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Biomedical subjects

J A Marshall

Publications and source records attributed to J A Marshall.

At least 19 recordsLinked to original sources
PubMed2002

A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Alleles↗
PubMed2002

The conformation of the mature dimeric human immunodeficiency virus type 1 RNA genome requires packaging of pol protein.

The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in trans as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.

Cell Line↗
PubMed2001

Synthesis of (-)-deoxypukalide, the enantiomer of a degradation product of the furanocembranolide pukalide.

A convergent stereoselective synthesis of (-)-deoxypukalide is described. This substance has not yet been found in Nature but is obtained through deoxygenation of pukalide, the first naturally occurring furanocembrane to be structurally elucidated. The route features a new intraannular furan synthesis that entails treatment of a 4-oxopropargylic beta-keto ester with silica gel. The product of this novel reaction, a 3-carboxy 2,5-bridged furan, is formed in 96% yield. The synthetic strategy was strongly directed by molecular mechanics calculations, which provided valuable insight into stereodefining steps including double bond stereochemistry and butenolide configuration.

Animals↗
PubMed2001

Synthesis of stereopentad analogues of the C14-C22 segment of callystatin A through additions of chiral allenylzinc reagents to stereotriads.

The addition of (P)- and (M)-allenylzinc reagents, prepared in situ through Pd-catalyzed metalation of (R)- and (S)-3-butyn-2-ol mesylates, to diastereomeric stereotriad aldehydes 8, 13, 18, and 23 of syn,syn, syn,anti, anti,anti, and anti,syn stereochemistry was examined. Additions to the former two aldehydes afforded the four anti adducts with high diastereoselectivity and negligible mismatching. Significant mismatching was observed with the latter two aldehydes and the (M)-allenylzinc reagent. An evaluation of possible transition states is presented in consideration of steric and dipolar control elements.

Aldehydes↗
PubMed2001

Synthesis of a C1-C21 subunit of the protein phosphatase inhibitor tautomycin: a formal total synthesis.

The synthesis of a C1-C21 subunit of tautomycin is described. The convergent route employs enantioenriched allenylstannane and zinc reagents derived from (S)-3-butyn-2-ol methanesulfonate. These reagents react with appropriate aldehyde segments to yield syn and anti adducts with high diastereoselectivity. The derived lithioalkynes are joined stepwise to a CO equivalent, (MeONMe)2C=O, to afford an intermediate ketone which is converted to the core spiroketal moiety of tautomycin upon acid treatment. Chain elongation by another addition of the aforementioned allenylzinc reagent to a spiroketal aldehyde proceeds with high diastereoselectivity to install the remaining stereocenters. The resulting homopropargylic alcohol adduct is converted to a methyl ketone through intramolecular hydrosilylation of the alkyne and Tamao oxidation of the derived five-membered siloxane. This ketone proved identical to an intermediate employed by Chamberlin in a prior total synthesis of tautomycin.

Antifungal Agents↗
PubMed2001

Soybean sterol composition and utilization by Phytophthora sojae.

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.

Molecular Structure↗
PubMed2001

Simultaneous isolation of verotoxin-producing strains of Escherichia coli O128:H2 and viruses in gastroenteritis outbreaks.

Three outbreaks of gastroenteritis from which the Verotoxin producing Escherichia coli serotype O128:H2 was isolated are reported. In addition Norwalk-like viruses were detected in patients from two of the outbreaks and astrovirus in the third outbreak. While it cannot be specifically determined which of these agents played the major role in these outbreaks, the findings suggest that the viral agents need to be considered in investigations of gastroenteritis outbreaks, regardless of whether bacterial enteropathogens have also been isolated. This study points to a strong need to investigate gastroenteritis outbreaks for both bacterial and viral agents and to review in detail the asymptomatic carriage rate of Verotoxin-producing bacteria and gastroenteritis-associated viral agents; these areas of public health significance have been largely neglected.

Australia↗
PubMed2001

Uptake of HIV and latex particles by fresh and cultured dendritic cells and monocytes.

