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PubMed1989

Didemnin B. Conformation and dynamics of an antitumour and antiviral depsipeptide studied in solution by 1H and 13C. n.m.r. spectroscopy.

The solution conformation of didemnin B, the most potent member of a family of depsipeptides that shows antitumour, antiviral, and immunosuppressive activity, has been studied in chloroform solution using n.m.r. spectroscopy. 1H and 13C spectra have been assigned from analysis of a number of two-dimensional homonuclear and heteronuclear chemical shift correlation experiments which confirm the recently corrected primary structure of the molecule. The conformation of the peptide has been deduced from measurements of the temperature dependence of the NH chemical shifts, analysis of coupling constant data and primarily through NOE effects observed in the rotating frame. Interproton distance bounds determined from a quantitative analysis of the ROE data provide 41 constraints from which a family of closely related structures were calculated using distance geometry algorithms. A type II beta-turn involving residues Thr6, Leu7, and Pro8 is well represented in the computed conformers as is a hydrogen bonding interaction between the NH of Leu3 and the carbonyl oxygen of Thr6. This latter interaction causes the linear portion of the structure to fold back over the depsipeptide ring, imparting to it a degree of structural stability as well as giving the molecule a somewhat globular character. Only one transannular hydrogen bond, between Ist1 NH and Leu3 carbonyl, stabilizes the conformation of the depsipeptide, which has an irregular non-planar configuration. The small temperature coefficients (less than 2.0 x 10(-3) ppm/degrees C) for the NHs of Ist1 and Leu3 are consistent with their involvement in these hydrogen bonding interactions. We find that many of the structural features observed in the crystalline form of didemnin B are conserved in solution. Analysis of the 13C spin-lattice relaxation rates of the protonated carbons reveals small variations in effective correlation times at specific sites in the molecule. The data suggests that the peptide segment encompassing residues Leu3 through to Thr6 is in a more motionally restricted part of the structure.

Amino Acid Sequence↗
PubMed1993

Hyperglycosylation of hen egg white lysozyme in yeast.

The large molecular size of N-glycosylated lysozyme with a polymannose chain was predominantly expressed in the yeast carrying the lysozyme expression plasmid in 9-fold greater secretion compared with the wild type. Complementary DNA encoding hen egg white lysozyme was subjected to site-directed mutagenesis to obtain the Asn-X-Ser/Thr sequence that is the signal for asparagine-linked (N-linked) glycosylation. At positions 49, 67, 70, and 103, the signal for N-linked glycosylation was created. Only the mutant lysozyme whose glycine 49 was substituted with asparagine was expressed in the two types of glycosylated forms, a small oligomannose chain (Man18GlcNAc2)-linked form and a large polymannose chain (Man310GlcNAc2)-linked form, whereas other mutants were not glycosylated. The secreted amount of polymannosyl lysozyme was much higher than that of the oligomannosyl lysozyme. Both types of glycosylated lysozymes were susceptible to endo-beta-N-acetylglucosaminidase cleavage of their carbohydrate chains. The average molecular masses of oligomannosyl and polymannosyl lysozymes were 18 and 71 kDa, respectively. The length of the polymannose chain was found to be 200-350 residues/molecule of lysozyme according to the estimation of the molecular mass distribution by low angle laser light scattering measurements. The protein conformation estimated by CD analysis was completely conserved in these glycosylated lysozymes. The enzymatic activities of oligomannosyl and polymannosyl lysozymes were 100 and 91%, respectively, of wild-type protein when glycol chitin was used as a substrate. In addition, the polymannosyl lysozyme revealed remarkable heat stability in that no coagulation was observed under conditions in which the wild-type lysozyme coagulated. Thus, this novel glycoprotein can be used as a reporter in studies of the processing and sorting of glycoproteins and as a model of the expression of foreign genes in yeast for the construction of stable enzymes.

Animals↗
PubMedDate not supplied

Genetic analysis of insulin signaling in Drosophila.

Studies in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans have revealed that components of the insulin signaling pathway have been highly conserved during evolution. Genetic analysis in Drosophila suggests that structural conservation also extends to the functional level. Flies carrying mutations that reduce insulin signaling have a growth deficiency phenotype similar to that seen in mice with disruptions of genes encoding insulin-like growth factors (IGFs) or the IGF-I receptor. Recent studies in flies have demonstrated a role for the insulin signaling pathway in the regulation of metabolism, reproduction and lifespan via modulation of central neuroendocrine pathways. Similarly, mice with loss of brain insulin receptors or insulin receptor substrate 2 deficiency exhibit neuroendocrine defects and female infertility. These parallels suggest that the insulin system has multiple conserved roles, acting directly to modulate growth and indirectly, via the neuroendocrine system, to modulate peripheral physiology in response to changes in nutrient availability.

