Yhong-Hee Shim

PGL-1, a Predicted RNA-Binding Component of Germ Granules, Is Essential for Fertility in C. elegans

Germ cells are distinct from somatic cells in their immortality, totipotency, and ability to undergo meiosis. Candidates for components that guide the unique germline program are the distinctive granules observed in germ cells of many species. We show that a component of germ granules is essential for fertility in C. elegans and that its primary function is in germline proliferation. This role has been revealed by molecular and genetic analyses of pgl-1. PGL-1 is a predicted RNA-binding protein that is present on germ granules at all stages of development. Elimination of PGL-1 results in defective germ granules and sterility. Interestingly, PGL-1 function is required for fertility only at elevated temperatures, suggesting that germline development is inherently sensitive to temperature.

p16 Hypermethylation in the early stage of hepatitis B virus- associated hepatocarcinogenesis

Abnormality of the p16 expression is involved in the pathogenesis of hepatocellular carcinoma (HCC), and hypermethylation of p16 gene is known as a major p16 inactivation mechanism. Cirrhotic nodule (CN) is now regarded as a preneoplastic lesion that is frequently associated with microscopic foci of HCC through dysplastic nodules (DNs). This observation clearly supports a multistep hepatocarcinogenesis from CNs through DNs. We thus examined the methylation status of p16 gene in HCCs surrounded by DNs and CNs to define the significance of p16 hypermethylation in the early stage of hepatocarcinogenesis. We tested 24 hepatitis B virus (HBV)-associated CNs, 37 DNs, and 18 HCCs within DNs that were microdissected from paraffin-embedded tissue sections. Frequency of p16 hypermethylation was significantly high in HCCs within DNs (15/18. 83.3%) and it increased from CNs (15/24. 62.5%) through DNs (26/37, 70.3%). Interestingly, 11 out of 12 (91.7%) HCC associated with methylation-positive DNs revealed hypermethylation of p16, and 18 out of 23 (78.2%) DNs associated with methylation-positive CNs showed p16 hypermethylation. These data suggest that p16 hypermethylation in the early stages, CNs and DNs may predispose to HCC. In addition, p16 methylation status of five cell lines with or without HBV infection was examined to test whether the high frequency of hypermethylation is related to HBV infection. HBV-infected cell lines were exclusively methylation-positive. These data suggest that high frequency of hypermethylation may be associated with hepatitis B virus infection.

Bfl-1S, a novel alternative splice variant of Bfl-1, localizes in the nucleus via its C-terminus and prevents cell death.

Bfl-1 is an antiapoptotic Bcl-2 family member and a mouse A1 homologue. The mouse A1 has been reported to have three isoforms, but little is known about human Bfl-1. By reverse–transcriptase polymerase chain reaction analysis, we have identified Bfl-1S (short form), an alternative splice variant of Bfl-1. The Bfl-1S primary sequence contains four conserved Bcl-2 homology (BH) domains and a positive-charged C-terminus containing KKRK amino acids. The expression of Bfl-1S mRNA was detected predominantly in normal lymph nodes and in B-lymphoid leukemia cells. Confocal microscopic analysis using green fluorescence protein fusion proteins demonstrated that Bfl-1S is localized in the nucleus by its C-terminus as an intrinsic nuclear localization sequence. Bfl-1S acts as an antiapoptotic agent in coexpression experiments with Bax, a proapoptotic molecule. The expression of Bfl-1S provided significant resistance against staurosporine (STS) treatments in Molt-4 human T-leukemia cells. Bfl-1S also significantly inhibited the cleavage of Bid, and of caspases 3 and 8 against STS treatment. These results indicate that Bfl-1S is a novel human Bcl-2 family member that possesses antiapoptotic function.

cdc-25.2, a C. elegans ortholog of cdc25, is required to promote oocyte maturation

Cdc25 is an evolutionarily conserved protein phosphatase that promotes progression through the cell cycle. Some metazoans have multiple isoforms of Cdc25, which have distinct functions and different expression patterns during development. C. elegans has four cdc-25 genes. cdc-25.1 is required for germline mitotic proliferation. To determine if the other members of the cdc-25 family also contribute to regulation of cell division in the germ line, we examined phenotypes of loss-of-function mutants of the other cdc-25 family genes. We found that cdc-25.2 is also essential for germline development. cdc-25.2 homozygous mutant hermaphrodites exhibited sterility as a result of defects in oogenesis: mutant oocytes were arrested as endomitotic oocytes that were not fertilized successfully. Spermatogenesis and male germline development were not affected. Through genetic interaction studies, we found that CDC-25.2 functions upstream of maturation-promoting factor containing CDK-1 and CYB-3 to promote oocyte maturation by counteracting function of WEE-1.3. We propose that cdc-25 family members function as distinct but related cell cycle regulators to control diverse cell cycles in C. elegans germline development.

