Potential Gene Products Research Articles

Nfat5 is involved in the hyperosmotic regulation of Tmem184b: a putative modulator of ibuprofen transport in renal MDCK I cells.

Nuclear factor of activated T cells 5 (NFAT5) is a transcription factor involved in the regulation of several genes involved in the response to extracellular hyperosmolality. Recently, the uptake of ibuprofen by an as yet unknown carrier was suggested in Madin‐Darby canine kidney (MDCK) I cells exposed to hyperosmolality. We therefore speculated that Nfat5 could be involved in the regulation of this ibuprofen carrier. Reverse transfection with siRNA against Nfat5 was used to knock down Nfat5 in MDCK I cells. The uptake of both radiolabelled taurine and ibuprofen was measured in MDCK I cells, first treated with siRNA against Nfat5 and afterwards cultivated with raffinose‐supplemented normal growth medium (500 mOsm) for 24 h. The siRNA transfection resulted in knockdown of Nfat5, and uptake of both taurine and ibuprofen was significantly decreased in transfected MDCK I cells. The decrease in ibuprofen uptake indicates that Nfat5 is involved in upregulation of the ibuprofen carrier. A transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 revealed 989 genes upregulated by Nfat5 during hyperosmotic exposure. From these genes, the gene product transmembrane protein 184b was found to be regulated by Nfat5, and Tmem184b was the only potential gene product involved in the uptake of ibuprofen in MDCK I cells.DatasetThe RNA sequencing dataset is available from the NCBI Gene Expression 452 Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE122074.

Acidianus Tailed Spindle Virus: a New Archaeal Large Tailed Spindle Virus Discovered by Culture-Independent Methods

The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria, and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content but rarely provide knowledge about virion morphology and/or cellular host identity. Here we describe a new virus, Acidianus tailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic data sets from a high-temperature (80°C) acidic (pH 2) hot spring located in Yellowstone National Park, followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included the identification of the large tailed spindle virion morphology, determination of the complete 70.8-kb circular double-stranded DNA (dsDNA) viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three-domain gene product containing an N-terminal leucine-rich repeat domain, followed by a likely posttranslation regulatory region consisting of high serine and threonine content, and a C-terminal ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related to Acidianus hospitalis, was determined by a combination of analysis of cellular clustered regularly interspaced short palindromic repeat (CRISPR)/Cas loci and dual viral and cellular fluorescence in situ hybridization (viral FISH) analysis of environmental samples and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches and provides a platform for predicting and confirming virus hosts. Virus discovery and characterization have been traditionally accomplished by using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In this research, we report a virus-centered approach to virus discovery and characterization, linking viral metagenomic sequences to a virus particle, its sequenced genome, and its host directly in environmental samples, without using culture-dependent methods. This approach provides a pathway for the discovery, isolation, and characterization of new viruses. While this study used an acidic hot spring environment to characterize a new archaeal virus, Acidianus tailed spindle virus (ATSV), the approach can be generally applied to any environment to expand knowledge of virus diversity in all three domains of life.

Galaxy Integrated Omics: Web-based Standards-Compliant Workflows for Proteomics Informed by Transcriptomics*

With the recent advent of RNA-seq technology the proteomics community has begun to generate sample-specific protein databases for peptide and protein identification, an approach we call proteomics informed by transcriptomics (PIT). This approach has gained a lot of interest, particularly among researchers who work with nonmodel organisms or with particularly dynamic proteomes such as those observed in developmental biology and host-pathogen studies. PIT has been shown to improve coverage of known proteins, and to reveal potential novel gene products. However, many groups are impeded in their use of PIT by the complexity of the required data analysis. Necessarily, this analysis requires complex integration of a number of different software tools from at least two different communities, and because PIT has a range of biological applications a single software pipeline is not suitable for all use cases. To overcome these problems, we have created GIO, a software system that uses the well-established Galaxy platform to make PIT analysis available to the typical bench scientist via a simple web interface. Within GIO we provide workflows for four common use cases: a standard search against a reference proteome; PIT protein identification without a reference genome; PIT protein identification using a genome guide; and PIT genome annotation. These workflows comprise individual tools that can be reconfigured and rearranged within the web interface to create new workflows to support additional use cases.