Animals↗
PubMed2005

Watchful waiting versus interval appendectomy for patients who recovered from acute appendicitis with tumor formation: a cost-effectiveness analysis.

BACKGROUND: Interval appendectomy after conservative treatment of appendicitis with tumor formation remains controversial. The purpose of this study was to evaluate the efficacy of interval appendectomy based on a cost-effectiveness analysis. METHODS: This was a retrospective study including appendicitis patients with tumor formation who received conservative treatment at first admission to Taipei Veterans General Hospital between January 1998 and December 2003. Demographic data, rate of recurrent appendicitis, and medical costs were analyzed. RESULTS: Of the 165 patients, 1 died after an acute myocardial infarction. The rate of recurrence of appendicitis after conservative treatment was 25.5%. The median cost of follow-up after conservative treatment was NT24,344 dollars. The median cost of interval appendectomy was NT47,746 dollars. The median cost of appendectomy after recurrent appendicitis was NT62,135 dollars. Routine interval appendectomy in all 164 patients would have cost NT7,830,344 dollars. The follow-up protocol with appendectomy after recurrence cost NT5,655,220 dollars. An additional NT2,175,124 dollars (38%) would have been needed for routine interval appendectomy compared with the follow-up policy, an extra NT13,263 dollars per person. CONCLUSION: Routine interval appendectomy would increase the cost per patient by 38% compared with follow-up and appendectomy after recurrence. Routine interval appendectomy is not a cost-effective intervention.

Acute Disease↗
PubMed1997

Lysine-ketoglutarate reductase and saccharopine dehydrogenase from Arabidopsis thaliana: nucleotide sequence and characterization.

We isolated the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, ED 1.5.1.9) from an Arabidopsis thaliana genomic DNA library based on the homology between the yeast biosynthetic genes encoding SDH (lysine-forming) or SDH (glutamate-forming) and Arabidopsis expressed sequence tags. A corresponding cDNA was isolated from total Arabidopsis RNA using RT-PCR and 5' and 3' Race. DNA sequencing revealed that the gene encodes a bifunctional protein with an amino domain homologous to SDH (lysine-forming), thus corresponding to LKR, and a carboxy domain homologous to SDH (glutamate-forming). Sequence comparison between the plant gene product and the yeast lysine-forming and glutamate-forming SDHs showed 25% and 37% sequence identity, respectively. No intracellular targeting sequence was found at the N-terminal or C-terminal of the protein. The gene is interrupted by 24 introns ranging in size from 68 to 352 bp and is present in Arabidopsis in a single copy. 5' sequence analysis revealed several conserved promoter sequence motifs, but did not reveal sequence homologies to either an Opaque 2 binding site or a Sph box. The 3'-flanking region does not contain a polyadenylation signal resembling the consensus sequence AATAAA. The plant SDH was expressed in Escherichia coli and exhibited similar biochemical characteristics to those reported for the purified enzyme from maize. This is the first report of the molecular cloning of a plant LKR-SDH genomic and cDNA sequence.

Amino Acid Sequence↗
PubMed2006

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

Amino Acid Sequence↗
PubMed1999

Analysis of the site occupancy constraints of primary amino acid sequences in the motif directing palmitylation of the vaccinia virus 37-kDa envelope protein.

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1-12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines; A, aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37.

Amino Acid Sequence↗
PubMed1997

Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding.

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase composed of the products of the UL5, UL52, and UL8 genes. UL5 possesses six motifs conserved among superfamily 1 of helicase proteins. Substitutions of conserved residues in each motif abolishes DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). Purified UL5.52 harboring a Gly to Ala change in motif V retains primase and helicase activities in vitro but exhibits a higher KM for single-stranded DNA and lower DNA-dependent ATPase activity (Graves-Woodward, K. L., and Weller, S. K. (1996) J. Biol. Chem. 272, 13629-13635). We have purified and characterized six other subcomplexes with residue changes in the UL5 helicase motifs. Each variant subcomplex displays at least wild type or greater levels of primase and DNA binding activities, but all are defective in helicase activity. Mutations in motifs I and II exhibit profound decreases in DNA-dependent ATPase activity. Mutations in motifs III-VI decrease DNA-dependent ATPase activity 3-6-fold. Since mutations in motifs III, IV, V, and VI do not eliminate ATP hydrolysis or DNA binding, we propose that they may be involved in the coupling of these two activities to the process of DNA unwinding. This analysis represents the first comprehensive structure-function analysis of the conserved motifs in helicase superfamily 1.