Blood dendritic cells (DC) efficiently carry HIV-1 and transmit infection to CD4+ T cells in the absence of productive infection of the APC. Fluorescent latex beads were used to define the endocytic pathways that may contribute to this non-infectious pathway of virus carriage. Beads between 14 nm and 2300 nm in diameter were taken up by uncultured blood DC, but uptake of beads larger than 280 nm was much reduced in the DC compared to monocytes. After culture, there was a reduction in bead carriage in DC compared to monocytes. In the DC, beads were found as small aggregates in class II containing compartments or as single beads just below the cell surface. Beads accumulated in monocytes as aggregates in class II negative compartments. Bead recycling occurred in DC, but not in the fresh or cultured monocytes. Electron microscopy of HIV-1-pulsed DC cultured with CD4+ T cells showed accumulation of apoptotic debris and virions within endosomes in the DC. The peripheral location and recycling of endocytosed material in DC provides a pathway for virion transfer from DC to T cells that does not occur in monocytes.

Cells, Cultured↗
PubMed2001

A novel method for counting eimerian oocysts at very low concentrations in aqueous suspensions.

A novel method for counting eimerian oocysts in samples of drinking water has been developed to fulfil the need for monitoring the delivery of very low concentrations of live anticoccidial vaccines to poultry via pipeline nipple-drinker systems. Advantages of the method are the ease of sample collection and processing, high degrees of accuracy and precision, and a sensitivity of one oocyst ml(-1). Results of a validation test are presented, with a protocol for the method and notes on its use. The coefficient of variation (CoV) of 10 sets of oocyst counts with nominal means of 10 to 160 oocysts ml(-1)ranged from about 16 per cent down to 6 per cent. The recovery efficiency for all 100 validation counts averaged 100.2 per cent with a range of 70-130 per cent. A practical example of field use of the method is given, including a modification to decrease the time taken for counting. In this case, when oocysts were pumped around a pipeline circuit of 129 m for 2.5 hours, the CoV of a mean of 112 oocysts ml(-1)(n = 10) was 12.4 per cent.

Animals↗
PubMed2001

Normal bone mineral density after treatment for childhood acute lymphoblastic leukemia diagnosed between 1991 and 1998.

OBJECTIVE: We investigated whether previous reports of reduced bone mineral density after management for childhood acute lymphoblastic leukemia (chALL) were confirmed in a more recently treated cohort. STUDY DESIGN: In a cross-sectional study 75 subjects who were given the diagnosis of chALL between January 1, 1991, and December 31, 1997 (69% standard, 31% high risk), at Denver Children's Hospital and who were 11 to 82 months post-diagnosis with no history of relapse, secondary malignancy, or transplant underwent whole body areal bone mineral densitometry (BMD(A) expressed as age- and sex-standardized z scores), a food frequency questionnaire, and a weight-bearing activity survey. RESULTS: Overall, the mean whole body BMD(A) z score was normal (+0.22 +/- 0.96). A significant positive association was found with whole body BMD(A) z score and years elapsed since the beginning of maintenance (linear regression coefficient = +0.2 Deltaz score/year; 95% CI = 0.09 to 0.3) after adjustment was done for risk status/age category, history of cranial radiation, and total days hospitalized. No association was found with high risk/older age at diagnosis, nutrient intake, chemotherapy dosage, or weight-bearing activity. CONCLUSION: Contrary to previous reports in which cranial radiation and longer hospitalizations were prominent components of therapy, our study suggests that more recently treated patients with chALL do not have persistent abnormalities of bone mineral density after completion of therapy.

Biomarkers↗
PubMed2001

Multiple outbreaks of Norwalk-like virus gastro-enteritis associated with a Mediterranean-style restaurant.

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

Caliciviridae Infections↗
PubMed2001

Heminested multiplex reverse transcription-PCR for detection and differentiation of Norwalk-like virus genogroups 1 and 2 in fecal samples.

The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 microl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

Caliciviridae↗
PubMed2001

Proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation.

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

Dimerization↗
PubMed2001

High level excretion of Norwalk-like virus following resolution of clinical illness.

We report the case of an elderly woman excreting high levels (about 5 x 10(5) virions per gram of faeces) of Norwalk-like virus (NLV) in the absence of any clinical symptoms of gastroenteritis. Analysis by reverse transcription, polymerase chain reaction and DNA sequencing was carried out on a 342-nucleotide region of open reading frame 1. This indicated that the NLV belonged to genogroup 2 and was more closely related to the Camberwell subgroup, the most common circulating in southeast Australia at present, than to the Norwalk and Mexico viruses.

Aged↗