Phenol Soluble Modulin (PSM) Variants of Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) Captured Using Mass Spectrometry-Based Molecular Networking

Molecular genetic analysis indicates that the problematic human bacterial pathogen methicillin-resistant Staphylococcus aureus possesses more than 2000 open reading frames in its genome. This number of potential gene products, coupled with intrinsic mechanisms of posttranslational modification, endows methicillin-resistant Staphylococcus aureus with a highly complex biochemical repertoire. Recent proteomic and metabolomic advances have provided methodologies to better understand and characterize the biosynthetic factors released by microbial organisms. Here, the emerging tool of mass spectrometry-based molecular networking was used to visualize and map the repertoire of biosynthetic factors produced by a community-associated methicillin-resistant Staphylococcus aureus strain representative of the epidemic USA300 clone. In particular, the study focused on elucidating the complexity of the recently discovered phenol soluble modulin family of peptides when placed under various antibiotic treatment stresses. Novel PSM truncated variant peptides were captured, and the type of variants that were clustered by the molecular networks platform changed in response to the different antibiotic treatment conditions. After discovery, a group of the peptides were selected for functional analysis in vitro. The peptides displayed bioactive properties including the ability to induce proinflammatory responses in human THP-1 monocytes. Additionally, the tested peptides did not display antimicrobial activity as previously reported for other phenol soluble modulin truncated variants. Our findings reveal that the PSM family of peptides are quite structurally diverse, and suggest a single phenol soluble modulin parent peptide can functionally spawn differential bioactivities in response to various external stimuli.

Abstract 2221: DNA methylation of microRNA genes in gastric carcinoma and its clinicopathological association

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric carcinoma (GC). DNA methylation of multiple CpG sites in promoter region of several tumor-related genes, such as hMLH1, p14, p16, and APC has been analyzed in GC so far. MicroRNAs (miRNA) are small, non-coding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies indicate that epigenetic silencing of miRNAs with tumor suppressor activity by CpG island methylation plays an important role in gastric carcinogenesis. To explore the role of epigenetic mechanisms in the down-regulation of miRNA genes (has-miR-9, has-miR-129, has-miR-137), we examined the presence of DNA methylation-associated silencing of miRNAs in GC and observed that aberrant methylation of these miRNAs is associated with expression of target gene products. Materials and Methods: The extent of promoter methylation of 4 miRNA genes (has-miR-9-1, has-miR-9-3, has-miR-129-2, has-miR-137) was assessed using methylation-specific polymerase chain reaction (MSP) in 100 GC tissues (50 early gastric cancers and 50 advanced gastric cancers) and corresponding non-tumor tissues. Twenty patients with benign gastric pathology were also included as a control group. The potential target gene products of miRNAs were studied by immunohistochemistry and the relationship between methylation profile of miRNAs promoter and clinicopathological parameters was analyzed. Results: Methylation of the has-miR-9-3 and has-miR-137 CpG island was frequently observed in tumor tissues (89% and 86%, respectively) and non-tumor tissues in 100 GC patients (70% and 78%), but not in normal gastric tissues (55% and 55%). Methylation of the has-miR-9-1 is slightly higher in tumor tissues (65%) than normal gastric tissue (45%). However, methylation level of the has-miR-129-2 was not shown significant difference in tumor tissues (97%) compared with non-tumor tissues (90%) and normal gastric tissues (90%). Expression of NF-κB and SOX4 protein, which are has-miR-9 and has-miR-129-2 potential target respectively, were inversely correlated with methylation level of miRNAs. The presence of H. pylori infection was significantly associated with aberrant methylation of miRNA genes (p<0.05). But, there was no significant correlation between aberrant methylation of miRNAs and other clinicopathological factors. Conclusion: These results suggest that specific miRNAs methylation in GC could be an important molecular mechanism causing loss of control on its target, and it may be correlated with the high transcriptional activity of target gene. Epigenetic silencing of some miRNAs may involve in the early stage of gastric carcinogenesis and could be used as an efficient diagnostic biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2011-2221

