Prediction of location of active sites in biologically active peptides

In a previous paper we demonstrated that the short-range compact regions in atrial natriuretic factor (-hANF) predicted by the average distance map (ADM) correspond to its active sites [Kikuchi,J. Protein Chem.11, 579–581 (1992)]. In the present paper we apply the same method to other bioactive peptides and peptidic enzyme inhibitors. We again observe that active sites in each peptide are contained in short-range compact regions predicted by the ADM for the peptide. This demonstrates that the ADM method predicts the possible location of active sites in biologically active peptides in general. The possibility of practical application of the present method to rational drug design is also discussed.     

Discrimination of folding types of globular proteins based on average distance maps constructed from their sequences

It has been shown that probable portions which form contacts in a protein can be predicted by means of an average distance map (ADM) as well as regular structures (-helices and -turns) defined as short-range compact regions (Kikuchiet al., 1988a,c). In this paper, we analyze the occurrence of those portions and short-range compact regions on ADMs for various proteins regarding their folding types. We have found out that each folding type of proteins shows characteristic distribution of such parts on ADMS. We also discuss the possibility of the prediction of folding types of proteins by ADMs.     

Neurotensin receptors: Binding properties,transduction pathways,and structure

Summary Neurotensin is a 13-amino acid peptide (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) originally isolated from hypothalami (Carraway and Leeman, 1973) and later from intestines (Kitabgiet al., 1976) of bovine. The peptide is present throughout the animal kingdom, suggesting its participation to important processes basic to animal life (Carrawayet al., 1982). Neurotensin and its analogue neuromedin-N (Lys-Ile-Pro-Tyr-Ile-Leu) (Minaminoet al., 1984) are synthesized by a common precursor in mammalian brain (Kislauskiset al., 1988) and intestine (Dobneret al., 1987). The central and peripheral distribution and effects of neurotensin have been extensively studied. In the brain, neurotensin is exclusively found in nerve cells, fibers, and terminals (Uhlet al., 1979), whereas the majority of peripheral neurotensin is found in the endocrine N-cells located in the intestinal mucosa (Orciet al., 1976; Helmstaedteret al., 1977). Central or peripheral injections of neurotensin produce completely different pharmacological effects (Table I) indicating that the peptide does not cross the blood-brain barrier. Many of the effects of centrally administered neurotensin are similar to those of neuroleptics or can be antagonized by simultaneous administration of TRH (Table I). The recently discovered nonpeptide antagonist SR 48692 (Gullyet al., 1993) can inhibit several of the central and peripheral effects of neurotensin (Table I).Like many other neuropeptides, neurotensin is a messenger of intracellular communication working as a neurotransmitter or neuromodulator in the brain (Nemeroffet al., 1982) and as a local hormone in the periphery (Hirsch Fernstromet al., 1980). Thus, several pharmacological, morphological, and neurochemical data suggest that one of the functions of neurotensin in the brain is to regulate dopamine neurotransmission along the nigrostriatal and mesolimbic pathways (Quirion, 1983; Kitabgi, 1989). On the other hand, the likely role of neurotensin as a parahormone in the gastrointestinal tract has been well documented (Rosell and Rökaeus, 1981; Kitabgi, 1982).Both central and peripheral modes of action of neurotensin imply as a first step the recognition of the peptide by a specific receptor located on the plasma membrane of the target cell. Formation of the neurotensin-receptor complex is then translated inside the cell by a change in the activity of an intracellular enzyme. This paper describes the binding and structural properties of neurotensin receptors as well as the signal transduction pathways that are activated by the peptide in various target tissues and cells.     

Construction and use of two α-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype α-hANP biosensor

Two α-human atrial natriuretic peptide (α-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of α-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate α-hANP-specific antibodies from a goat anti-α-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an α-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological α-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This α-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.     

