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Biomedical subjects

L Berthiaume

Publications and source records attributed to L Berthiaume.

At least 19 recordsLinked to original sources
PubMed1992

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the "tac" promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.

Adrenal Glands↗
PubMed1992

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations.

Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV3) and chilo iridescent virus (CIV), two other well known viruses of the family Iridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A2. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.

Animals↗
PubMed1992

Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles.

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of an important disease in hatchery-reared salmonid fish in North America, Europe and Japan. It belongs to the family Birnaviridae and shows a high degree of antigenic heterogeneity. However, genomic variations between the 10 identified serotypes have not yet been studied. In order to correlate genomic heterogeneity with the different serotypes, oligonucleotides were synthesized according to the published sequence of the Jasper strain (serotype A9). They were used as primers for the amplification of a 359 bp cDNA fragment of the viral genome using the polymerase chain reaction. Fragments amplified from 37 strains were digested with five different restriction enzymes. Restriction fragment profiles obtained an agarose gels showed heterogeneity not only between strains of different serotypes, but also among those belonging to serotype A1. A cluster analysis of the restriction patterns showed that IPNV strains can be divided into three major groups, corresponding approximately to serotypes A1, A2 and A3, and 10 subgroups which do not correlate with the serotyping of the strains.

Animals↗
PubMed1992

Antigenic and genomic differences of two Jasper strains of infectious pancreatic necrosis virus.

Strains of infectious pancreatic necrosis virus (IPNV-Jasper) obtained from two different laboratories were compared serologically with polyclonal and monoclonal antibodies. Nucleotide sequence and restriction endonuclease patterns of 359-bp fragment of genome segment A cDNA were also compared. Substantial differences were found in both analyses that will support the fact that the two Jasper strains are not identical.

Animals↗
PubMed1991

Carboxyl terminus region modulates catalytic activity of recombinant maize aldolase.

Site-directed mutagenesis was utilized to study the functional role of the COOH-terminal region in recombinant maize aldolase. A single mutation was created in each of the last nine amino acids of the COOH terminus and characterized kinetically. Point mutations in the COOH-terminal region were found to influence both the rate of fructose 1,6-bisphosphate and fructose 1-phosphate cleavage. Catalytic efficiency, kcat/Km, was not affected by the mutations within experimental error consistent with this region of the COOH terminus modulating product release. Concentrations of the carbanion-enamine enzyme intermediate complex produced upon substrate cleavage increased with the severity of the point mutation. A condensation assay was developed to directly measure fructose 1,6-bisphosphate synthesized by aldolases in the presence of high triose phosphate concentrations. The maximal rate of aldol condensation of triose phosphates, D-glyceralehyde-3-P and dihydroxyacetone-P, was affected by the point mutations to the same extent as the maximal rate of substrate cleavage. Interpretation of the data is consistent with point mutations in the COOH terminus predominantly affecting the proton exchange with the dihydroxyacetone-P enzymatic complex at the carbanion-enamine step and that this step is probably rate-limiting in the catalytic mechanism of recombinant maize aldolase. The role of the COOH-terminal region in aldolases is thus consistent with a sequence dependent modulation of catalytic activity.

Amino Acid Sequence↗
PubMed1989

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications.

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.

Amino Acid Sequence↗
PubMed1989

A new bacteriophage of Corynebacterium glutamicum isolated from swine waste.

A bacteriophage for Corynebacterium glutamicum strain LP-6 was isolated from swine waste. It belongs to the Siphoviridae family or Bradley morphologic group B, has a narrow host range, and is sensitive to chloroform and resistant to carbon tetrachloride. The phage is unstable (96% inactivation) in swine waste stored for 4 months at 22 C. The DNA has a molecular weight of approximately 20 Md, cohesive ends, and numerous restriction endonuclease sites. The phage differs from other known C glutamicum phages.

Animals↗
PubMed1988

Electron microscopic evidence for bridges between bovine respiratory syncytial virus particles.

Electron microscopic examination of ultrathin sections of a continuous cell line of ovine kidney (OK) origin, infected by bovine respiratory syncytial virus (BRSV), revealed the presence of well defined bridges between virus particles. This is the first report of this novel structure. Observation of ultrathin sections of human RSV Long strain also grown on OK cells did not show inter-particle bridges and therefore suggested that this structure could be specific to BRSV. The biological significance of these bridges is not clear at this time; a possibility is that the bridges are formed by the fusion protein of BRSV which is known to cause cell fusion. Besides the structural implications, the importance is in relation to purification strategies for this virus, which must now take into account that most of the viral particles occur in large aggregates.