Genome organization of epidemic Acinetobacter baumannii strains

BackgroundAcinetobacter baumannii is an opportunistic pathogen responsible for hospital-acquired infections. A. baumannii epidemics described world-wide were caused by few genotypic clusters of strains. The occurrence of epidemics caused by multi-drug resistant strains assigned to novel genotypes have been reported over the last few years.ResultsIn the present study, we compared whole genome sequences of three A. baumannii strains assigned to genotypes ST2, ST25 and ST78, representative of the most frequent genotypes responsible for epidemics in several Mediterranean hospitals, and four complete genome sequences of A. baumannii strains assigned to genotypes ST1, ST2 and ST77. Comparative genome analysis showed extensive synteny and identified 3068 coding regions which are conserved, at the same chromosomal position, in all A. baumannii genomes. Genome alignments also identified 63 DNA regions, ranging in size from 4 o 126 kb, all defined as genomic islands, which were present in some genomes, but were either missing or replaced by non-homologous DNA sequences in others. Some islands are involved in resistance to drugs and metals, others carry genes encoding surface proteins or enzymes involved in specific metabolic pathways, and others correspond to prophage-like elements. Accessory DNA regions encode 12 to 19% of the potential gene products of the analyzed strains. The analysis of a collection of epidemic A. baumannii strains showed that some islands were restricted to specific genotypes.ConclusionThe definition of the genome components of A. baumannii provides a scaffold to rapidly evaluate the genomic organization of novel clinical A. baumannii isolates. Changes in island profiling will be useful in genomic epidemiology of A. baumannii population.

Cytoplasmic- and extracellular-proteome analysis of Diplodia seriata: a phytopathogenic fungus involved in grapevine decline.

BackgroundThe phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome.ResultsIntracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism.ConclusionsThis study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity process. In fact, several of the identified proteins have been reported as pathogenicity factors in other phytopathogenic fungi. Moreover, this proteomic analysis supposes a useful basis for deepening into D. seriata knowledge and will contribute to the development of the molecular biology of this fungal strain as it has been demonstrated by cloning the gene Prx1 encoding mitochondrial peroxiredoxin-1 of D. seriata (the first gene to be cloned in this microorganism; data not shown).

Complete genome sequence of a Megalocytivirus (family Iridoviridae) associated with turbot mortality in China

BackgroundTurbot reddish body iridovirus (TRBIV) causes serious systemic diseases with high mortality in the cultured turbot, Scophthalmus maximus. We here sequenced and analyzed the complete genome of TRBIV, which was identified in Shandong province, China.ResultsThe genome of TRBIV is a linear double-stranded DNA of 110,104 base pairs, comprising 55% G + C. Total 115 open reading frames were identified, encoding polypeptides ranging from 40 to 1168 amino acids. Amino acid sequences analysis revealed that 39 of the 115 potential gene products of TRBIV show significant homology to other iridovirus proteins. Phylogenetic analysis of conserved genes indicated that TRBIV is closely related to infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), orange-spotted grouper iridovirus (OSGIV), and large yellow croaker iridovirus (LYCIV). The results indicated that TRBIV belongs to the genus Megalocytivirus (family Iridoviridae).ConclusionsThe determination of the genome of TRBIV will provide useful information for comparative study of Megalocytivirus and developing strategies to control outbreaks of TRBIV-induced disease.

Determination of Nucleotide Sequences of Mitochondrial Plasmid pGT704 of Paramecium caudatum

Many kinds of linear plasmids, or plasmid-like DNAs, in mitochondria have been reported in higher plants and fungi. Nevertheless, such plasmids were not previously known from the animal kingdom. Paramecium is the first example in which a mitochondrial plasmid-like DNA so far discovered among ciliates. In natural stocks, P. caudatum has one of three types of DNA : type I (6.4 kb), type II, a set of four DNAs (8.2 kb; 4.1 kb; 2.8kb; and 1.4 kb) and type III (8.6 kb). Paramecium caudatum stock GT704 only has type-II DNA. The 8.2 and 2.8 kb DNAs are dimers of the 4.1 and 1.4 kb DNA, respectively. The aim of this study was to determine the sequence of mitochondrial plasmid-like DNA in P. caudatum stock GT704 in order to identify potential gene products and other nucleotide sequences that might have relationship with integration of DNA and also the maintanance of DNA itself in mitochondria. The methods used in this study were isolation of the DNA, cloning and sequencing then followed by analysis of sequences obtained using available public domain. The dimers were found to be easily dissociated to monomers when treated with heat underconditions of low ionic strength. These results suggest that each monomer has a sticky end of single- stranded DNA that has complementary nucleotide sequences, and so attaches to each other at the end by base-pairing to form a dimer, leaving a nick that can be filled by ligation using ligase. Nucleotide sequences of some part of the DNA have been obtained randomly. Sequence analysis suggests that these dimers DNAs have 2 ORFs that code for putative RNA and DNA polymerases. It is assumed that the origin of this mitochondrial plasmid-like DNA is transposable element or some viruses since the motif EVI-1 (ectopic viral integration site-1) was found in the sequence. It is also assumed that this DNA can integrate into mtDNA since it also has some sequences homolog to the sequences of mt- DNA of some ciliates.