Prediction of the Tertiary Structure of the Complement Control Protein Module

Complement control protein (CCP) modules, or short consensus repeats (SCR), exist in a wide variety of complement and adhesion proteins, principally the selectins. We have predicted the three-dimensional structure of a CCP module based upon secondary structural information derived by two-dimensional NMR [Barlow et al. (1991), Biochemistry 30, 997–1004]. Accordingly, the CCP is predicted to contain seven -strands with extensive hydrogen-bonding interactions, and shows a compact, globular structure. Comparison of this model to the X-ray structure of a kringle domain suggests that the CCP unit is more compact than a kringle structure, and that despite their similarities in size and disulfide bond format, the two are not homologous. Although the function of CCP domains is unknown, it is hoped that the structural model presented herein will facilitate further inquiry into how they contribute to so many systems of biological importance.     

Mass Spectral Identification of Vc1.1 and Differential Distribution of Conopeptides in the Venom Duct of Conus victoriae. Effect of Post-Translational Modifications and Disulfide Isomerisation on Bioactivity

Molluscs of the genus Conus (cone shells) are carnivorous, feeding on marine worms, small fish and other marine molluscs. They capture their prey by injecting venom containing hundreds of neurally active peptide components. These peptides are classed as conotoxins and consist of small disulfide-bonded peptides exhibiting a high degree of post-translational modifications (PTMs). The functional roles of these modifications remain largely unknown. Two of the most frequently observed modifications are γ-carboxylation of glutamate and hydroxylation of proline (Buczek et al. Cell Mol Life Sci 62:3067, 2005). Vc1.1 is an α-conotoxin from Conus victoriae (Sandall et al. Biochemistry 42(22):6904–6911, 2003) and the only form of this peptide which has been detected in the venom is the γ-glutamate and hydroxyproline (Vc1.1.P6O:E14Gla) version of the molecule (Jakubowski et al. Toxicon 47(6):688–699, 2006). In order to investigate the role of PTMs, we did mass spectral profiling of the venom duct of C. victoriae looking at changes in mass and the number of peptides detected. We synthesised a number of predicted Vc1.1-PTM peptides together with the three possible disulfide isoforms of Vc1.1 and assessed the possible functional role of the PTM conopeptides by measuring the in vitro activity at the cognate neuronal nicotinic acetylcholine receptors (nAChRs). In addition we looked for their presence Vc1.1 venom by mass spectrometry and by this approach we were able to detect unmodified Vc1.1 in C. victoriae venom for the first time.     

Location and nucleotide sequence of a tobacco chlorophlast DNA segment capable of replication in yeast

Summary A 1.7 Kbp EcoRI fragment of Nicotiana tabacum chloroplast DNA cloned in YIp5, consisting of pBR322 and the yeast ura3 gene, possessed ars (autonomously replicating sequences) activity in Saccharomyces cerevisiae. This fragment was located in the small single copy region proximal to the 23S rRNA gene.Sequences responsible for potential ars activity were narrowed to about 350 base pairs, where clusters of nucleotides similar to a consensus sequence (11 bp) essential for several yeast ars (Broach et al. 1982), to the stem-and-loop structure typical of yeast ars3 (Feldmann et al. 1981), and regions surrounding the replication origin of mitochondrial DNAs of HeLa Cells (Crews et al. 1979) and yeast (de Zamaroczy et al. 1981) can be observed.Abbreviations ctDNA chloroplast DNA - Kbp kilobase pairs     

The sequence of an atriopeptigen: A precursor of the bioactive atrial peptides

The high molecular weight fraction (atriopeptigen-APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides (Thibault, et al. (1984) FEBS Letters 167, 352–356, and Kangwa, et al., Biochem. Biophys. Res. Commun 119, 933–940) provide the complete primary structure of an 111 residue APG.     

