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Biomedical subjects

N Gilbert

Publications and source records attributed to N Gilbert.

At least 19 recordsLinked to original sources
PubMed2002

Comparison of commercial assays for the quantification of HBV DNA load in health care workers: calibration differences.

Until recently, carriers of hepatitis B virus (HBV) were allowed to undertake exposure prone procedures providing their serum did not contain HBeAg. However, the recent description of hepatitis B transmission events occurring from HBV-infected health care workers who conduct exposure prone procedures demonstrated that the then current Department of Health guidelines needed to be revised. As part of a series of studies carried out to determine if viral load measurements are a more secure means of assessing the conduct of exposure prone procedures, the suitability of commercially available assays for HBV DNA detection and quantification were investigated. This study describes a comparative analysis on the performances of three assays each based on a different methodology. The assays included the QUANTIPLEX HBV DNA Assay (bDNA), (Chiron Diagnostics Ltd.), the AMPLICOR HBV Monitor Test, (Roche Diagnostics Systems) and the Digene Hybrid Capture System HBV DNA Assay (Digene Corporation). Calibration curves from experiments using the Eurohep ad and ay HBV DNA standard controls indicated a close correlation between the three assays over the dynamic ranges claimed by the manufacturers, although the Quantiplex assay did appear to be over-reporting. This became more apparent when testing patients undergoing anti-viral therapy where the Quantiplex assay consistently over-reported by 0.5 log(10) when compared with the Amplicor assay. The results of this study indicate that based on its dynamic range, the Amplicor HBV Monitor test is the most appropriate assay for the routine investigation of anti-HBe carriers, which will have lower levels of HBV DNA. The investigation also highlights the need for using accepted standard HBV DNA control sera. This will be essential when using an assay to establish whether health care workers who are hepatitis B carriers can be allowed to perform exposure prone procedures under the new guidelines of the UK Department of Health.

Calibration↗
PubMed2001

Distinctive higher-order chromatin structure at mammalian centromeres.

The structure of the higher-order chromatin fiber has not been defined in detail. We have used a novel approach based on sucrose gradient centrifugation to compare the conformation of centromeric satellite DNA-containing higher-order chromatin fibers with bulk chromatin fibers obtained from the same mouse fibroblast cells. Our data show that chromatin fibers derived from the centromeric domain of a chromosome exist in a more condensed structure than bulk chromatin whereas pericentromeric chromatin fibers have an intermediate conformation. From the standpoint of current models, our data are interpreted to suggest that satellite chromatin adopts a regular helical conformation compatible with the canonical 30-nm chromatin fiber whereas bulk chromatin fibers appear less regularly folded and are perhaps intermittently interrupted by deformations. This distinctive conformation of the higher-order chromatin fiber in the centromeric domain of the mammalian chromosome could play a role in the formation of heterochromatin and in the determination of centromere identity.

3T3 Cells↗
PubMed2001

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens.

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.

Alleles↗
PubMed2001

Human L1 retrotransposition: cis preference versus trans complementation.

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

Base Sequence↗
PubMed2000

Evolutionary inventions and continuity of CORE-SINEs in mammals.

We characterized short interspersed elements (SINEs), of the CORE-suprafamily in egg-laying (monotremes), pouched (marsupials) and placental mammals. Five families of these repeats distinguished by the presence of distinct LINE-related 3'-segments shared tRNA-like promoter and the central core region. The putative active elements were reconstructed from the alignment of genomic repeats representing molecular fossils of sequences that amplified in the past and since then underwent multiple mutations. Their mode of proliferation by retroposition was indicated by the presence of: (1) internal RNA PolIII promoter; (2) simple sequence repeated tail; (3) direct repeats; and (4) subfamilies recording the evolution of elements. The copy number of CORE-SINEs in placental genomes was estimated at about 300,000; they were highly divergent and apparently ceased to amplify before radiation of these lineages. On the other hand, among almost half a million fossil elements present in marsupials and monotremes, the youngest subfamilies could still be retropositionally active. CORE-SINEs terminate in sequence repeats of a few nucleotides similar to their 3'-segment LINE-homologues, CR1, L2 and Bov-B. These three LINE elements fall into clades distinct from that of L1 elements which, similar to their co-amplifying SINEs, end in a poly(A) tail. We propose a model in which new CORE-families, with distinct 3'-segments, are created at the RNA level due to template switching between LINE and CORE-RNA during reverse transcription. The proposed mechanism suggests that such an adaptation to the changing amplification machinery facilitated the survival and prosperity of CORE-elements over long evolutionary periods in different lineages.

