Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single- stranded DNA and RNA phages.      

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Facile analysis and purification of deblocked N-terminal pyroglutamyl peptides with a strong cation-exchange sulfoethyl aspartamide column

A high-performance strong cation-exchange Sulfoethyl Aspartamide column was used to analyze and purify five N-terminal pyroglutamyl peptides after treatment with Pyroglutamate Aminopeptidase. The resulting deblocked N-1 peptides possess an increased positive charge and are therefore retained to a greater extent by the column. Salt gradient elution in a pH 3 mobile phase was then used to recover the desired peptides and the purified deblocked peptides were directly subjected to N-terminal sequence analysis. The same digests were also chromatographed on a C18 reversed-phase column using standard trifluoroacetic acid-acetonitrile gradient elution. The elution order for the parent peptide and the N-1 peptide on the reversed-phase column was reversed from that on the Sulfoethyl Aspartamide column and the resolution of the two peptides obtained on the reversed-phase column was less than that observed on the cation-exchange column. In addition, the Sulfoethyl Aspartamide column was shown to be useful to monitor the extent of N-terminal glutamine cyclization formed during peptide purification and storage.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VKII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region

To characterize the L chain V region repertoire of IgG anti-Haemophilus influenzae type b capsular polysaccharide (Hib-PS) antibodies, clonal antibodies were purified from immune serum and internal amino acid sequences of VKII anti-Hib-PS L chains obtained. We examined VKII L chains because it is the most common VL family expressed in the anti-Hib-PS response. Comparison of VKII amino acid sequences, including the entire CDR2 and CDR3 regions, of five anti-Hib-PS clonal antibodies from four unrelated individuals revealed complete identity with the exception of a single CDR3 residue from one antibody. When the sequence of these antibodies was compared with known VKII genes and myeloma proteins, it was found to be identical to the human VKII gene, A2, whose genomic sequence is presented here. In addition, all five of the VKII anti-Hib-PS antibodies examined contain an arginine inserted at the V-J junction. Finally, in contrast to the extraordinary homology of the VKII-encoded residues, there is variability in the JK gene utilization by these antibodies. These results demonstrate that the most common L chain V region in IgG anti-Hib-PS antibodies is the product of a single germ-line gene. The invariant arginine insertion suggests that this residue has an important role in Ag binding.     

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of an Internal Deletion Mutant of the Arabidopsis thaliana ABI3 Gene

An Arabidopsis thaliana mutant that produces green seeds thatare highly insensitive to exogenous ABA, non-dormant and severelydesiccation intolerant was isolated from a population of fastneutron-irradiated seeds. Molecular and genetic analysis ofthis mutant shows that these phenotypes are caused by an internaldeletion of approximately one third of the ABI3 gene. Thereforeabi3 mutants with the above phenotypes are representative ofnull alleles at this locus. (Received December 3, 1993; Accepted January 22, 1994)     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Cleavage specificity of human skin type IV collagenase (gelatinase). Identification of cleavage sites in type I gelatin, with confirmation using synthetic peptides

Type IV collagenase (gelatinase) readily cleaves denatured collagen into very small peptides. Large cyanogen bromide fragments (25 kDa) of type I collagen are degraded at the same rate as the complete alpha-chain. A number of the gelatinolytic cleavage sites of alpha 1(I)CB7 and alpha 1(I)CB8, representing 50% of the collagen alpha-chain, were determined by sequence analysis of product peptides. In addition to the expected cleavage between glycine and hydrophobic residues, several other cleavage sites were identified. These sites were Gly-Glu, Gly-Asn, and Gly-Ser. Basic residues were found adjacent to the cleavage site in several cases. Hexapeptides containing these unexpected cleavage sites were synthesized, and Km and kcat values were determined. All but one of the Km values were in the submillimolar range, and turnover numbers for the peptides uncharged at the carboxyl terminus were on the order of 10,000/h. Of particular significance was the finding that hydroxyproline occurs 5 residues from the cleavage site in all carboxyl-terminal product peptides and also occurs 5 residues from the cleavage site in seven of nine amino-terminal product peptides. A requirement for hydroxyproline may be of importance in determining the specificity of this enzyme for denatured collagenous substrates.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Identity and phylogenetic placement of Spirogyra species (Zygnematophyceae,Charophyta) from California streams and elsewhere1

Diversity of the filamentous green algae in the genus Spirogyra (Zygnematophyceae) was investigated from more than 1,200 stream samples from California. We identified 12 species of Spirogyra not previously known for California (CA), including two species new to science, Spirogyra californica sp. nov. and Spirogyra juliana sp. nov. Environmental preferences of the Californian species are discussed in the light of their restricted distribution to stream habitats with contrasting nutrient levels. We also investigated the systematic relationships of Spirogyra species from several continents using the chloroplast‐encoded genes ribulose‐1,5‐bisphosphate carboxylase/hydrogenase large subunit (rbcL) and the beta subunit of the ATP synthase (atpB). Californian species were positioned in most major clades of Spirogyra. The phylogeny of Spirogyra and its taxonomic implications are discussed, such as the benefits of combining structural and molecular data for more accurate and consistent species identification. Considerable infraspecific genetic variation of globally distributed Spirogyra species was observed across continental scales. This finding suggests that structurally similar species from distant regions may be genetically dissimilar and that Spirogyra may contain a large number of cryptic species. Correlating the morphological and genetic variation within the genus will be a major challenge for future researchers.