Effect of dilute solutions of biologically active substances on cell membranes

The article presents data on changes in physicochemical properties of different biological membranes (plasmatic, microsomal, synaptosomes) under the action of biologically active substances, which are different in their chemical structure and the mechanism of action (natural and synthetic antioxidants, thyrotropin - releasing hormone, phorbol esters), in the wide range of concentrations (10^?22?10^?3 M). Dose dependences of the effect of biologically active substances on the activity of membrane-bound enzymes, lipid peroxidation, the structural state of the various regions of the lipid bilayer of membranes have been obtained and analyzed in terms of their formal generality of polymodality, number and position of the maxima, a sign change of the effect. An attempt to explain the mechanism of each of the observed peaks in these curves has been made. The maximum in the range of relatively high “physiological” concentrations (10^?3–10^?7 M) is associated with introduction of biologically active substances into biomembranes. In this study maxima in the range of ultra-low doses (10^?11–10^?16 M) and “apparent” concentrations (10^?18 M), where the presence of biologically active substance molecule in a reaction volume is probabilistic in nature, are explained by physicochemical properties of diluted biologically active substances solutions. This conclusion is based on our data on the changes in IR spectra of aqueous solutions of biologically active substances and the results obtained by academician A.I. Konovalov et al. concerning the physicochemical properties of dilute solutions of biologically active substances (conductivity, surface tension, charge), due to the formation of so-called “nanoassociates” from biologically active substance molecule and numerous number of water molecules. The nanoassociates formation and biological effect disappear if the low concentration solutions are kept in a special shielded permalloy container protecting its contents from external electromagnetic field. Thus, nanoassociates are the material carriers of the unique ability of the ultra-low doses of biologically active substances to exhibit biological effects.     

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Background  

Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An^LC_L domain catalyzes a peptide bond between two L-amino acids, a^DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β -hydroxy-carboxylic acid (typically a β -hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.     

A conformational analysis of biologically active RGD-containing cyclopentapeptides

The theoretical conformational analysis of the biologically active RGD-containing pentapeptide cyclo(-Arg-Gly-Asp-Phe-DVal-), an inhibitor of laminin P1 interaction with its receptor, was performed. The space of permissible torsional angles of the backbone of the molecule was studied by the Monte Carlo method. From the large number of predicted low-energy conformers with various packings of the cyclic moiety of this pentapeptide, only those were selected that corresponded to stable structures of the model linear tripeptide Ac-Ala-Gly-Asp-NHMe. This peptide simulated the spatial possibilities of the backbones of RGD-containing fragments of laminin, vitronectin, and fibronectin. We selected several dozen structures that may be potential biologically active conformers, but only a few of them were capable of forming stable intramolecular hydrogen bonds. We assumed that a biologically active conformer of cyclo(-Arg-Gly-Asp-Phe-DVal-) can be present in significant amounts in an equilibrium mixture in solution along with other conformers without necessarily dominating among them.     

184 Small molecule identification against novel MDRA protein of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an obstinate pathogen causing tuberculosis (TB) in Homo sapiens. One third of the World population is affected by Mtb (James et al., 2008). The multidrug-resistant protein-A (MDRA) belongs to ABC transporter family. The protein MDRA and the membrane integral protein MDRB together form the efflux pump (MDRA₂B₂ complex) that confers resistance by transport of the drugs out of the cell. The MDRB protein expression depends on the expression of MDRA (Baisakhee et al., 2002). In the present study, MDRA 3-D model (Figure) was generated with the help of comparative homology modeling techniques using pair-wise sequence alignment. The predicted 3-D model was subjected to refinement and validated. The active site of the protein was predicted. The virtual screening (VS) studies were performed at MDRB binding site with an in-house library of small molecules to identify a lead molecule that can inhibits the MDRA protein. The results of VS project competitive inhibitors of MDRB, for its binding with MDRA, and its drug-resistant activity. Hence, the MDRA protein may be treated as a novel target for the development of new chemical entities for tuberculosis therapy (Bhargavi et al., 2010; Malkhed et al., 2011).     