Animals↗
PubMed1999

Saskatchewan physicians' attitudes and knowledge regarding assessment of medical fitness to drive.

BACKGROUND: Although legislation has been introduced in Saskatchewan for mandatory reporting by physicians of patients considered medically unfit to drive, little is known about physicians' attitudes, knowledge or resources with regard to evaluating medical fitness to drive. METHODS: The objective of this study was to determine Saskatchewan physicians' attitudes, knowledge, training, resources and current educational needs with regard to evaluating medical fitness to drive. A questionnaire survey of all physicians in the province who were identified as likely to be involved in determining medical fitness to drive was conducted between October and December 1996. RESULTS: Of the 1102 physicians who received a questionnaire, 690 (62.6%) responded, of whom 167 were excluded because they were not involved in assessing fitness to drive. Thus, 523 (55.9%) of the 935 eligible physicians surveyed completed the questionnaire. Most (57.6% [298/517]) of the respondents indicated that they do not hesitate to report patients medically unfit to drive; however, 59.5% (307/516) felt that the physician-patient relationship is negatively affected by reporting. Overall, 85.5% (444/519) of the respondents felt that restricted licensing is a fair alternative for people who might otherwise be denied a full licence. The availability of restricted licensing positively influenced the decision to report for 60.3% (313/519) of the respondents. Significantly more rural physicians than urban physicians believed that the need to drive was greater for rural residents than for urban dwellers (81.2% [95/117] v. 64.2% [257/400], p < 0.001). Physician knowledge regarding specific medical conditions and fitness to drive was generally poor. The resource most commonly used in determining medical fitness to drive was the Physicians' Guide to Driver Examination (71.1% [361/508] of respondents). The most useful continuing medical education methods indicated by physicians for assessing medical fitness to drive included conference presentations, workshops and journal articles. INTERPRETATION: Most of the Saskatchewan physicians surveyed supported restricted licensing, and the availability of restricted licensing made them more likely to report patients considered medically unfit to drive. The physician-patient relationship was felt to be negatively affected by reporting.

Attitude of Health Personnel↗
PubMed1999

CORE-SINEs: eukaryotic short interspersed retroposing elements with common sequence motifs.

A 65-bp "core" sequence is dispersed in hundreds of thousands copies in the human genome. This sequence was found to constitute the central segment of a group of short interspersed elements (SINEs), referred to as mammalian-wide interspersed repeats, that proliferated before the radiation of placental mammals. Here, we propose that the core identifies an ancient tRNA-like SINE element, which survived in different lineages such as mammals, reptiles, birds, and fish, as well as mollusks, presumably for >550 million years. This element gave rise to a number of sequence families (CORE-SINEs), including mammalian-wide interspersed repeats, whose distinct 3' ends are shared with different families of long interspersed elements (LINEs). The evolutionary success of the generic CORE-SINE element can be related to the recruitment of the internal promoter from highly transcribed host RNA as well as to its capacity to adapt to changing retropositional opportunities by sequence exchange with actively amplifying LINEs. It reinforces the notion that the very existence of SINEs depends on the cohabitation with both LINEs and the host genome.

Animals↗
PubMed1997

Plant S1 SINEs as a model to study retroposition.

The S1 element is a plant SINE (Short INterspersed Element) that was first described and studied in Brassica napus and is widely distributed among Cruciferae, especially in species of the Brassiceae tribe. We propose that S1 amplification in Cruciferae could represent a good eukaryotic model to study retroposition. This is based on the fact that S1 elements share clear structural and evolutionary characteristics with mammalian SINEs but are present in a much lower copy number (500 loci by haploid genome for the S1 element in B. napus compared to 700,000 loci by haploid genome for the Alu element in human). This low copy number allows the characterization of a large portion of SINEs from a given plant species. This can lead to a more precise understanding of the evolutionary history of SINE amplification and can more easily allow an evaluation of the impact of retroposition on the evolution of that species. It can also lead more rapidly to the characterization of genomic elements active in transcription and retroposition so that the cellular control of these elements can be addressed. Finally, we show that the study of S1 insertion sites can reveal information on the RNA reverse transcription and integration step of the retroposition process.

