Biomedical subjects

N J Fitch

Publications and source records attributed to N J Fitch.

12 recordsLinked to original sources

PubMed2000

Human brain cytochrome P450 1B1: immunohistochemical localization in human temporal lobe and induction by dimethylbenz(a)anthracene in astrocytoma cell line (MOG-G-CCM).

CYP1B1, a new member of human cytochrome P450 family 1, is involved in the xenobiotic detoxification metabolism and possibly activation of numerous procarcinogens and promutagens. Localization of CYP1B1 in human temporal lobe and its induction in astrocytoma cell line (MOG-G-CCM) by 7,12-dimethylbenz(a)anthracene (DMBA) was investigated using antibodies against human CYP1B1. A single band of approximately 58 kDa size in both human temporal lobe and in MOG-G-CCM was detected by Western blot analysis. Treatment of MOG-G-CCM cells with DMBA resulted in approximately 2.8-fold induction of CYP1B1. CYP1B1 immunoreactivity was detected at the blood-brain interface areas of the temporal lobe as evidenced by co-localization with CD34 antigen. These results suggest that this enzyme may be important in brain xenobiotic metabolism acting as an enzymatic barrier.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

In vivo compartmentalization of human immunodeficiency virus: evidence from the examination of pol sequences from autopsy tissues.

High rates of mutation and replication of human immunodeficiency virus (HIV) allow for the continuous generation of diverse genetic variants in vivo. Selective pressures within the microenvironments of different anatomic compartments result in the emergence of dominant quasispecies which can be distinguished by their envelope sequences. It is not known whether comparable tissue-specific selective pressures lead to the independent evolution of pol sequences within different tissue compartments, nor is it known how differing rates of virus turnover in tissues might affect the pace of such evolution. These issues are of importance for the formulation of a model for the emergence of drug resistance in vivo and for a general understanding of virus trafficking and virus turnover. Regions of the HIV type 1 reverse transcriptase (RT) which carry the majority of the known resistance codons to RT inhibitors (700 nucleotides from each clone) were cloned and sequenced directly from autopsied brain, spleen, and lymph node specimens from four subjects who had received zidovudine therapy. Clones from proviral DNA (143) and from viral cDNA (14) were analyzed. In three of four subjects, a discordance in distribution of resistance codons was noted. Moreover, brain-derived sequences appeared to be phylogenetically distinct from spleen- and lymph node-derived sequences even after exclusion of resistance codons from analysis. In each case, evidence for differential immune selective pressure, based on comparison of inferred amino acid sequences corresponding to known major histocompatibility complex class I cytotoxic T-lymphocyte epitopes, was found. These observations support the concept of anatomically distinct, independently evolving quasispecies (virodemes).

Acquired Immunodeficiency Syndrome↗

PubMed1997

Human cysteine dioxygenase type I (CDO-I; EC 1.13.11.20): 5' flanking region and intron-exon structure of the gene.

AIM: The elucidation of the structure of the 5' flanking region and the exonic organisation of the human cysteine dioxygenase type I gene. METHODS: Material for sequence studies was generated by polymerase chain reaction (PCR) methods using human genomic DNA, a cosmid clone containing the entire gene, and a commercial gene walking kit. RESULTS AND CONCLUSIONS: The gene was found to be--12 kb in length and made up of five exons (418, 78, 155, 170, and 731 base pairs, respectively). The immediate 5' flanking region did not contain the canonical TATAA box. One DNA source (Clontech promoter finder kit) contained a liver specific nuclear factor response element approximately 550 base pairs from the transcription start site, whereas a second source did not. This and other cis acting factors in the 5' flanking region were identified by computer analysis. The physiological significance of such elements requires detailed experimental evaluation.

Base Sequence↗

PubMed1996

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

CD4-Positive T-Lymphocytes↗

PubMed1995

The effects of ethanol, 3,5,3'-triiodothyronine (T3) and oestradiol (E2) on thyroid hormone nuclear receptor expression in human hepatocyte primary cultures.

The possibility that ethanol regulated the expression of thyroid hormone receptor mRNA levels was explored in a model involving human hepatocyte primary culture. Ethanol (20, 50 and 100 mM for 24 h was found to have no effect on the steady-state levels of either triiodothyronine (T3) receptor alpha 1 or alpha 2 mRNAs. In contrast both T3 (1, 10 and 100 nM) and oestradiol (0.1, 1 and 10 ng/ml) treatments affected either one or both of these mRNA levels in a complex manner, showing that the model was capable of responding to other stimuli. Triiodothyronine receptor beta 1 mRNA was not assayed. The hypermetabolic effects of long-term ethanol consumption in humans appears not to be due to the direct effect of ethanol on the regulation of these receptors.

Culture Techniques↗

PubMed1994

Cell surface CD4 downregulation and resistance to superinfection induced by a defective provirus of HIV-1.

Proviral sequences encoding defective HIV-1 regulatory genes have been detected previously in infected individuals; however, the role of these defective genomes in pathogenesis is unclear. The hypothesis that such replication-defective genomes might induce downregulation of the cellular receptor for HIV-1 (CD4) was tested. CEM cells were stably transfected with a provirus that contains a mutation in the splice site immediately 5' of the rev coding sequence. This mutant expresses early HIV-1 mRNAs but is defective for replication. Cells expressing this defective provirus displayed markedly reduced surface CD4. This downregulation of CD4 was dependent on an intact nef gene and was sufficient to cause resistance to superinfection by wild-type virus. These findings indicate that Nef as expressed by replication-defective HIV-1 can downregulate CD4. This perturbation of the T lymphocyte cell membrane is a potential basis for pathogenicity of defective HIV-1.