The potential suitability of Jiangsu Province, east China for the invasive red imported fire ant, Solenopsis invicta

The red imported fire ant Solenopsis invicta Buren (RIFA), an invasive pest that has diverse detrimental impacts on the communities it invades, was recently discovered in China and has the potential to colonize numerous other regions. Using the model of Korzukhin et al. as modified by Morrison et al. and the biological and ecological characteristics of RIFA, we show that Jiangsu Province is a potentially suitable establishment area of RIFA. An isotherm map made by ArcMap, a Geographic Information System, indicated that several regions of Jiangsu Province (Changzhou, Liyang, Wuxian Dongshan, Nanjing and Lvsi) are at higher risk of S. invicta infestation, especially from late July and early August. Quarantine officials should be vigilant for any accidental introductions of this pest in the susceptible regions and time.     

Genetics of subtilin and nisin biosyntheses

Several peptide antibiotics have been described as potent inhibitors of bacterial growth. With respect to their biosynthesis, they can be devided into two classes: (i) those that are synthesized by a non-ribosomal mechanism and (ii) those that are ribosomally synthesized. Subtilin and nisin belong to the ribosomally synthesized peptide antibiotics. They contain the rare amino acids dehydroalanine, dehydrobutyrine, meso-lanthionine, and 3-methyl-lanthionine. They are derived from prepeptides which are post-translationally modiffied and have been termed lantibiotics because of their characteristic lanthionine bridges (Schnell et al. 1988). Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain gram-positive bacteria (Mattick & Hirsch 1944, 1947; Rayman & Hurst 1984). It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and other lantibiotics are based on depolarization of energized bacterial cytoplasmic membranes. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. A trans-negative membrane potential of 50 to 100 mV is necessary for pore formation by nisin (Ruhr & Sahl 1985; Sahl et al. 1987). Nisin occurs as a partially amphiphilic molecule (Van de Ven et al. 1991). Apart from the detergent-like effect of nisin on cytoplasmic membranes, an inhibition of murein synthesis has also been discussed as the primary effect (Reisinger et al. 1980). In several countries nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morall (1971), and its structural gene was isolated in 1988 (Buchman et al. 1988; Kaletta & Entian 1989). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidin in nisin A is replaced by asparagin in nisin Z (Mulders et al. 1991; De Vos et al. 1993). Subtilin is produced by Bacillus subtilis ATCC 6633. Its chemical structure was first unravelled by Gross & Kiltz (1973) and its structural gene was isolated in 1988 (Banerjee & Hansen 1988). Subtilin shares strong similarities to nisin with an identical organization of the lanthionine ring structures (Fig. 1), and both lantibiotics possess similar antibiotic activities. Due to its easy genetic analysis B. subtilis became a very suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin, and the proteins involved share significant homologies over the entire proteins (for review see also De Vos et al. 1995b). The respective genes have been identified adjacent to the structural genes, and are organized in operon-like structures (Fig. 2). These genes are responsible for post-translational modification, transport of the modified prepeptide, proteolytic cleavage, and immunity which prevents toxic effects on the producing bacterium. In addition to this, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which consists of a histidin kinase and a response regulator protein.     

Purification and properties of a myotoxin from Conus geographus venom

Previous studies (R. Endean et al. (1974), Toxicon12: 131–138) indicate that whole venom from the marine mollusc Conus geographus directly inhibits skeletal muscle, leaving peripheral nerves, cardiac and smooth muscle unaffected. A quantitative bioassay has been used to detect and measure biologically active myotoxin. By using chromatography on phosphocellulose, purified myotoxin is obtained which has the same physiological effects as whole venom. The LD₅₀ of purified toxin is 12 μg/kg in mice, death occurring as a result of flaccid paralysis and respiratory failure. A biochemical characterization of the purified myotoxin indicates that it is a peptide of 13 amino acids containing two disulfide bonds. This and related peptide myotoxins from Conus should be of exceptional interest in muscle physiology.     