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

BackgroundPenicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei.ResultsWhole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous Aspergillus proteins.ConclusionThis study clearly demonstrates the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in P. marneffei.

Hydrogen peroxide production by Lactobacillus johnsonii NCC 533 and its role in anti-Salmonella activity

The human intestinal isolate Lactobacillus johnsonii NCC 533 (La1) is a probiotic strain with well-documented antimicrobial properties. Previous research has identified the production of lactic acid and bacteriocins as important factors, but that other unidentified factors are also involved. We used the recently published genome sequence of L. johnsonii NCC 533 to search for novel antipathogen factors and identified three potential gene products that may catalyze the synthesis of the known antimicrobial factor hydrogen peroxide, H(2)O(2). In this work, we confirmed the ability of NCC 533 as well as eight different L. johnsonii strains and Lactobacillus gasseri to produce H(2)O(2) when resting cells were incubated in the presence of oxygen, and that culture supernatant containing NCC 533-produced H(2)O(2) was effective in killing the model pathogen Salmonella enterica serovar Typhimurium SL1344 in vitro.

Synaptic arrangement of the neuroligin/beta-neurexin complex revealed by X-ray and neutron scattering.

Neuroligins are postsynaptic cell-adhesion proteins that associate with their presynaptic partners, the neurexins. Using small-angle X-ray scattering, we determined the shapes of the extracellular region of several neuroligin isoforms in solution. We conclude that the neuroligins dimerize via the characteristic four-helix bundle observed in cholinesterases, and that the connecting sequence between the globular lobes of the dimer and the cell membrane is elongated, projecting away from the dimer interface. X-ray scattering and neutron contrast variation data show that two neurexin monomers, separated by 107 A, bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer. Using these data, we developed structural models that delineate the spatial arrangements of different neuroligin domains and their partnering molecules. As mutations of neurexin and neuroligin genes appear to be linked to autism, these models provide a structural framework for understanding altered recognition by these proteins in neurodevelopmental disorders.

The implications of alternative splicing in the ENCODE protein complement.

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.

Experimental Confirmation of Global Murine Cytomegalovirus Open Reading Frames by Transcriptional Detection and Partial Characterization of Newly Described Gene Products

Murine cytomegalovirus (MCMV) and human CMV (HCMV) share many features making the mouse system a potential small-animal model for HCMV. Although the genomic DNA sequence and the predicted open reading frames (ORFs) of MCMV have been determined, experimental evidence that the ORFs are actually transcribed has been lacking. We developed an MCMV global-DNA microarray that includes all previously predicted ORFs and 14 potential ones. A total of 172 ORFs were confirmed to be transcribed, including 7 newly discovered ORFs not previously predicted. No gene products from 10 previously predicted ORFs were detected by either DNA microarray analysis or reverse transcriptase PCR in MCMV-infected mouse fibroblasts, although 2 of those were expressed in a macrophage cell line, suggesting that potential gene products from these open reading frames are silenced in fibroblasts and required in macrophages. Immunohistochemical localization of the six newly described ORF products and three recently identified ones in cells transfected with the respective construct revealed four of the products in the nucleus and five in mitochondria. Analysis of two ORFs using site-directed mutagenesis showed that deletion of one of the mitochondrion-localized gene products led to significantly decreased replication in fibroblasts.

Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Identification of carboxypeptidase E and γ-glutamyl hydrolase as biomarkers for pulmonary neuroendocrine tumors by cDNA microarray

Pulmonary neuroendocrine tumors vary dramatically in their malignant behavior. Their classification, based on histological examination, is often difficult. In search of molecular and prognostic markers for these tumors, we used cDNA microarray analysis of human transcripts against reference RNA from a well-characterized immortalized bronchial epithelial cell line, BEAS-2B. Tumor cells were isolated by laser-capture microdissection from primary tumors of 17 typical carcinoids, small cell lung cancers, and large cell neuroendocrine carcinomas. An unsupervised, hierarchical clustering algorithm resulted in a precise classification of each tumor subtype according to the proposed histological classification. Selection of genes, using supervised analysis, resulted in the identification of 198 statistically significant genes ( P <.004) that also accurately discriminated between 3 predefined tumor subtypes. Two-by-two comparisons of these genes identified classifier genes that distinguished each tumor subtype from the others. Changes in expression of selected differentially expressed genes for each tumor subtype were internally validated by real-time reverse-transcription polymerase chain reaction. Expression of 2 potential classifier gene products, carboxypeptidase E (CPE) and γ-glutamyl hydrolase (GGH), was validated by immunohistochemistry and cross-validated on additional archival samples of pulmonary neuroendocrine tumors. Kaplan-Meier survival analysis revealed that immunostaining for CPE was a statistically significant predictor of good prognosis, whereas GGH expression correlated with poor prognosis. Thus, cDNA microarray analysis led to the identification of 2 novel biomarkers that should facilitate molecular diagnosis and further study of pulmonary neuroendocrine tumors.