CD4 Antigens↗

PubMed1994

Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.

The replication competence of human immunodeficiency virus type 1 genomes containing mutations in the nef open reading frame was evaluated in continuous cell lines. Mutants that contained a deletion in the nef open reading frame, premature termination codons, or missense mutations in the N-terminal myristoylation signal were constructed. The replication of these mutants was tested in three ways. First, plasmid genomes were used to transfect T-lymphoblastoid cells. Second, low-passage posttransfection supernatants were used to infect cells with a relatively low virus input. Third, high-titer virus stocks were used to infect cells with a relatively high virus input. These experiments demonstrated a 100- to 10,000-fold decrement in p24 production by the nef mutants compared with that by the wild-type virus. The greatest difference was obtained after infection with the lowest virus input. The myristoylation signal was critical for this positive effect of nef. To investigate the mechanism of the positive influence of nef, nef-positive and nef-minus viruses were compared during a single cycle of replication. These single-cycle experiments were initiated both by infection with high-titer virus stocks and by transfection with viral DNA. Single-cycle infection yielded a three- to fivefold decrement in p24 production by nef-minus virus. Single-cycle transfection yielded equal amounts of p24 production. These results implied that nef does not affect replication after the provirus is established. In support of these results, viral production from cells chronically infected with nef-positive or nef-minus viruses was similar over time. To determine whether the effect of nef was due to infectivity, end point titrations of nef-positive and nef-minus viruses were performed. nef-positive virus had a greater infectivity per picogram of HIV p24 antigen than nef-minus virus. These data indicated that the positive influence of nef on viral growth rate is due to an infectivity advantage of virus produced with an intact nef gene.

Amino Acid Sequence↗

PubMed1993

The structure of the human thyroxine binding globulin (TBG) gene.

A portion of the human X-chromosome (> 5 kb) encoding the translated portion of the thyroxine-binding globulin (TBG) gene was sequenced. The primary templates for sequencing were isolated from a human X-chromosome library (two positive plaques from 400,000 screened initially with a TBG cDNA probe) or were produced by PCR amplification using leucocyte genomic DNA as the amplification template. Potential hormone response elements (HREs) were identified at either end of the gene. These HREs have sequences based on the consensus half-site of thyroid hormone response elements, although it is unclear whether the structures are functional HREs. Other potential regulatory elements also were identified towards the 3' end of the gene.

Animals↗

PubMed1991

An inherited non-amyloidogenic transthyretin variant, [Ser6]-TTR, with increased thyroxine-binding affinity, characterized by DNA sequencing.

Amplification and sequencing of the four transthyretin (TTR) exons of a subject with a variant TTR with four-fold increased affinity for thyroxine revealed a heterozygous G to A point mutation at base 7 of exon 2. This results in a serine for glycine change at residue 6 of the mature TTR monomer. No other mutations were found in any exon. Amplification and MspI digestion of TTR exon 2 from the leucocyte DNA of eight members of the subject's family revealed that all but one member were also heterozygous for [Ser6]-TTR.

DNA↗

PubMed1991

Familial nephropathic non-neuropathic amyloidosis: clinical features, immunohistochemistry and chemistry.

Classification of familial amyloidosis by the chemical nature of the fibrillar protein has become possible. Most such amyloidogenic proteins so far recognized are variant transthyretins, but two kindreds with the same apolipoprotein AI modification have been reported. We describe the clinical features of another such family in whom petechial skin rash appeared to be a marker for the disease, which was non-neuropathic and of the Ostertag-type. Immunohistochemistry showed the protein to be apolipoprotein AI, but allele-specific DNA amplification indicated that it was not the Arg26 variant previously identified.

Amyloidosis↗

PubMed1990

Thyroxine-binding prealbumin gene polymorphism: a population study.

The human thyroxine-binding prealbumin (TBPA) gene was examined for restriction fragment length polymorphism (RFLP) in normal subjects and a subject with euthyroid hyperthyroxinaemia, due to increased thyroxine binding by TBPA, using 16 restriction enzymes. Only Taq I and Msp I were shown to detect RFLPs. In a male of the normal population and one of his daughters, an additional Taq I site was found in the 3'-flanking region of the TBPA gene. The RFLP in a subject with euthyroid hyperthyroxinaemia was due to the deletion of a MspI site. All three subjects with RFLPs were heterozygous.

Adult↗

PubMed1989

Is oleic acid the thyroxine binding inhibitor in the serum of ill patients?

The possibility that oleic acid is the thyroxine binding inhibitor in the serum of seriously ill patients was investigated. 3H-Oleic acid was shown to bind directly to human thyroxine-binding globulin (TBG) by the techniques of one and two-dimensional immunoelectrophoresis in combination with autoradiography. However, no correlation was seen between serum thyroxine concentration and oleic acid concentration in two groups of patients, one of which underwent routine cholecystectomy, whilst the other group was admitted to an intensive therapy unit (mortality 75%). No correlation was seen between serum total thyroxine concentration and either stearic, palmitic, linoleic or arachidonic acid concentrations in these groups. Therefore, it was concluded that oleic acid was unlikely to be the circulating inhibitor of thyroxine binding.

Blood Proteins↗