Caenorhabditis elegans proteomics comes of age

Caenorhabditis elegans, a free‐living soil nematode, is an ideal model system for studying various physiological problems relevant to human diseases. Despite its short history, C. elegans proteomics is receiving great attention in multiple research areas, including the genome annotation, major signaling pathways (e.g. TGF‐β and insulin/IGF‐1 signaling), verification of RNA interference‐mediated gene targeting, aging, disease models, as well as peptidomic analysis of neuropeptides involved in behavior and locomotion. For example, a proteome‐wide profiling of developmental and aging processes not only provides basic information necessary for constructing a molecular network, but also identifies important target proteins for chemical modulation. Although C. elegans has a simple body system and neural circuitry, it exhibits very complicated functions ranging from feeding to locomotion. Investigation of these functions through proteomic analysis of various C. elegans neuropeptides, some of which are not found in the predicted genome sequence, would open a new field of peptidomics. Given the importance of nematode infection in plants and mammalian pathogenesis pathways, proteomics could be applied to investigate the molecular mechanisms underlying plant– or animal–nematode pathogenesis and to identify novel antinematodal drugs. Thus, C. elegans proteomics, in combination of other molecular, biological and genetic techniques, would provide a versatile new tool box for the systematic analysis of gene functions throughout the entire life cycle of this nematode.

Proteomic Analysis of Caenorhabditis elegans

Proteomic studies of the free-living nematode Caenorhabditis elegans have recently received great attention because this animal model is a useful platform for the in vivo study of various biological problems relevant to human disease. In general, proteomic analysis is carried out in order to address a specific question with respect to differential changes in proteome expression under certain perturbed conditions. In this chapter, we focus on gel-based proteomic analysis of C. elegans subjected to two specific stress conditions during development: induction of the dauer state for whole body protein expression and a temperature shift for egg protein expression. Utilizing these differently perturbed C. elegans protein samples, two-dimensional electrophoresis and differential in-gel electrophoresis methods have led to the discovery of remarkable aspects of the worms biology. We also provide numerous details about the technical points and protocols necessary for successful experimentation.

Regulation of sperm-specific proteins by IFE-1, a germline-specific homolog of eIF4E, in C. elegans

AbsteactIFE-1 is one of the five C. elegans homologs of eIF4E, which is the mRNA 5′ cap-binding component of the translation initiation complex eIF4F. Depletion of IFE-1 causes defects in sperm, suggesting that IFE-1 regulates a subset of genes required for sperm functions. To further understand the molecular function of IFE-1, proteomic analysis was performed to search for sperm proteins that are downregulated in ife-1(ok1978); fem-3(q20) mutants relative to the fem-3(q20) control. The fem-3(q20) mutant background was used because it only produces sperm at restrictive temperature. Total worm proteins were subjected to 2D-DIGE, and differentially expressed protein spots were further identified by MALDI-TOF mass spectrometry. Among the identified proteins, GSP-3 and Major Sperm Proteins (MSPs) were found to be significantly down-regulated in the ife-1(ok1978) mutant. Moreover, RNAi of gsp-3 caused an ife-1-like phenotype. These results suggest that IFE-1 is required for efficient expression of some sperm-specific proteins, and the fertilization defect of ife-1 mutant is caused mainly by a reduced level of GSP-3.

A mutation of cdc-25.1 causes defects in germ cells but not in somatic tissues in C. elegans

By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.

C. elegans: an invaluable model organism for the proteomics studies of the cholesterol-mediated signaling pathway.

With the availability of its complete genome sequence and unique biological features relevant to human disease, Caenorhabditis elegans has become an invaluable model organism for the studies of proteomics, leading to the elucidation of nematode gene function. A journey from the genome to proteome of C. elegans may begin with preparation of expressed proteins, which enables a large-scale analysis of all possible proteins expressed under specific physiological conditions. Although various techniques have been used for proteomic analysis of C. elegans, systematic high-throughput analysis is still to come in order to accommodate studies of post-translational modification and quantitative analysis. Given that no integrated C. elegans protein expression database is available, it is about time that a global C. elegans proteome project is launched through which datasets of transcriptomes, protein–protein interaction and functional annotation can be integrated. As an initial target of a pilot project of the C. elegans proteome project, the cholesterol-mediated signaling pathway will be an excellent example since, like in other organisms, it is one of the key controlling pathways in cell growth and development in C. elegans. As this field tends to broaden to functional proteomics, there is a high demand to develop the versatile proteome informatics tools that can mange many different data in an integrative manner.

Apigenin inhibits larval growth of Caenorhabditis elegans through DAF-16 activation.

Treatment of Caenorhabditis elegans with apigenin, 5,7,4′‐trihydroxyflavone, induces larval growth inhibition. To understand the molecular basis of apigenin‐induced larval growth inhibition, the effects of apigenin on DAF‐16 activity were examined. DAF‐16 was activated through nuclear translocation and the mRNA level of sod‐3, one of the known DAF‐16 target genes, was increased upon apigenin treatment. DAF‐16 activity was required for the growth inhibition, since the larval growth retardation upon apigenin treatment was suppressed in daf‐16 mutants. These results indicate that apigenin acts as a stressor that activates DAF‐16, which in turn inhibits larval growth.