The DY sub-region of the sheep MHC contains an A/B gene pair

The major histocompatibility complex (MHC) class II region of ruminants appears to have a structure broadly similar to that of the human class II or HLA-D region. Restriction fragment length polymorphism (RFLP) studies of class II genes in cattle (Andersson et al. 1988; Anderson and Rask 1988; Sigurdardottir et al. 1988, 1991 b), and in sheep (Scott et al. 1987), have provided an estimate of the number and type of class II genes in these species. The subsequent cloning and sequencing of sheep and cattle class II genes (Muggli-Cockett and Stone 1989; Groenen et al. 1990; van der Poel et al. 1990; Andersson et al. 1991; Scott et al. 1991 a, b; Ballingall et al. 1992; Sigurdardottir et al. 1991 a, 1992), have demonstrated that they are highly homologous to their human counterparts. Of more interest, therefore, are loci within the ruminant MHC which differ from the HLA class II region.Three distinguishing features of the ruminant class II region described to date are, firstly, the apparent absence of a DP-like isotype, secondly, the variability in the number of DQ genes between haplotypes (Andersson and Rask 1988), and thirdly, the presence of class II genes presumed to be unique to the ruminant (Andersson et al. 1988). The presence of two such genes, designated DYA and DYB, was deduced from RFLP studies of cattle DNA. These genes were shown to segregate together with the DOB gene in one region separated by a recombination distance of 17 cM from the region which contains the DQA, DQB, DRB, DRA, and C4 loci (Andersson et al. 1988). Subsequently, Bota-DYA was cloned from a phage library and sequenced (van der Poel et al. 1990; Acc. Nos. m30119 and m30118). The sequence of part of a similar gene in the goat, obtained by PCR by using primers derived from the cattle sequence, has recently been reported (Mann et al. 1993; Acc. No. m94325). However, there has been no report of the cloning of a B gene partner for the DYA gene. A novel cattle class II B gene designated Bota-DIB was cloned from a phage library and sequenced by Stone and Muggli-Cockett (1990). This was shown to be a single copy gene of limited polymorphism, which on the basis of RFLP analysis was probably not Bota-DYB but did appear to be distinct from other known cattle class II genes. The species distribution of this B gene was shown to be restricted to Cervidae, Giraffidae, and Bovidae (Stone and Muggli-Cockett 1993). However, it is not known whether any of these novel genes are functional.Expressed human class II genes usually occur as A/B gene pairs situated close to each other on the chromosome. This is also the case with Bota-DQ genes (Groenen et al. 1990) and Ovar-DQ genes (Deverson et al. 1991; Wright and Ballingall 1994). We used the techniques of cosmid cloning and DNA-mediated gene transfection to determine whether there is a sheep equivalent of the Bota-DYA gene, whether there is a DYB gene partner, and whether there is a protein product.A cosmid library was constructed from DNA prepared from a Finnish Landrace ram. The library was screened with Ovar-DQA, Ovar-DQB, HLA-DQA, and HLA-DQB gene probes at low stringency. A cosmid clone, 365, was obtained which hybridized weakly to both the Ovar gene probes. Restriction maps of the clone were produced for the enzymes Eco R1, Bam HI, Hin dIII, Sac I and Sma I. When the maps were compared to those published for the phage clones containing the Bota-DYA (van der Poel et al. 1990) and the Bota-DIB gene (Stone and Muggli-Cockett 1990), there was an imperfect match (Figure 1 shows the Eco RI maps). However, the sequence data for the A and B genes in cosmid 365 are more convincing. The sequences of exons 2 and 3 of the A gene in cosmid 365 and the Bota-DYA gene, together with the partial sequence from the third exon of the Cahi-DYA gene are shown in Figure 2 A. The predicted amino acid translations of these genes together with those of other published sheep MHC class II A genes are shown in Figure 2 B. The A gene in cosmid 365 had all the salient features of an MHC class II A gene. It showed a high sequence similarity to the cattle and caprine DYA genes and much less so to the Ovar-DRA gene (Ballingall et al. 1992; Acc. No z11600) and the Ovar-DQA1 and DQA2 (Scott et al. 1991 a; Acc. Nos. m33304 and m33305), as detailed in Table 1. The cosmid A gene showed low sequence similarity to the sheep DNA (formerly DZA) gene (unpublished observations). The A gene described here is clearly the sheep homologue of the Bota-DYA gene.The sequences of the second, third, and fourth exons of the B gene in cosmid 365 are shown in Figure 3 A together with those of the Bota-DIB gene (Stone and Muggli-Cockett 1990). Unfortunately, the presence of a Bam HI site in exon 2 of the sheep gene caused a truncation at this point, during the cloning procedure and so a part of exon 2, the whole of exon 1, and all the upstream regulatory elements were missing. The predicted amino acid translations of exons 2, 3, and 4 are shown together with those of an Ovar-DQB (Scott et al. 1991 a; Acc. No. m33323) and an expressed Ovar-DRB gene (Ballingall et al. 1992; Acc. No. z11522) in Figure 3 B.     