Adenosine Triphosphatases↗
PubMed2003

A composite motif of the Drosophila morphogenetic protein bicoid critical to transcription control.

Bicoid is a molecular morphogen-controlling embryonic patterning in Drosophila. It is a homeodomain-containing protein that activates specific target genes during early embryogenesis. Our recent studies have identified a domain of Bcd located outside its homeodomain and referred to as a self-inhibitory domain that can dramatically repress its own ability to activate transcription. Here we present evidence that the self-inhibitory function is evolutionarily conserved. A systematic analysis of this domain reveals a composite 10-amino acid motif with interdigitating residues that regulate Bcd activity in opposite manners. Mutations within the Bcd motif can exert their respective effects when the self-inhibitory domain is grafted to an entirely heterologous activator, but they do not affect DNA binding in vitro or subcellular localization of Bcd in cells. We further show that the self-inhibitory domain of Bcd can interact with Sin3A, a component of the histone deacetylase co-repressor complex. Our study suggests that the activity of Bcd is intricately controlled by multiple mechanisms involving the actions of co-repressor proteins.

Amino Acid Sequence↗
PubMed2004

Predicting allergenic proteins using wavelet transform.

MOTIVATION: With many transgenic proteins introduced today, the ability to predict their potential allergenicity has become an important issue. Previous studies were based on either sequence similarity or the protein motifs identified from known allergen databases. The similarity-based approaches, although being able to produce high recalls, usually have low prediction precisions. Previous motif-based approaches have been shown to be able to improve the precisions on cross-validation experiments. In this study, a system that combines the advantages of similarity-based and motif-based prediction is described. RESULTS: The new prediction system uses a clustering algorithm that groups the known allergenic proteins into clusters. Proteins within each cluster are assumed to carry one or more common motifs. After a multiple sequence alignment, proteins in each cluster go through a wavelet analysis program whereby conserved motifs will be identified. A hidden Markov model (HMM) profile will then be prepared for each identified motif. The allergens that do not appear to carry detectable allergen motifs will be saved in a small database. The allergenicity of an unknown protein may be predicted by comparing it against the HMM profiles, and, if no matching profiles are found, against the small allergen database by BLASTP. Over 70% of recall and over 90% of precision were observed using cross-validation experiments. Using the entire Swiss-Prot as the query, we predicted about 2000 potential allergens. AVAILABILITY: The software is available upon request from the authors.

Algorithms↗
PubMed1993

Analysis of the murine All-1 gene reveals conserved domains with human ALL-1 and identifies a motif shared with DNA methyltransferases.

A series of translocation break points found in a subset of human acute leukemias have one of the breaks on human chromosome 11q23. This region has recently been cloned and a large gene, ALL-1, with homology to the Drosophila trithorax gene has been identified. This paper describes the cloning, sequencing, and mapping of the mouse homolog of ALL-1. We have found a motif present in All-1 that shows homology to the zinc-binding domain of DNA (cytosine-5) methyltransferases (EC 2.1.1.63). Sequence analysis of the murine All-1 gene has identified distinct regions of homology with the human ALL-1 gene; these highly conserved domains may define regions of functional significance in mammals. In addition, we have identified alternatively spliced forms of All-1 within one of the zinc-finger domains, suggesting that there may be different targets and/or functions for All-1 proteins. Finally, we report that All-1 resides in the proximal portion of mouse chromosome 9 and is a candidate for a mutation that results in skeletal transformations during embryonic development.

Alternative Splicing↗
PubMed2005

Structural implications of novel diversity in eucaryal RNase P RNA.

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.

Acanthamoeba castellanii↗
PubMed1998

DQ-haplotype analysis in rhesus macaques: implications for the evolution of these genes.