Base Sequence↗
PubMed1996

A transcriptional analysis of the S1Bn (Brassica napus) family of SINE retroposons.

S1Bn is a plant short interspersed element (SINE) whose amplification probably involves the reverse transcription of an RNA intermediate. In this report, we identified and characterized S1Bn transcripts from different Brassica napus tissues. Despite the presence of a consensus internal POL III promoter in a large number of genomic S1Bn elements, we observed that S1Bn transcripts are rare in B. napus cells. The use of two very sensitive methods (RT-PCR and RACE PCR) allowed the characterization of 102 independent S1Bn cDNA clones from three different tissues (shoot, root and callus). From this analysis, we conclude that the majority of S1Bn transcripts probably result from a small number of cotranscriptional events where an S1Bn element is transcribed due to its presence in a POL II transcriptional unit. Specific POL III RNA transcripts, initiating at the first 5' nucleotide of the DNA element, are also present in the tested tissues and possibly result from the transcriptional activity of as few as three genomic elements. Two of these transcripts could represent master transcripts responsible for the amplification of S1Bn subfamilies. We also observed that the population of specific POL III transcripts varies among the three tested tissues and that some transcripts appear completely tissue-specific.

Base Sequence↗
PubMed1996

Hepatitis B virus surface mutations associated with infection after liver transplantation.

BACKGROUND/AIMS: Liver transplantation for chronic liver disease due to hepatitis B virus infection is associated with a high risk of graft infection, graft failure and death. Many centres restrict this procedure to those seronegative for HBV-DNA (by hybridisation assay) and use prophylactic polyclonal human hepatitis B specific immunoglobulin to prevent infection of the graft, despite the very high cost. METHODS: We describe three patients who underwent liver transplantation for chronic HBV-related disease in whom death was due to fibrosing cholestatic hepatitis following graft infection with hepatitis B virus, despite receiving hepatitis B specific immunoglobulin. Variation within the immunodominant a epitope of HBsAg was sought by analysis of hepatitis B virus sequences and the use of a point mutation assay, following amplification from serum by the polymerase chain reaction. RESULTS: Prior to transplantation, Cases 1 and 2 had mutations at nucleotide 1902 (codon 145), resulting in G-C substitutions, which persisted at a low level after transplantation. In Case 2 a second mutant type with a G-A substitution at nucleotide 1902, became the predominant viral type post transplant. Case 3 had exclusively wild type virus before and after transplantation. The emergence of mutant type virus in Case 2 may have occurred because of immune pressure exerted by high titre anti-HBs detectable for more than 7 months. Cases 1 and 3 received only brief courses of anti-HBs therapy. The mutant viral surface antigen was not detected by a monoclonal antibody-based assay, and therefore the choice of HBsAg assay for post-transplant monitoring of patients who receive liver grafts for hepatitis B virus disease is important. CONCLUSIONS: A search for mutations affecting the a determinant prior to liver transplantation for HBV-related liver disease may help to identify those at risk of failure of prophylaxis. Monoclonal antibodies specific to the codon 145-mutant surface antigen might prevent graft infection, but other mutations might then emerge.

Base Sequence↗
PubMed1995

Haemoglobin-based blood substitutes and sepsis.

An important concern that has received little attention is the possible increased susceptibility to bacterial infections of patients infused with cell-free haemoglobin-based blood substitutes. We show that pyridoxalated polymerised human haemoglobin promotes fulminating Escherichia coli septicaemia in mice, which draws attention to the potential danger of such products in the clinic.

Animals↗
PubMed1995

Three-dimensional co-location of RNA polymerase I and DNA during interphase and mitosis by confocal microscopy.

The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells. For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.

Carcinoma, Squamous Cell↗
PubMed1994

Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy.

