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Biomedical subjects

K W Brown

Publications and source records attributed to K W Brown.

At least 37 records · Page 2Linked to original sources
PubMed1997

Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector.

Cytomegalovirus-based mammalian expression vectors are widely used to drive the expression of transfected genes in cultured cells. Immunofluorescent staining of the WT1 protein in 3T3 and 293 cell clones, stably transfected with a cyomegalovirus (CMV) expression vector carrying a cDNA coding for the tumour suppressor protein WT1, showed extreme cell to cell variation in the amount of recombinant protein expressed, indicative of cell cycle dependence. This was investigated further by Western blot and FACS analysis which showed that WT1 protein expression was highest in S phase and almost absent in G0/G1. Northern blot analysis of cell clones expressing sense or antisense WT1 cDNAs regulated by the CMV promoter/enhancer showed that RNA expression was also cell cycle-dependent. Western blotting of cells expressing a luciferase reporter gene driven by the CMV promoter/enhancer also showed apparent cell cycle-dependent expression. We further demonstrated that the expression of these gene constructs was serum responsive with a 10-fold increase in expression occurring 2 h after the addition of serum. These results show that the CMV promoter/enhancer system varied in its response to serum and the cell cycle state. Therefore, care must be taken when interpreting any phenotypic alterations (or lack of them) produced in cells transfected with CMV-based expression vectors.

3T3 Cells↗
PubMed1997

Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate.

Induction of a mucosal immune response is generally thought to require introduction of an immunogen directly onto the mucosal surface. It has been observed, however, that live, attenuated mutants of the alphavirus, Venezuelan equine encephalitis virus (VEE), induce protection from virulent challenge at the respiratory mucosa even after parenteral inoculation. In this report, we propose a mechanism by which subcutaneous immunization with a molecularly cloned, attenuated double mutant of VEE is able to stimulate the production of mucosal anti-VEE IgA. Our results showed that the immunizing virus spread to, and replicated within, lymphoid tissues throughout the mouse. Several tissues known to be inductive sites of the mucosal immune system were found to be positive for the presence of VEE RNA by 48 hr postimmunization. Moreover, this mucosal lymphotropism resulted in the production of virus-specific IgA antibody detectable in vaginal secretions of immunized mice.

Administration, Intranasal↗
PubMed1997

Does unhappiness make you sick? The role of affect and neuroticism in the experience of common physical symptoms.

The relative strength of both affective state and the personality trait neuroticism in predicting common physical symptoms was tested. The authors used an event-sampling design to overcome methodological limitations of past research in the area. Contrary to much previous research, neuroticism was found to be unrelated to reports of physical symptoms, although it was found to be related to unpleasant affective state. Unpleasant affect bore a strong concurrent relation to the frequency of reported symptoms. Temporal relations between experiences of unpleasant affect and subsequent symptoms were found for some individuals, but wide individual variability was seen in both the strength and direction of this linkage. The findings suggest that when individuals are asked to report their subjective experiences of physical illness without the necessity to retrospect over significant periods of time, unpleasant affective state is more strongly related to experiences of symptoms than is the trait neuroticism.

Adaptation, Psychological↗
PubMed1996

Localization of a novel t(1;7) translocation associated with Wilms' tumor predisposition and skeletal abnormalities.

Cytogenetic analysis of predisposition syndromes has played a critical role in the elucidation of the genetics of Wilms' tumor (WT). Therefore, we became interested in a patient who presented with a WT and a nephrogenic rest in the contralateral kidney (suggestive of a predisposition) and a de novo t(1;7)(q42;p15) constitutional translocation as the only visible cytogenetic abnormality. He also had bilateral radial aplasia and other skeletal abnormalities, but there was no manifestation of any syndrome previously associated with WT. In the tumor, the translocation was retained, and the other 7p region was lost by the formation of an isochromosome i(7q). Here, we report the localization of the chromosome 7 breakpoint within a yeast artificial chromosome (YAC) contig by using fluorescence in situ hybridization (FISH), localizing the breakpoint between markers sWSS355 and sWSS1449. A number of YACs span the breakpoint and, thus, contain the region that is disrupted by the translocation. This may represent the site of a novel tumor suppressor gene that is involved in WT and also in normal renal development.

Bone and Bones↗
PubMed1996

Imprinting mutation in the Beckwith-Wiedemann syndrome leads to biallelic IGF2 expression through an H19-independent pathway.

