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Biomedical subjects

D W Brown

Publications and source records attributed to D W Brown.

At least 91 records · Page 5Linked to original sources
PubMed1996

Aspergillus has distinct fatty acid synthases for primary and secondary metabolism.

Aspergillus nidulans contains two functionally distinct fatty acid synthases (FASs): one required for primary fatty acid metabolism (FAS) and the other required for secondary metabolism (sFAS). FAS mutants require long-chain fatty acids for growth, whereas sFAS mutants grow normally but cannot synthesize sterigmatocystin (ST), a carcinogenic secondary metabolite structurally and biosynthetically related to aflatoxin. sFAS mutants regain the ability to synthesize ST when provided with hexanoic acid, supporting the model that the ST polyketide synthase uses this short-chain fatty acid as a starter unit. The characterization of both the polyketide synthase and FAS may provide novel means for modifying secondary metabolites.

Aflatoxins↗
PubMed1996

Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens.

Four methods for extraction of the RNA genome of small round structured viruses (SRSVs) from faecal specimens by reverse transcription polymerase chain reaction (RT-PCR) were evaluated. The efficiency of recovery of viral RNA and removal of amplification inhibitors were compared. RNA extraction using the metal chelating agent Chelex-100 and Sephadex G200 column chromatography were the most sensitive, detecting a 10(-4) dilution of a known SRSV positive specimen. Guanidinium thiocyanate (GTC) with adsorption of viral RNA onto silica was 10-fold less sensitive. The fourth method, based on PEG precipitation followed by phenol/chloroform extraction with the addition of the detergent cetyltrimethylammonium bromide (CTAB), only detected a 1 in 10 dilution of the positive specimen. The CTAB method was 2- to 50-fold less sensitive than the GTC/silica method when dilution series of three further SRSV positive specimens were tested. Thirty-six SRSV negative faecal specimens were spiked with virus and RT-PCR performed following RNA extraction by each of the four techniques in order to assess the ability of these methods to remove amplification inhibitors. The GTC/silica method successfully removed inhibitors from all samples whereas partial or complete inhibition was observed in seven (19%) specimens following extraction by the CTAB method, 17 (47%) by the Sephadex method, and 20 (56%) by the Chelex method. We conclude that, of these four methods, the GTC/silica method is the most appropriate for the extraction of viral RNA from faecal samples prior to RT-PCR for detecting SRSVs.

Base Sequence↗
PubMed1996

Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans.

Sterigmatocystin (ST) and the aflatoxins (AFs), related fungal secondary metabolites, are among the most toxic, mutagenic, and carcinogenic natural products known. The ST biosynthetic pathway in Aspergillus nidulans is estimated to involve at least 15 enzymatic activities, while certain Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius strains contain additional activities that convert ST to AF. We have characterized a 60-kb region in the A. nidulans genome and find it contains many, if not all, of the genes needed for ST biosynthesis. This region includes verA, a structural gene previously shown to be required for ST biosynthesis, and 24 additional closely spaced transcripts ranging in size from 0.6 to 7.2 kb that are coordinately induced only under ST-producing conditions. Each end of this gene cluster is demarcated by transcripts that are expressed under both ST-inducing and non-ST-inducing conditions. Deduced polypeptide sequences of regions within this cluster had a high percentage of identity with enzymes that have activities predicted for ST/AF biosynthesis, including a polyketide synthase, a fatty acid synthase (alpha and beta subunits), five monooxygenases, four dehydrogenases, an esterase, an 0-methyltransferase, a reductase, an oxidase, and a zinc cluster DNA binding protein. A revised system for naming the genes of the ST pathway is presented.

Aflatoxins↗
PubMed1996

Development of a dual target-PCR for detection and characterization of measles virus in clinical specimens.

A polymerase chain reaction (PCR) method was developed for detecting measles virus (MV) RNA in a variety of clinical samples using primer pairs in the nucleocapsid (N) and matrix (M) genes in one reaction (dual target-PCR). The dual target-PCR detected MV RNA in tissue culture fluid containing 1 TCID50 of the MV Loss strain, and was as sensitive as a single target-PCR. Specificity was confirmed by the failure of the dual target-PCR to amplify products from the infected tissue culture containing related paramyxoviruses. Thirty-two of 35 (91.4%) samples collected from 23 patients with confirmed measles infection by the detection of measles specific IgM were found to be positive for MV RNA by PCR. Direct sequencing of the PCR amplicons revealed three different genotypes among the MV strains that were detected in 12 patients. The dual target-PCR method is suitable for the diagnosis of measles infection and, based on the sequencing of the PCR product DNA, for investigating the molecular epidemiology of MV strains.

Adult↗
PubMed1996

Evaluation of a degenerate primer for the PCR detection of human caliciviruses.

