Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.     

Low-power inductive RF plasma sources for technological applications

The objective of the present paper is to develop an analytic theory of cylindrical low-power RF plasma sources operating at an industrial frequency (f=13.56 MHz, ω=8.52×10⁷ s^?1). Inductive surface exciters of electromagnetic fields (exciting antennas) are considered that are positioned either at the side surface of the cylinder or at one of its end surfaces. In the latter case, the plasma flows out of the source through the opposite end surface of the cylinder. A study is made of elongated systems in which the length L of the cylinder exceeds its diameter 2R and of planar disk-shaped systems with L<2R. Simple analytic expressions are derived for electromagnetic fields excited by the antenna in the source plasma. The equivalent plasma resistance and the equivalent RF power deposited in the plasma are calculated for systems with prescribed parameters, i.e., in a non-self-consistent model. Up to now, such sources have been investigated mainly through the numerical solution of the complicated general electrodynamic equations. In the Introduction, the problem is formulated in general terms and the geometry of the sources, as well as the characteristic parameters of the source plasma, is discussed. In Section 2, plasma sources operating without an external magnetic field are investigated. In Section 3, helicon plasma sources in a sufficiently strong external magnetic field are considered. Analytic predictions are compared with the results from solving the problem numerically without using the helicon approximation. Section 4 gives a brief discussion of an electron cyclotron resonance-based RF plasma source. In the Conclusion, the main results of the paper are summarized and the technological efficiency of the sources under consideration is estimated at a qualitative level.     

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

Arisostatins A induces apoptosis through the activation of caspase-3 and reactive oxygen species generation in AMC-HN-4 cells

A microbial secondary metabolite, arisostatins A (As-A), was originally discovered as a substance carrying the antibiotic activity against Gram-positive bacteria and shown to possess potent anti-tumor properties. The mechanism by which arisostatins A initiates apoptosis remains poorly understood. In the present report we investigated the effect of arisostatins A on activation of the apoptotic pathway in HN-4 cells. Arisostatins A was shown to be responsible for the inhibition of HN-4 cell growth by inducing apoptosis. Treatment with 4 microM arisostatins A for 24h produced morphological features of apoptosis and DNA fragmentation in HN-4 cells. Arisostatins A caused dose-dependent apoptosis and DNA fragmentation of HN-4 cells used as a model. Treatment with caspase inhibitor significantly reduced the arisostatins A-induced caspase 3 activation. In addition, arisostatins A-induced apoptosis was associated with the generation of reactive oxygen species (ROS), which was prevented by an antioxidant NAC (N-acetyl-cysteine). These data indicate that cytotoxic effect of arisostatins A on HN-4 cells is attributable to the induced apoptosis and that arisostatins A-induced apoptosis is mediated by caspase-3 activation pathway, loss of mitochondrial transmembrane potential (DeltaPsi(m)), and release of cytochrome c into cytosol.     

Helicobacter muricola sp. nov., a novel Helicobacter species isolated from the ceca and feces of Korean wild mouse (Mus musculus molossinus)

A slowly growing microaerophilic Helicobacter strain was isolated from the ceca and fecal pellets of Korean wild mice (Mus musculus molossinus). This bacterial strain possessed a pair of nonsheathed bipolar flagella, was positive for urease, catalase and oxidase, and reduced nitrate to nitrite. It proved susceptible to nalidixic acid and resistant to cephalodine, and did not hydrolyze hippurate. On the basis of phenotypic characteristics and 16S rRNA gene sequence analysis, the isolate represents a new species of the genus Helicobacter, for which the name Helicobacter muricola sp. nov. is proposed; the type strain of the new species is w-06T.     

Identification of a novel microtubule-associated protein that regulates microtubule organization and cytokinesis by using a GFP-screening strategy

Microtubules play critical roles in a variety of cell processes, including mitosis, organelle transport, adhesion and migration, and the maintenance of cell polarity. Microtubule-associated proteins (MAPs) regulate the dynamic organization and stability of microtubules, often through either cell-specific or cell division stage-specific interactions. To identify novel cytoskeletal-associated proteins and peptides that regulate microtubules and other cytoskeletal and adhesive structures, we have developed a GFP cDNA screening strategy based on identifying gene products that localize to these structures. Using this approach, we have identified a novel MAP, GLFND, that shows homology to the Opitz syndrome gene product [6], localizes to a subpopulation of microtubules that are acetylated, and protects microtubules from depolymerization with nocodazole. Expression of an N-terminal deletion binds microtubules but alters their organization. During the cell cycle, GLFND dissociates from microtubules at the beginning of mitosis and then reassociates at cytokinesis. Furthermore, ectopic expression of GLFND inhibits cell division and cytokinesis in CHO cells. These observations make GLFND unique among MAPs characterized thus far.