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Biomedical subjects

M A Purdy

Publications and source records attributed to M A Purdy.

33 records · Page 2Linked to original sources
PubMed1992

Recent developments in the molecular cloning and characterization of hepatitis C and E viruses.

The molecular properties of the genomes of both hepatitis C virus (HCV) and hepatitis E virus (HEV), the major etiologic agents of non-A, non-B hepatitis, are briefly described. The organization of the genome of each of these viruses is discussed and compared to those of other related or distantly related viruses that contain single-stranded, positive-sense RNA genomes. In the case of HEV, the reactivity of expressed proteins and genetic divergence of geographically distinct isolates are also described within the context of sequence variation, type-common epitopes and type-specific epitopes.

Cloning, Molecular↗
PubMed1992

Acute sporadic hepatitis E in Sudanese children: analysis based on a new western blot assay.

A newly developed Western blot assay for antibody to hepatitis E virus (anti-HEV) was used to evaluate 39 cases of acute pediatric hepatitis and 39 control patients in Khartoum, Sudan. The mean age of cases was 6.5 years (range, 2-14); 64% were male. Acute hepatitis A (IgM anti-HAV-positive) was diagnosed in 13 cases, acute hepatitis B (IgM anti-HBc-positive) in 1, and acute hepatitis E (positive for IgM anti-HEV) in 23 (59%). None of the cases with IgM anti-HAV or IgM anti-HBc had IgM anti-HEV; 3 controls had IgM anti-HEV. Acute hepatitis E was associated with recent contact with a family member or acquaintance with jaundice and the presence of indoor plumbing. The newly developed hepatitis E assay appeared to be specific for the diagnosis of acute icteric non-A, non-B hepatitis. Hepatitis E was found to be the most common cause of acute sporadic hepatitis in children living in an urban area of Africa.

Acute Disease↗
PubMed1991

Hepatitis E virus (HEV): the novel agent responsible for enterically transmitted non-A, non-B hepatitis.

A normally endemic form of viral hepatitis is the cause of major epidemic outbreaks in developing countries. This disease has a global distribution and has been referred to as water-borne, epidemic or enterically transmitted non-A, non-B hepatitis (ET-NANBH). Although the fecal-oral route of transmission predominates, person-to-person routes of exposure were also suggested in some epidemiologic studies. The disease has been documented as having an extremely high mortality in pregnant women (approximately 20%). Sporadic cases of ET-NANBH, as well as imported travel exposures, have been reported in developed countries. Molecular cloning was hampered by the lack of a tissue culture system for virus propagation, however, an available animal model and a newly developed non-specific amplification procedure were used to clone and identify an exogenous cDNA (ET1.1) from a Burma-isolate infected animal. Molecular clones were also identified by immunoscreening of a cDNA library made from a fecal specimen collected from a Mexican outbreak of ET-NANBH. The isolation and sequencing of a set of overlapping cDNA clones had led to the recognition that this form of hepatitis is caused by a virus unlike any of the other viral hepatitis agents. The molecular characterization of HEV will lead to important pathobiologic insights and hasten the development of potentially useful diagnostic and therapeutic products for ET-NANBH.

Cloning, Molecular↗
PubMed1991

Application of two RNA extraction methods prior to amplification of hepatitis E virus nucleic acid by the polymerase chain reaction.

Amplification of the enterically-transmitted non-A, non-B hepatitis virus (HEV) RNA using conventional reverse transcriptase reactions followed by the polymerase chain reaction (PCR) of the cDNA has not been successful. However, after application of two different RNA capture/extraction methods we were able to amplify HEV nucleic acid from clinical samples and specimens from experimentally infected animals. The first procedure, adapted from an immune electron microscopy (IEM) technique, incorporated an immunocapture step with concentration of the virus-antibody complexes by pelleting in a Beckman airfuge. In the second method, glass powder (or size-fractionated silicon dioxide) was used to capture the RNA from its surrounding milieu by adsorption of the nucleic acid to the silicate particles. Since conventional immunoassays for HEV antigen or antibody are not currently available, the use of these RNA extraction methods, coupled with PCR techniques, will be valuable in screening clinical specimens and in further defining the course of disease using animal infectivity studies.

