ProteoMix: an integrated and flexible system for interactively analyzing large numbers of protein sequences

ProteoMix is a suite of JAVA programs for identifying, annotating and predicting regions of interest in large sets of amino acid sequences, according to systematic and consistent criteria. It is based on two concepts (1) the integration of results from different sequence analysis tools increases the prediction reliability; and (2) the integration protocol is critical and needs to be easily adaptable in a case-by-case manner. ProteoMix was designed to analyze simultaneously multiple protein sequences using several bioinformatics tools, merge the results of the analyses using logical functions and display them on an integrated viewer. In addition, new sequences can be added seamlessly to an analysis performed on an initial set of sequences. ProteoMix has a modular design, and bioinformatics tools are run on remote servers accessed using the Internet Simple Object Access Protocol (SOAP), ensuring the swift implementation of additional tools. ProteoMix has a user-friendly interactive graphical user interface environment and runs on PCs with Microsoft OS. AVAILABILITY: ProteoMix is freely available for academic users at http://bio.gsc.riken.jp/ProteoMix/     

Determination of trivalent methylated arsenicals in rat urine by liquid chromatography-inductively coupled plasma mass spectrometry after solvent extraction

A method for the determination of trivalent arsenicals in urine was examined. Trivalent arsenicals, extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, were determined by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The trivalent methylated arsenicals monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and trimethylarsine (TMA) were detected in urine of rats that had received dimethylarsinic acid (DMA(V)) or monomethylarsonic acid (MMA(V)) at concentration of 200 microg ml(-1) in drinking water for 24 weeks. This method is the first to permit quantification of trivalent methylated arsenicals in urine without significant changes in concentration during storage or pretreatment.     

A polyclonal antibody directed against syringylpropane epitopes of native lignins

With a view to visualizing the ultrastructural distribution of syringyl lignins in secondary plant cell walls, a polyclonal antibody raised from a synthetic DHP polymer consisting only of syringyl propane units was prepared. To test the reactivity of the antiserum, a mini-dot-blot immunoassay reducing the amounts of substrates and antiserum was developed. A characteristic attribute of the S-antiserum appears to be its specific recognition of sequences of three or more consecutive syringyl units. On ultra-thin sections of model plants of Arabidopsis thaliana, Populus and tobacco, the antiserum allowed us to demonstrate a higher concentration of syringyl epitopes in fibres than in vessels. Variations in the distribution pattern of these epitopes between the three plants examined suggest that the synthesis of syringyl lignins in angiosperms depends on the species.     

Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium,Microlunatus phosphovorus

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.     

Integration of cytochrome b5 into endoplasmic reticulum membrane: participation of carboxy-terminal portion of the transmembrane domain

Integration of cytochrome b(5) (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.     

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.     

Regulation of galactoside transport by the PTS

Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed. It is now well known that non-phosphorylated IIA(Glc) inhibits the transport via direct binding to the transporter. Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIA(Glc). It is concluded that there are multiple interaction sites for IIA(Glc) in these transporters.     

Molecular cloning, characterization, and expression of wheat cystatins

We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47-68% amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63-67% similalities to one another while 93% of amino acids were identical between WC1 and WC3. This suggested that WCI, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.     

Axial protocadherin is a mediator of prenotochord cell sorting in Xenopus

Prenotochord cell sorting is regarded as one of the first cell sorting events in early chordate development. We recently demonstrated that this sorting event occurs in vitro, although the mediator of this activity remains unidentified. Herein, we report the isolation of a full-length cDNA clone of Axial protocadherin (AXPC), the homologue of human protocadherin-1 (PCD1). AXPC encodes a transmembrane protein (AXPC) that is expressed exclusively in the notochord at the neurula stage and in the pronephros, somites, heart, optic vesicle, otic vesicle, and distinct parts of the brain at the tailbud stage. Cell dissociation and reaggregation assays and in vivo microinjection experiments demonstrated that cells overexpressing a membrane-tethered form of AXPC (MT-AXPC) acquired the same adhesive properties as prenotochord cells. Moreover, microinjection of either mRNA encoding the dominant negative form of AXPC (DN-AXPC) or morpholino oligonucleotides interferes with the sorting activity of prenotochord cells and normal axis formation. This study suggests that AXPC is necessary and sufficient for prenotochord cell sorting in the gastrulating embryo, and may also mediate sorting events later in development.