CFTR: Domains, Structure, and Function

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.     

Sequence and characterization of twoHSP70 genes in the colonial protochordateBotryllus schlosseri

Two genes belonging to the heat shock protein 70 gene family have been cloned from the colonial protochordateBotryllus schlosseri. The two intronless genes(HSP70.1 andHSP70.2) exhibit 93.6% sequence identity within the predicted coding region, and 83.3% and 81.7% sequence identity in the 5′ and 3′ flanking regions, respectively. The predicted amino acid sequences are 95% identical and contain several signatures characteristic of cytoplasmic eukaryoticHSP70 genes (Gupta et al. 1994; Rensing and Maier 1994). Northern blotting and sequence analysis suggest that both genes are heat-inducible merebees of theHSP70 gene family. Given these characteristics,HSP70.1 andHSP70.2 appear to be good candidates for protochordate homologues of the major histocompatibility complex-linkedHSP70 genes of human, mouse, and rat (Milner and Campbell 1990; Walter et al. 1994). Further experiments to determine whether there is functional evidence for such similarity are in progress. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers US 1901 (HSP70.1) and US 1902 (HSP70.2)     

Identification of cold-inducible inner membrane proteins of the psychrotrophic bacterium, Shewanella livingstonensis Ac10, by proteomic analysis

Shewanella livingstonensis Ac10 is a psychrotrophic Gram-negative bacterium that grows at temperatures close to 0°C. Previous proteomic studies of this bacterium identified cold-inducible soluble proteins and outer membrane proteins that could possibly be involved in its cold adaptation (Kawamoto et al. in Extremophiles 11:819–826, 2007). In this study, we established a method for separating the inner and outer membranes by sucrose density gradient ultracentrifugation and performed proteomic studies of the inner membrane fraction. The cells were grown at temperatures of 4 and 18°C, and phospholipid-enriched inner membrane fractions were obtained. Two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting analysis of the proteins identified 14 cold-inducible proteins (more than a 2-fold increase at 4°C). Six of these proteins were predicted to be inner membrane proteins. Two predicted periplasmic proteins, 5 predicted cytoplasmic proteins, and 1 predicted outer membrane protein were also found in the inner membrane fraction, suggesting their association with the inner membrane proteins and/or lipids. These cold-inducible proteins included proteins that are presumed to be involved in chemotaxis (AtoS and PspA), membrane protein biogenesis (DegP, SurA, and FtsY), and morphogenesis (MreB). These findings provide a basis for further studies on the cold-adaptation mechanism of this bacterium.     

Determination of the mouse homologous region for the rat dmy locus

The autosomal recessive mutation dmy causes neurological changes which are characterized by a severe demyelination in the spinal cord, associated with paralysis of the hind limbs. Kuramoto et al. have already mapped the mutation between Hh1ltts and Agtr1a loci on rat chromosome 17 (Kuramoto et al. 1996). Toward the identification of the dmy mutation, we determined here the corresponding genomic region on mouse chromosome 13 and constructed its fine genetic and physical maps. We are currently producing further backcross progeny to narrow down the dmy interval on the rat genome. Determination of the homologous region on the mouse genome should help identifying the causative gene.