Numerous outbreaks of gastroenteritis have been associated with Norwalk virus and Small Round Structured Viruses (SRSVs). These single-stranded RNA viruses, recently classified in the Caliciviridae, have been divided into three genogroups. Antigenic relationships also have been established among the different strains. As both an in vitro culture system and an animal model are lacking for these viruses, virus detection depends primarily on electron microscopy, immunological assays or molecular detection. In this study we first analyzed the genetic homology of the RNA polymerase region for 40 SRSV strains. From a consensus sequence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate primer in combination with three previously described primers in PCR reactions. A panel of 15 stools containing SRSVs, typed when possible by solid phase immune electron microscopy (SPIEM), were selected to represent all three genogroups and four different SPIEM antigenic types. Serial dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to characterize the amplicons obtained. Virus-specific amplicons were obtained with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1000-fold between primer sets. Amplicons from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most sensitive primer pair and SPIEM type were seen. This study showed that a single degenerate primer could be used in cDNA synthesis for a variety of SRSVs but that the sensitivity of the RT-PCR assay depended upon the second primer and virus-specific probes used.

DNA Primers↗
PubMed1996

Evaluation of an antigen capture ELISA based on recombinant mexico virus capsid protein.

BACKGROUND: The diagnosis of gastrointestinal infections caused by small round structured viruses (SRSV) has relied upon electron microscopy and antigen/antibody assays based on Norwalk virus. We investigated cases of gastroenteritis associated with SRSVs employing a new sandwich enzyme-linked immunosorbent assay (ELISA) using hyperimmune animal anti-sera against recombinant Mexico virus capsid protein (rMXV). STUDY DESIGN: One hundred and thirty-five specimens from 86 episodes of gastroenteritis associated with SRSVs, collected in the UK between October 1993 and September 1994, were tested in the rMXV assay. RESULTS: Forty-seven (35%) specimens from 35 of 86 (41%) episodes were positive in the rMXV ELISA and these could further be divided into high and low reactors. Sequencing of a 266-base region of the RNA polymerase gene revealed that strains highly reactive in the rMXV assay demonstrated a high degree of similarity to MXV (97-99% at the nucleotide level), whereas low-reactive strains consist of Mexico-like strains and a heterogeneous group of viruses exhibiting 70-75% similarity to MXV. CONCLUSION: Our results indicate that the rMXV ELISA is predominantly a type specific assay, although some cross reactivity with other genogroup 2 SRSVs was observed. MXV was responsible for 26% of SRSV-associated gastrointestinal infections investigated in the UK during one year's surveillance.

Journal Article↗
PubMed1996

Autism, Asperger's syndrome and the Crick-Mitchison theory of the biological function of REM sleep.

Autism, Asperger's syndrome and other autistic syndromes are developmental brain disorders that cause serious impairments in communication, social interaction, empathy, mood and play. In addition to such deficits, the autistic syndromes involve pathologically high levels of repetitive, stereotypic, ritualistic, compulsive or obsessive behavior, together with extreme resistance to change. According to the Crick-Mitchison theory of the biological function of rapid eye movement sleep, normal brain development in the fetus and infant depends on undisrupted function of a 'reverse learning' mechanism during rapid eye movement sleep. Could abnormalities in this hypothetical reverse learning during rapid eye movement sleep in the fetus explain some aspects of the autistic syndromes? Does the Crick-Mitchison theory suggest if a drug could interfere with rapid eye movement sleep and cross the placental barrier, then that drug might cause developmental brain disorders in the fetus? Should all pregnant women completely avoid caffeine or any agent that might disrupt serotonergic or cholinergic systems?

Autistic Disorder↗
PubMed1996

Diagnosis of parvovirus B19 infection by detection of specific immunoglobulin M antibody in saliva.

Serum and saliva samples were simultaneously collected from patients with B19 infection. Specimens were collected in a period of 1 to 18 days after the onset of symptoms. Saliva samples were collected with a commercial device, OraSure. The quality of these samples was evaluated by determining the concentration of total immunoglobulin G (IgG) by an enzyme immunoassay. The concentration of IgG in these samples ranged from 4.8 to > 250 mg/liter. B19 infection was confirmed for 20 patients by testing sera in a 1: 100 dilution by an IgM capture enzyme immunoassay (MACEIA) and an IgM capture hemadherence test (MACHAT). Saliva samples from these IgM-positive patients were tested neat by MACEIA and MACHAT. IgM could be detected in 11 of 20 (55%) samples by MACEIA and in 15 of 18 (83%) samples by MACHAT. Serum and saliva samples from a further 17 patients with rash were also tested. All of these specimens were unreactive by both assays. These results show that saliva may be a convenient alternative to serum for the diagnosis of recent B19 infection.

Antibodies, Viral↗
PubMed1995

Detection and characterisation of bisegmented double-stranded RNA viruses (picobirnaviruses) in human faecal specimens.