Adsorption↗
PubMed1991

Hepatitis E virus genome. Molecular features, expression of immunoreactive proteins and sequence divergence.

The strategy for molecular cloning of hepatitis E virus (HEV), the major etiologic agent of enterically-transmitted non-A, non-B hepatitis, is briefly described. The organization of the HEV genome is discussed and compared to those of two other vertebrate viruses that contain single-stranded, positive-sense, polyadenylated RNA genomes with three overlapping ORFs. Serologic cross-reactivity of expressed proteins and genetic divergence of HEV isolates are also described within the context of sequence variation, type-common epitopes, and type-specific epitopes.

Cloning, Molecular↗
PubMed1990

Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis.

Major epidemic outbreaks of viral hepatitis in underdeveloped countries result from a type of non-A, non-B hepatitis distinct from the parenterally transmitted form. The viral agent responsible for this form of epidemic, or enterically transmitted non-A, non-B hepatitis (ET-NANBH), has been serially transmitted in cynomolgus macaques (cynos) and has resulted in typical elevation in liver enzymes and the detection of characteristic virus-like particles (VLPs) in both feces and bile. Infectious bile was used for the construction of recombinant complementary DNA libraries. One clone, ET1.1, was exogenous to uninfected human and cyno genomic liver DNA, as well as to genomic DNA from infected cyno liver. ET1.1 did however, hybridize to an approximately 7.6-kilobase RNA species present only in infected cyno liver. The translated nucleic acid sequence of a portion of ET1.1 had a consensus amino acid motif consistent with an RNA-directed RNA polymerase; this enzyme is present in all positive strand RNA viruses. Furthermore, ET1.1 specifically identified similar sequences in complementary DNA prepared from infected human fecal samples collected from five geographically distinct ET-NANBH outbreaks. Therefore, ET1.1 represents a portion of the genome of the principal viral agent, to be named hepatitis E virus, which is responsible for epidemic outbreaks of ET-NANBH.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10.

The complete sequence of the RNA segment that codes for a major outer capsid protein (VP5) of bluetongue virus serotype 10 has been determined from overlapping cDNA clones inserted into pBR322. The segment 5 RNA of the virus (M5 RNA) is deduced to be 1638 base pairs long (1.05 X 10(6) daltons) and has an open reading frame in one strand capable of coding for a protein with a calculated size of 59 163 daltons (526 amino acids) and a net charge of -4.5 at neutral pH.

Amino Acids↗
PubMed1985

The complete sequence of bluetongue virus serotype 10 segment 3 and its predicted VP3 polypeptide compared with those of BTV serotype 17.

The complete sequence of the large RNA segment 3 (L3) of bluetongue virus serotype 10 (BTV-10) has been determined from DNA copies of the viral RNA cloned in the E. coli plasmid pBR322. The L3 viral RNA is 2772 nucleotides long with a single open reading frame of 2706 nucleotides. The L3 predicted primary gene product (VP3) is 103 342 daltons and has a net charge at neutral pH of -5. The sequence of the L3 RNA species differs by 126 point mutations from that of BTV-17 (i.e., 95.5% homology; see M. Purdy, J. Petre and P. Roy, J. Virol. 51, 754-759, 1984). The predicted L3 primary gene products of the two viruses differ by 9 amino acids. These differences correspond to 9 point mutations and represent 0.15% of the sites where nucleotide substitution could cause an amino acid change. By contrast, another 114 point mutations in the genome correspond to 6.5% of the available sites where nucleotide substitutions could be silent (i.e., where a nucleotide substitution may not cause an amino acid change). Three point mutations are in the 3' non-coding region of the RNA species. The quantitative differences between the coding and silent mutations are interpreted as representing the result of gene product conservation.

Amino Acid Sequence↗
PubMed1985

Complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies.