The Beckwith-Wiedemann syndrome (BWS) is genetically linked to chromosome 11p15.5, and a variety of observations suggest that deregulation of imprinted genes in this region is causally involved in the pathogenesis of the disease. It has been shown that in some patients without cytogenetic abnormalities the otherwise repressed maternal copy of the insulin-like growth factor 2 (IGF2) gene is expressed, leading to biallelic expression of IGF2. In some of these cases, this is accompanied by repression and DNA methylation of the maternal (otherwise active) copy of the neighbouring H19 gene. Hence, it is attractive to think that mutations may interfere with some aspect of H19 imprinting, thus leading to an inactive maternal allele, and indirectly to activation of the maternal IGF2 allele as reported in mice with an H19 gene deletion. However, no mutations have been identified so far in these patients. The only known mutations associated with BWS are maternally transmitted translocations, which are clustered in two locations centrometric to IGF2. The first cluster is 200-400 kb from IGF2 and the second is several megabases away. Hence, genes located far from the translocation breakpoints are potentially deregulated by them. Here we provide the first evidence of alteration of imprinting in a translocation family, with biallelic expression of IGF2 and altered DNA replication patterns in the IGF2 region. Interestingly, H19 imprinting was normal, suggesting an H19-independent pathway to biallelic IGF2 transcription. DNA methylation in IGF2 remained monoallelic, suggesting that the mutation in this family had uncoupled allele-specific methylation from expression.

Alleles↗
PubMed1996

Caudate nucleus area in drug-induced parkinsonism.

This study examined caudate nuclei areas in 10 schizophrenic subjects with and 25 schizophrenic subjects without parkinsonian symptoms. The subjects with parkinsonian symptoms were found to have statistically significantly smaller right caudate nuclei and a trend towards smaller left caudates. Cortical measurements did not differ between the groups. These results are consistent with the suggestion that antipsychotic drugs exert some neurotoxic effects, but should be viewed as preliminary and require replication.

Aged↗
PubMed1996

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.

Animals↗
PubMed1996

Caudate nucleus morphology in tardive dyskinesia.

OBJECTIVE: The objective of this project was to test whether there are differences in the size of the caudate nucleus in schizophrenic in-patients with and without tardive dyskinesia. METHOD: The study was cross-sectional in design, examining group differences between institutionalised schizophrenic patients with and without tardive dyskinesia, using non-enhanced computerised tomography scans of the brain. The group comprised 15 schizophrenic patients with persistent tardive dyskinesia and 21 in-patient schizophrenic controls who were group-matched for demographic variables. RESULTS: The dyskinetic subjects had a significantly larger left caudate nucleus and tended to have a larger right caudate nucleus than the controls. There were no differences between the groups on any of the measures of cerebral atrophy. CONCLUSIONS: The findings can be understood within the context of models of neostriatal function. It is possible that a larger caudate nucleus could be used to identify patients at risk of developing tardive dyskinesia.

Adult↗
PubMed1995

Identification of an antisense WT1 promoter in intron 1: implications for WT1 gene regulation.

Expression of the Wilms' tumour suppressor gene, WT1, is tightly regulated during nephrogenesis, and loss of function of its gene product correlates with malignancy. By using luciferase reporter gene constructs containing DNA sequences from the first intron of the WT1 gene, we have identified an antisense WT1 promoter operational in the opposite direction relative to the 5' promoter. Transcription directed by the promoter is negatively regulated by upstream elements, but is activated by expression of WT1. This effect of WT1 is reciprocal to that observed on the 5' promoter, suggesting that antisense promoter activity is involved in WT1 gene regulation. By mimicking expression of the transcript regulated by the antisense promoter, we demonstrate that cellular levels of WT1 can be effectively down-regulated by antisense mRNA complementary to sequences in the first exon of WT1.

Base Sequence↗
PubMed1995

Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second-site suppressor mutation in E1.

The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a small proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at E1 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and E1 proteins in the alphavirus spike. They also have implications for the design of recombinant live virus vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation.

Amino Acid Sequence↗
PubMed1995

Twin analysis of odor identification and perception.

Findings from the first twin analysis using the University of Pennsylvania Smell Identification Test (UPSIT) and a phenyl ethyl alcohol (PEA) threshold detection test are presented. A genetic influence on odor identification was suggested for males, but not for females, consistent with previous twin research on physical measures. In addition, females scored significantly higher on the UPSIT than males. A curvilinear age trend for odor identification was detected for males, but not for females. In contrast, neither genetic, age, nor gender effects were suggested for PEA sensitivity. Performance on the olfactory tests was unrelated to scores on standard measures of intelligence.