The prevalence of picobirnaviruses (PBVs) in human stools was investigated by polyacrylamide gel electrophoresis (PAGE) analysis of 832 faecal specimens collected between 1982 and 1993 from patients in various clinical groups. Similar prevalences (9-13%) were detected in patients with or without gastroenteritis and throughout the age range of 3 to > 65 years. Two methods for the extraction of nucleic acid, a phenol/chloroform method and a guanidinium thiocynate (GTC)/silica method, were compared. Detection of PBVs by PAGE was three times more sensitive following RNA extraction by the GTC/silica method. Characterisation of three strains was carried out. Segment sizes ranged from 1.625 to 1.95 kilo base pairs (Kbp) and 2.2 to 2.5 Kbp for the fast and slow migrating bands, respectively. The nuclic acid was shown to be double-stranded RNA (dsRNA) by nuclease digestion. PBV-like particles were detected by electron microscopy in two PAGE-positive stools. Virion diameters ranged from 35 to 41 nm and a buoyant density of 1.38-1.4 g/ml in caesium chloride (CsCl) was demonstrated. These findings suggest that PBVs are widespread in humans in the United Kingdom. However, no disease association could be demonstrated.

Adolescent↗
PubMed1995

Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis.

A limitation to date of reverse transcriptase polymerase chain reactions (RT-PCRs) for the detection of small, round structured viruses (SRSVs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (N1/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)-labelled oligonucleotides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from approximately 5% of amplified strains did not hybridise. The N1/E3 primer pair were shown to be SRSV-specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology. Hybridisation of PCR products with the individual oligonucleotides relating to SRSV SPIEM types UK 1-4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT-PCR and offers the potential for direct detection in food and environmental samples.

Animals↗
PubMed1995

Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans.

A picobirnavirus with an atypical genome profile was detected by polyacrylamide gel electrophoresis (PAGE) in 37% (20/54) of human faecal samples also containing oocysts of Cryptosporidium typical of C. parvum. This virus shares many of the characteristics of the previously described picobirnaviruses, but has a significantly smaller genome (1.75 and 1.55 Kbp).

Animals↗
PubMed1995

Monoclonal antibody blocking tests for the detection of HSV-1- and HSV-2-specific humoral responses: comparison with western blot assay.

Monoclonal antibody blocking radioimmunoassays (MAb block RIAs) which detect specific humoral responses to each of the two human herpes simplex virus (HSV) types are described. RIAs were compared with Western blot assay (WBA) in a blind study of 64 sera obtained from clinically well-documented cases of genital herpes. WBA and MAb block RIA each detected HSV-1 antibodies in 16/17 (94%) sera from confirmed HSV-1 genital infections (first episodes and recurrent infections). Detection of HSV-2 antibody in 21 sera from HSV-2 first episodes was more effective by WBA which identified homologous antibody in 19 (96%), whereas MAb block RIA detected HSV-2 antibody in 16 (76%). HSV-2 antibody was detected in 24/25 (96%) sera from recurrent HSV-2 infections by WBA and by MAb block RIA, the highest degree of concordance for both methods. In addition, the MAb block RIA may be more effective in detecting the presence of HSV-1 antibody in sera from recurrent HSV-2 cases. Prevalence of HSV-1 and HSV-2 antibody was measured by the MAb block RIAs in 3 UK human study populations which consisted of 100 children/young adolescents, 104 adult blood donors and 80 genito-urinary medicine clinic attenders.

Adolescent↗
PubMed1995

Felonious sex crime: the possibility of unilateral cerebral irradiation for the offenders.

Some felonious sex crimes might result from compulsions that the perpetrator finds impossible to control. Under judicial systems derived from British law, the court should be extraordinarily careful in mandating any medical procedure. However, if a rapist or child molester voluntarily asks a physician for help, then medical ethics might allow destruction of part of the patient's brain for the purpose of controlling compulsive behaviour. The part of the human brain primarily associated with volition might be near the anterior cingulate sulcus. Could unilateral irradiation of an area near the anterior cingulate sulcus partially destroy the patient's volition? By leaving intact the patient's will based on language and by partially destroying the patient's will based on intuition, could the physician diminish the patient's will to commit felonious sex crimes? Would some patients reject chemical castration but accept unilateral irradiation of an area near the anterior cingulate sulcus?

Adult↗
PubMed1995

Werner syndrome and genetic obesity: speculation.

Werner syndrome (WS) is a rare autosomal recessive disease with poor growth, premature aging, scleroderma-like skin changes, endocrine abnormalities, and deficiencies of adipose tissue. Could there be a genetic obesity syndrome which offers an instructive contrast to at least one form of WS? At least one form of WS might result from an enzyme defect that causes hypertriglyceridemia, hyperinsulinism, and hyperglucagonism; the defective enzyme might play a key role in the utilization of tryptophan, riboflavin (vitamin B2), or other vitamins or in the synthesis of prostaglandins that inhibit insulin secretion. At least one form of genetic obesity might result from an enzyme defect that causes hypotriglyceridemia and hyperinsulinism without hyperglucagonism; the defective enzyme might be unable to bind properly to a product that inhibits some step in the process of conversion of free fatty acid (FFA) CoA into ketoacids.

Glucagon↗