction coefficients) and interesting electrochemical properties, resulting in its improved open circuit potential and high overall light-to-electric power conversion efficiency of 5.3% (AM1.5, 75 mW/cm²). These facts indicate that the carbazole-based phenanthrenyl ruthenium complex is a promising candidate for improving the conversion efficiency of dye-sensitized solar cells.     

A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in a single assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patterns were observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using the culture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determined to be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.     

A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.     

Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.     

Size-fractionated chlorophyll a and primary productivity in the Chukchi Sea and its northern Chukchi Plateau

Investigations on chlorophyll a and primary productivity were carried out in the Chukchi Sea and its northern Chukchi Plateau during the 2^nd Chinese National Arctic Research Expedition in the summer of 2003. The results showed that chlorophyll a concentrations were 0.009–30.390 μg/dm³ at the surveyed waters; the surface chlorophyll a concentrations were 0.050–4.644 μg/dm³ and the average value was (0.875±0.981) μg/dm³ in the surveyed area. In the Chukchi Sea Shelf, chlorophyll a concentrations at the depth from 10 m to bottom were higher than that in the surface water, and the concentrations were lower at the depth below 75 m in the Chukchi Plateau. Chlorophyll a concentrations descended in 3 sequential samplings on Transect R, with average values of (2.564±1.496) μg/dm³, (1.329±0.882) μg/dm³ and (0.965±0.623) μg/dm³, respectively. The potential primary productivity ((2.305± 1.493) mgC/(m³·h)) in the Chukchi Sea was higher than that ((0.527±0.374) mgC/(m³·h)) in the Chukchi Plateau. The results of the size-fractionated chlorophyll a and primary productivity showed that microplankton accounted for the majority of the total chlorophyll a (63.13%) and primary productivity (65.16%) at the survey stations. The contributions of the nanoplankton and picoplankton to the total chlorophyll a and primary productivity were roughly the same.     

Identification of a putative oocyte-specific small nuclear ribonucleoprotein polypeptide C in gibel carp

Previous studies have demonstrated that germinal vesicle of amphibian oocyte contains small nuclear ribonucleoprotein polypeptide C (SNRPC). In this study, a putative member of SNRPC was identified from Carassius auratus gibelio oocyte cDNA library. Its full-length cDNA has an open reading frame of 201 nt for encoding a peptide of 66 aa, a short 5'-UTR of 19 nt and a long 3'-UTR of 347 nt including a polyadenylation signal and poly- (A) tail, and the deduced amino acid sequence has 47% identity with the C-terminal of the zebrafish small nuclear ribonucleoprotein polypeptide C. Western blot analysis revealed its oocyte-specific expression. Immunofluorescence localization indicated that its gene product localized to numerous nucleoli within the oocytes and showed dynamic changes with the nucleoli during oocyte maturation. RT-PCR and Western blot analysis further revealed its constant presence in the oocytes and in the embryos until hatching. The data suggested that the newly identified CagOSNRPC might be a nucleolar protein.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.