Role of TRP channels in endothelial pathophysiology—evidence for vascular TRPs as a potential target for drug therapy

Proteins related to the Drosophila transient receptor potential (TRP) gene product are key players in Ca 2+ homeostasis of mammalian cells. TRP proteins form cation channels, which function as sensors for a variety of stimuli including oxidative stress. The canonical TRP proteins TRPC3 and TRPC4 are likely to play a role in endothelial physiology and pathophysiology. Here, we report that overexpression of TRPC3 or TRPC4 generates a redox-sensitive cation conductance in human embryonic kidney cells (HEK293). Redox activation of these TRPC species appears to be due to oxidant-induced promotion of phospholipase C activity and/or sensitisation of TRPC proteins to regulation by phospholipase C. Our results suggest TRPC species as attractive, novel targets for cardiovascular therapy.

Reduced expression of cell cycle regulator p18(INK4C) in human hepatocellular carcinoma.

Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18INK4C, a member of the INK4 family of CdkIs, is a potential tumor-suppressor gene product. However, the expression of p18INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18INK4C- positivity. Further, the kinase activity of Cdk4 was found to be higher in p18INK4C-negative HCCs than in p18INK4C- positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18INK4C- positive HCCs, p18INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18INK4C - negative than in p18INK4C - positive HCCs. In conclusion, the loss of p18INK4C expression may play a role in the differentiation and development of HCC through the up-regulation of Cdk4 activity.

Proteins with Class α/β Fold Have High-level Participation in Fusion Events

Now that complete genome sequences are available for a variety of organisms, the elucidation of potential gene products function is a central goal in the post-genome era. Domain fusion analysis has been proposed recently to infer the functional association of the component proteins. Here, we took a new approach to the analysis of the structural features of the proteins involved in fusion events. An exhaustive survey of fusion events within 30 completely sequenced genomes and subsequent structure annotations to the component proteins at a SCOP superfamily level with hidden Markov models was carried out. A domain fusion map was then constructed. The results revealed that proteins with the class α/β fold are frequently involved in fusion events, around 86% of the total 676 assigned single-domain fusion pairs including at least one component protein belonging to the α/β fold class. Moreover, the domain fusion map in our work may offer an attractive framework for designing chimeric enzymes following Nature's lead, and may give useful hints for exploring the evolutionary history of proteins.

Ibogaine signals addiction genes and methamphetamine alteration of long-term potentiation.

The mapping of the human genetic code will enable us to identify potential gene products involved in human addictions and diseases that have hereditary components. Thus, large-scale, parallel gene-expression studies, made possible by advances in microarray technologies, have shown insights into the connection between specific genes, or sets of genes, and human diseases. The compulsive use of addictive substances despite adverse consequences continues to affect society, and the science underlying these addictions in general is intensively studied. Pharmacological treatment of drug and alcohol addiction has largely been disappointing, and new therapeutic targets and hypotheses are needed. As the usefulness of the pharmacotherapy of addiction has been limited, an emerging potential, yet controversial, therapeutic agent is the natural alkaloid ibogaine. We have continued to investigate programs of gene expression and the putative signaling molecules used by psychostimulants such as amphetamine in in vivo and in vitro models. Our work and that of others reveal that complex but defined signal transduction pathways are associated with psychostimulant administration and that there is broad-spectrum regulation of these signals by ibogaine. We report that the actions of methamphetamine were similar to those of cocaine, including the propensity to alter long-term potentiation (LTP) in the hippocampus of the rat brain. This action suggests that there may be a "threshold" beyond which the excessive brain stimulation that probably occurs with compulsive psychostimulant use results in the occlusion of LTP. The influence of ibogaine on immediate early genes (IEGs) and other candidate genes possibly regulated by psychostimulants and other abused substances requires further evaluation in compulsive use, reward, relapse, tolerance, craving and withdrawal reactions. It is therefore tempting to suggest that ibogaine signals addiction gene products.