Adolescent↗
PubMed1995

Imprinting mutations in the Beckwith-Wiedemann syndrome suggested by altered imprinting pattern in the IGF2-H19 domain.

Regional regulations of parental imprinting in the IGF2-H19 domain of imprinted genes was studied in the Beckwith-Wiedemann syndrome (BWS). We identified BWS patients who had inherited a normal biparental chromosome complement of the chromosome 11p15.5 region (where IGF2 and H19 reside), but had an altered pattern of allelic methylation of both genes, with the maternal chromosome carrying a parental imprinting pattern. In fibroblasts, IGF2 was expressed from both parental alleles and H19 was not expressed, precisely as predicted from the altered pattern of allelic methylation. Interestingly, DNA replication patterns of the 11p15.5 region remained asynchronous as in controls. Our results therefore provide the first example of the dissociation of regional control of DNA replication from regional control of allelic methylation and expression in imprinting. We suggest that the altered pattern of allelic methylation and expression arises in the germline or in the early embryo from defects in resetting or setting of imprinting in maternal germline. Potential candidate regions for mutations include the previously identified translocation breakpoint clusters and the H19 gene itself. The finding of possible 'imprinting mutations' in BWS raises the prospect of identifying genetic factors that control imprinting in this region.

Base Sequence↗
PubMed1994

Germline and somatic abnormalities of chromosome 7 in Wilms' tumor.

Although a gene (WT1) located at chromosome 11p13 is implicated in the development of Wilms' tumor (WT), there is evidence that genes on other chromosomes are also involved. A WT patient presented with a constitutional balanced translocation between chromosomes 1 and 7, t(1;7)(q42;p15), the breakpoints of which could represent a WT predisposition gene in this patient. Cytogenetic analysis of the tumor from this patient revealed an acquired abnormality of the other chromosome 7, resulting in an isochromosome of the long arm and a 46,XY,t(1;7)(q42;p15)c,i(7)(q10) karyotype. The regions of the translocation breakpoints were investigated in a series of 24 WTs using Southern blot analysis. This confirmed the monosomy of 7p and trisomy of 7q in the tumor of the translocation patient, and in addition a loss of chromosome 7p alleles was identified in a WT of a bilaterally affected patient. In addition, two WTs were shown to have an extra copy of chromosome 7 alleles. Multiple copies of chromosome 1q alleles, probably resulting from secondary changes, were observed in two WTs, one of which was also associated with a trisomy of chromosome 7. These results indicate that 7p may contain a tumor suppressor gene involved in WT development, and that duplications of 7q also may play a role in WT development.

Alleles↗
PubMed1994

Autoregulation of the human WT1 gene promoter.

The human Wilms tumour suppressor gene, WT1, encodes a zinc-finger protein which can function as a transcriptional activator or suppressor. This study reports the analysis of the human WT1 gene promoter, and demonstrates that high levels of WT1 expression lead to autosuppression of the WT1 promoter. Deletion analyses of the promoter region implicate sequences 5' and 3' of the transcriptional start site as being crucial in WT1 autosuppression. Loss or alteration of this function of WT1 may be important in tumourigenesis.

DNA Mutational Analysis↗
PubMed1994

Somatic allelic loss at the DCC, APC, nm23-H1 and p53 tumor suppressor gene loci in human prostatic carcinoma.

We present a restriction fragment length polymorphism (RFLP) analysis of 29 benign and 30 malignant prostatic tumors, using polymorphic DNA probes to the putative tumor suppressor genes DCC (Deleted in Colorectal Carcinoma; chromosome 18q21.3), nm23-H1 (17q21.3), APC (Adenomatous Polyposis Coli; 5q21) and p53 (17p13). Six of 23 evaluable cancers (26%) showed loss of heterozygosity (LOH) at DCC; 5 were advanced stage and one was clinically localized (p < 0.05). Mapping 18q deletions, another (advanced) cancer showed LOH at a locus distal to DCC (18q22), but no LOH at DCC. Three of 15 evaluable cancers (20%), all advanced, showed LOH at APC. Three of eight (38%) cancers, of which 2 were advanced, showed LOH at p53. One high grade/stage cancer of 21 (5%) showed LOH at nm23-H1 (and also at DCC). Combining data, allelic losses at either DCC, APC, or p53 genes were seen in 13% of localized cancers, but in 71% of advanced cancers (p < 0.002). Allelic loss involving nm23-H1 is rare in prostatic carcinoma. We suggest that loss of tumor suppressor genes DCC and/or an unidentified gene located distally on chromosome 18q, APC, or p53 may influence progression in prostatic carcinoma.

Adenocarcinoma↗