Animals↗
PubMed1985

An enteric coronavirus of the rabbit: detection by immunoelectron microscopy and identification of structural polypeptides.

The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particles sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.

Animals↗
PubMed1979

Influenza viruses in birds: rapid identification by counterimmunoelectrophoresis.

Counterimmunoelectrophoresis with an antiserum raised in rabbits against the M protein of the avian N virus proved to be particularly useful for large-scale identification of influenza A virus isolates. Of a total of 231 hemagglutinating agents isolated from 1,656 rectal swabs collected from shore and open-country birds, 158 could be identified as influenza A viruses by counterimmunoelectrophoresis, and 75 were serologically related to Newcastle disease virus by hemagglutination inhibition with an antiserum to Newcastle disease virus. Two isolates contained a mixture of influenza A virus and Newcastle disease virus; although the Newcastle disease virus virus particles outnumbered the influenza A virus particles in a ratio of 1,000:1, as seen by electron microscopy, the latter could be readily detected by counterimmunoelectrophoresis. This type of assay appears to be of potential use for epidemiological surveillance of influenza virus isolated from humans and animals. It combines specificity, sensitivity, and simplicity.

Animals↗
PubMed1978

Morphological and quantitative study of spirochetes in the feces of normal and infected SPF pigs during the incubation period of swine dysentery.

Spirochetes found in feces of normal and infected pigs during the eight days preceding the onset of the clinical signs of swine dysentery have been studied using electron and phase contrast microscopy. According to their dimensions, diameter, length and pitch, three morphological groups have been described: small, intermediate and large spirochetes. On the basis of their axial filaments arrangement, eight types have been observed. Compared to the control pigs, there was no increase in the total number of spirochetes in infected pigs, except at day 6 before the onset of clinical signs. However, infected pigs have shown a wide spectrum of different morphological types of spirochetes in comparison to control, uninfected pigs, which showed only the type "1-2-1" spirochete of axial filaments arrangement. This last type was predominant in infected swine during the entire incubation period as compared to other spirochete types described.

Animals↗
PubMed1978

The experimental infection of piglets with a porcine reovirus.

A strain (Quebec) of reovirus isolated from the faeces of a pig with dysentery was neutralised by reovirus type 1 antiserum. Four of eight hysterectomy-produced, colostrum-deprived (HPCD) piglets dosed orally with the third cell culture passage of the virus developed diarrhoea and showed focal areas of villous atrophy in the small intestine. The virus was isolated from the intestinal tract of all eight specific pathogen free piglets, but not from three control animals. Nine germ-free piglets dosed orally with the eight cell culture passage of the virus showed neither clinical signs nor lesions, but virus was recovered from their intestinal tracts for 14 days after infection. No virus was isolated from four control germ-free piglets.

Animals↗
PubMed1977

[Formation of spheriods by cultured human diploid cells].

When human lung diploid fibroblasts are grown in micro-wells in presence of the organic buffer HEPES in an atmosphere of 5% of CO2, the cells migrate and form spheroids. Histologic examination reveals the presence of fibroblastic and epithelioid cells in these spheroids. Signs of degeneration are seen in both cell types under the electron microscope. Following dissociation of the spheroids and their subculture in absence of HEPES, the cells reverse to their original fibroblast-like morphology and grow in monolayer.

Cell Aggregation↗
PubMed1976

Herpes encephalitis. Rapid diagnosis and treatment with antiviral drugs.

The use of specific IgM antibodies and direct electron-microscopic examination of brain biopsies or vesicle fluid was tested as means of raped diagnosis in 6 cases of herpes simplex encephalitis seen consecutively in Montreal. In 2 of 3 biopsies herpes viruses were seen by negative staining of a cell extract within 1 hr. In the negative case, the biopsy was done almost 1 month after onset. In 2 additional cases herpes virus particles were found directly in the fluid of isolated vesicles. In the last 2 cases, who survived, the diagnosis of herpes encephalitis rested upon the demonstration of a greater than 4-fold rise in complement fixing herpes simplex virus antibodies in convalescent sera and upon the appearance late in the course of the encephalitis of specific antibodies in the cerebrospinal fluid. The early appearance of specific IgM antibodies contributed to the diagnosis in 4 of the 6 cases. Antiviral therapy was attempted in alternate cases (3 cases) but was not successful. Brain biopsy is rarely performed for diagnostic purposes but when prompt antiviral therapy is contemplated, the examination of the biopsy material for herpes virus particles by electron microscopy in negative staining and thin sections can rapidly and reliably confirm the diagnosis.

Aged↗