The gelatinase A (72 kDa type IV collagenase) is a matrix metallo-proteinase which degrades basement membrane collagens. Various studies emphasize its role in stromal invasion of cancers, but there is some controversy about its origin. Gelatinase A was localized by immunohistochemistry using confocal microscopy in 15 human mammary carcinomas. In addition, the cells responsible for the synthesis of this enzyme were detected by in situ hybridization. Most invasive and non-invasive tumour cells were labelled by immunohistochemistry. Of particular interest was the pattern observed in some pre-invasive areas. Gelatinase A was found in fibroblasts in close contact with pre-invasive tumour clusters. Confocal observation allowed a more precise localization of gelatinase A to the periphery of tumour clusters along the basement membranes and in peritumour fibroblasts. The malignant epithelial cells were negative by immunohistochemistry in these areas. By in situ hybridization, mRNAs encoding gelatinase A were detected only in fibroblasts in close contact with pre-invasive and well differentiated tumour clusters. These findings support the hypothesis that peritumour fibroblasts produce gelatinase A and that breast cancer cells may bind this enzyme to their cell surface and/or internalize it.

Amino Acid Sequence↗
PubMed1994

The relation between climatic temperature and sudden infant death syndrome differs among communities: results from an ecologic analysis.

We examined the negative relation between temperature and the sudden infant death syndrome (SIDS) in 22 communities in seven countries. We estimated the percentage increase in SIDS rate for a 1 degree C drop in climatic temperature. The relation differed substantially among communities. In New Zealand and Australia (10 communities), the association was consistently strong; in Europe (seven communities), it varied from strong to weak; and in the USA (five communities), it was moderate or weak. We postulate that low climatic temperature indirectly increases the incidence of SIDS, particularly in countries where outdoor climatic temperature modifies the indoor temperature and clothing habits.

Australia↗
PubMed1994

Power and limits of laser scanning confocal microscopy.

In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out-of-focus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and non-destructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three-dimensional reconstitution of nuclear components using a cubic representation and a ray-tracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3-D data set: self-shadowing effects of thick specimens, spherical aberrations due to the sub-optimum use of the objective lenses and photo-bleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for a 3-D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers and a possible means to attenuate photodamage to biological specimens.

Animals↗
PubMed1994

Three-dimensional co-localization of nucleolar argyrophilic components and DNA in cell nuclei by confocal microscopy.

Silver dots deposited specifically on proteins of the nucleolar organizer regions (Ag-NOR proteins) after a one-step silver staining technique were visualized in cells in culture, in cells in smears, and in tissue sections, with a scanning laser confocal microscope working in the reflectance mode. After specific labeling of DNA with the fluorescent dye chromomycin A3, DNA and silver dots could be observed either individually or simultaneously. Therefore, it was possible to study the three-dimensional organization of nucleolar silver-stained structures relative to DNA with a high X, Y, and Z resolution. Our results showed that the argyrophilic components are organized as a twisted necklace structure within interphase nucleoli of cells in culture. We also demonstrated a striking three-dimensional symmetric disposition of NORs within the two sets of chromosomes in telophase cells. Similar results were obtained for cells in smears, although their three-dimensional organization was somewhat disturbed due to air-drying. We also demonstrated that silver dots cannot be visualized in the reflectance mode within sections of paraffin-embedded tissues. However, their simultaneous demonstration in non-confocal transmitted light, together with that of DNA in confocal mode, appeared very useful to study their localization within nuclei and mitotic chromosomes.

Adenocarcinoma↗
PubMed1992

Climatic temperature and variation in the incidence of sudden infant death syndrome between the Australian states.

OBJECTIVE: To describe the relationship between climatic temperature and the incidence of sudden infant death syndrome (SIDS) for the Australian States and examine the extent to which differences in climatic temperature might explain the regional variation of SIDS in Australia. DESIGN: Case series study. A generalised linear model was used to model the association between monthly average temperature and the incidence of SIDS. SETTING: The report is population based. Data are available from all Australian States. SUBJECTS: Cases of SIDS from birth to less than 12 months of age occurring in Queensland (1981-1987), New South Wales (1981-1987), Victoria (1984-1987), Tasmania (1975-1989), South Australia (1980-1989), and Western Australia (1980-1988). RESULTS: Every one degree Celsius decrease in average monthly temperature within the range 9 degrees C to 25 degrees C is associated with a 10.6% (95% confidence interval, 9.6%-11.7%) increase in the incidence of SIDS. Climatic temperature accounts for 84% of the interstate variation in the rate of SIDS. After controlling for the effect of temperature, a significant overall difference in SIDS incidence remains (P less than 0.0001) for the Australian States. CONCLUSION: Climatic temperature accounts for most but not all of the regional variation of SIDS incidence in the Australian States. The remaining variation may reflect differences in the maternal and infant characteristics of the State populations.

Australia↗