The DQA1 and DQB1 alleles of 258 rhesus monkeys (Macaca mulatta) of different origin were typed by PCR-RFLP. Five novel MamuDQA1 and five novel -DQB1 alleles were detected and 15 Mamu-DQA1-DQB1 haplotypes were identified. Haplotype analysis confirmed the conservation of the DQA1*01-DQB1 *06 haplotypes in evolution. The most conspicuous finding was the tight linkage between the Mamu-DQA1 and -DQB1 alleles. Almost in every case the Mamu-DQA1 allele was linked to only one particular Mamu-DQB1 allele. Although there also are constraints in the formation of DQ haplotypes in humans, such tight linkages are not observed. These findings support the hypothesis of some kind of co-evolution between DQA1 and DQB1 alleles and may reflect a stronger force of natural selection in macaques than in humans.

Amino Acid Sequence↗
PubMed2005

The phage-shock-protein response.

The phage-shock-protein (Psp) system responds to extracytoplasmic stress that may reduce the energy status of the cell. It is conserved in many different bacteria and has been linked to several important phenotypes. Escherichia coli psp mutants have defects in maintenance of the proton-motive force, protein export by the sec and tat pathways, survival in stationary phase at alkaline pH, and biofilm formation. Yersinia enterocolitica psp mutants cannot grow when the secretin component of a type III secretion system is mislocalized, and have a severe virulence defect in animals. A Salmonella enterica psp mutation exacerbates some phenotypes of an rpoE null mutant and the psp genes of S. enterica and Shigella flexneri are highly induced during macrophage infection. PspA, the most abundant of the Psp proteins, is required for most of the phenotypes associated with the Psp system. Therefore, PspA is probably an effector that may play a role in maintaining cytoplasmic membrane integrity and/or the proton-motive force. However, PspA is not required for the ability to tolerate secretin mislocalization, which suggests an important physiological role for other Psp proteins. This article summarizes our current understanding of the Psp system: inducing signals, the underlying signal transduction mechanisms, the physiological roles it may play, and a genomic analysis of its conservation.

Bacterial Proteins↗
PubMed1997

Proviral organization and sequence analysis of feline immunodeficiency virus isolated from a Pallas' cat.

The nucleotide sequence and genomic organization have been determined for a highly cytopathic feline immunodeficiency virus (FIV) isolated from a Pallas' cat. The 9747-bp provirus of this virus, FIV-Oma, has typical lentivirus organization with LTRs, gag, pol, and env open reading frames (ORFs), putative vif and rev ORFs, and an ORF similar to ORF2/ORFA of domestic cat FIV isolates. Although the FIV-Oma provirus is 300 to 600 bp longer than other FIV proviruses, these additional bases are distributed throughout the genome. Phylogenetic analysis of a conserved region of the pol gene suggests that FIV-Oma is more closely related to some of the puma and lion lentiviruses than it is to domestic cat FIV isolates; however, many regions of the genome exhibit extensive nucleotide sequence divergence. None of the eight molecular proviral clones isolated from a genomic library are infectious, but we have constructed an infectious, cytopathic clone of FIV-Oma from subcloned and PCR-amplified fragments of these proviral clones. This clone will be useful for identifying the genetic determinants of FIV-Oma's biological activities.

Amino Acid Sequence↗
PubMed1988

The perils of prudence: how conservative risk assessments distort regulation.

An analysis of several agencies' risk assessments reveals that the present conservative approach through several stages of estimates of amounts of regulated substances, the exposure levels, and cancer risks often tends to distort the actual final regulation. Changes in this process are desirable and possible and would reduce overestimation of cancer risk assessments. These possible changes are discussed.

Animals↗
PubMed1999

Is hepatitis C virus genotype 4 predominant in Saudi Arabia?

Hepatitis C virus (HCV) genotype 4 is believed to be predominant in the Middle East including Saudi Arabia (SA). We attempted to genotype 80 HCV isolates from different parts of SA by direct sequencing of a variable 222bp fragment from the NS5B region. The phylogenetic analysis of the NS5B sequences was complemented by direct sequence analysis of the conserved 5'-NCR region for HCV type-specific polymorphism. All 80 NS5B sequences separated into 3 clades which comprised 6 type 1b variants, 30 type 4 variants (24 of type 4a and 6 of type 4c or d) and 44 type 3 variants. Apart from two definitive type 3b variants the other 42 type 3 NS5B sequences formed 4 clusters with low similarity to type 3a-f HCV sequences from the database. The precise subtyping of these 42 type 3 variants awaits sequencing of longer HCV RNA stretches. Our results indicate that HCV type 4 may not be the only dominant genotype in SA.

Endemic Diseases↗
PubMed1997

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

Amino Acid Sequence↗