The complete sequence of the RNA which encodes the major outer-shell-neutralizing antigen (VP2) of bluetongue virus serotype 10 was determined from overlapping cDNA clones inserted into pBR322. The segment L2 RNA was 2,926 base pairs long (1.87 X 10(6) daltons) and had, in one strand, an open reading frame capable of coding for a protein that had a calculated size of 111,122 daltons (956 amino acids) and a +11.5 net charge. The coding strands of both the L2 gene and the group-specific L3 gene of bluetongue virus serotype 17 (M. Purdy, J. Petre, and P. Roy, J. Virol. 51:754-759, 1984) had common sequences of some six nucleotides at their 5' termini (namely, GUUAAA...) and eight nucleotides (namely, ...ACACUUAC) at their 3' termini. Both had short 5' noncoding regions with AUG codons at residues 20 to 22 (L2) and 18 to 20 (L3). The sequences flanking these AUG codons were similar (A/GCCAUGG). The 3' noncoding regions were longer (36 nucleotides for L2, 49 nucleotides for L3). The predicted amino acid sequence of the L2, compared with the similarly sized L3 gene product, was rich in cysteine residues and charged amino acids.

Antigens, Viral↗
PubMed1985

Cloning and nucleotide sequencing of bluetongue virus genomes.

This is the 1st report of molecular cloning of bluetongue virus (BTV) gene using DNA recombinant techniques. A number of complete and overlapping clones of BTV genes have been cloned into pBR322 by standard procedures with some modification. The full length clones were confirmed by matching the terminal cDNA nucleotide sequences with those previously determined for the termini of double-stranded genomic RNA (Rao et al., 1983). The complete sequence of the cDNA clone of RNA segment 3 of US serotype 17 as determined by the method of Maxam and Gilbert (1980) has been presented here. The clone is 2,772 nucleotides (1.78 X 10(6) daltons) long excluding the 3' polyadenylic acid sequence and has an open reading frame which encodes a protein of some 901 amino acids (103,412 daltons).

Amino Acid Sequence↗
PubMed1983

Effect of growth phase and cell envelope structure on susceptibility of Salmonella typhimurium to the lactoperoxidase-thiocyanate-hydrogen peroxide system.

The lactoperoxidase-thiocyanate-hydrogen peroxide system was found to have both bacteriostatic and bactericidal activities against strains of Salmonella typhimurium. The bactericidal activity was clearly dependent on the permeability of the bacterial cell envelope. The deep rough mutant TA1535, with the most permeable cell envelope, was killed both at neutral and acid pH, whereas very little or no killing was observed with the intact cells of the parent strain hisG46. The delta gal mutant, TA1530, representing an intermediate in cell envelope permeability, was inhibited to a much lesser extent than TA1535. Bacteria in log phase of growth were more sensitive to the bactericidal effects than were those in stationary phase. Growth phase had little influence on the bacteriostatic effects. The hisG46 strain produced significant quantities of acid in the presence of glucose. This acid production was inhibited by the lactoperoxidase-thiocyanate-hydrogen peroxide system, and, in contrast to results obtained with several strains of streptococci, this inhibition was not reversed by addition of a reducing agent (2-mercaptoethanol).

Cell Membrane Permeability↗
PubMed1979

Enhancement of post-ultraviolet killing in Escherichia coli K-12 through the action of gyrase inhibitors: evidence for associated gyrase-recBC deoxyribonuclease function.

This work in conjunction with the results presented in an earlier report (M. A. Purdy and K. L. Yielding, Antimicrob. Agents Chemother. 10:182--184, 1976) showed the following. (i) Nalidixic acid and novobiocin could inhibit post-ultraviolet and post-X-ray survival, implicating gyrase function in deoxyribonucleic acid repair. (ii) The inhibition of post-ultraviolet survival requires the action of functional recBC deoxyribonuclease. (iii) Structural changes in the gyrase could (a) cause recBC mutants to exhibit enhancement of post-ultraviolet killing in the presence of novobiocin, (b) increase the ultraviolet sensitivity of recBC mutants, and (c) enhance the thermal lability of a recBCts mutant.

Deoxyribonucleases↗
PubMed1976

Nalidixic acid inhibition of post-ultraviolet recovery in Escherichia coli K-12: requirement for recBC function.

Various repair-deficient mutants of Escherichia coli K-12 were tested for the impairment of post-ultraviolet survival by nalidixic acid. These studies have shown that the target of this nalidixic acid effect is dictated by the recB and recC genes and may be due to the binding of nalidixic acid to exonuclease V. Furthermore, this effect cannot be correlated with either recombination proficiency or excision repair alone.

Dose-Response Relationship, Radiation↗