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Biomedical subjects

J E Hewitt

Publications and source records attributed to J E Hewitt.

47 records · Page 3Linked to original sources
PubMed1991

Structure and sequence of the human homeobox gene HOX7.

A cosmid containing the human sequence HOX7, homologous to the murine Hox-7 gene, was isolated from a genomic library, and the positions of the coding sequences were determined by hybridization. DNA sequence analysis demonstrated two exons that code for a homeodomain-containing protein of 297 amino acids. The open reading frame is interrupted by a single intron of approximately 1.6 kb, the splice donor and acceptor sites of which conform to known consensus sequences. The human HOX7 coding sequence has a very high degree of identity with the murine Hox-7 cDNA. Within the homeobox, the two sequences share 94% identity at the DNA level, all substitutions being silent. This high level of sequence similarity is not confined to the homeodomain; overall the human and murine HOX7 gene products show 80% identity at the amino acid level. Both the 5' and 3' untranslated regions also show significant similarity to the murine gene, with 79 and 70% sequence identity, respectively. The sequence upstream of the coding sequence of exon 1 contains a GC-rich putative promoter region. There is no TATA box, but a CCAAT and numerous GC boxes are present. The region encompassing the promoter region, exon 1, and the 5' region of exon 2 have a higher than expected frequency of CpG dinucleotides; numerous sites for rare-cutter restriction enzymes are present, a characteristic of HTF islands.

Amino Acid Sequence↗
PubMed1991

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11.

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.

Amino Acid Sequence↗
PubMed1991

Glycosylation is not required for ligand or receptor binding by expressed rat intrinsic factor.

A cDNA clone encoding rat intrinsic factor (IF), pIFQ, has been inserted into the eukaryotic expression vector pSVL and used to transfect COS-1 cells. The IF produced by the transfected cells was secreted nearly exclusively into the medium at concentrations of 0.1-0.2 micrograms/ml. Tunicamycin treatment (1-10 micrograms/ml) completely blocked N-linked glycosylation but had no effect on IF secretion. The secreted glycosylated IF retained all the properties of native IF, i.e., high affinity for cobalamin (Cbl) and for the IF-Cbl receptor and relative resistance to low pH and to proteolysis. The nonglycosylated IF also retained these properties except that it was more protease sensitive. The protease degradation was prevented by the presence of the ligand Cbl. The presence of carbohydrate may play a role in protecting IF from digestion by pancreatic proteases in the intestinal lumen.

Animals↗
PubMed1990

Isolation and characterization of a cDNA encoding porcine gastric haptocorrin.

A cDNA encoding the gastric haptocorrin was isolated from a porcine gastric mucosal lambda gt11 cDNA library using oligonucleotide probes. The 1.4-kb cDNA contains a 1.25-kb open reading frame and 178 nucleotides of 3' noncoding region. Although no initiator methionine is present, primer extension analysis indicated that the transcription initiation site is only 100 bp upstream of the 5' end of this clone. Northern blot analysis showed that a single mRNA species of 1.6 kb exists in hog gastric mucosa. There was no cross-hybridization between this cDNA and the mRNA for haptocorrin in rat submaxillary gland or gastric RNA by Northern blot analysis. This lack of cross-reactivity was also seen on Southern blots, where cow, sheep and dog but neither rat nor mouse genomic DNAs showed cross-hybridizing bands. Comparison of the deduced amino acid sequence of this cDNA with that previously reported for rat intrinsic factor [Dieckgraefe, B.K. et al. (1988) Proc. Natl Acad. Sci. USA 85, 46-50] showed considerable similarity, suggesting that these cobalamin-binding proteins may have a common evolutionary origin.

Amino Acid Sequence↗
PubMed1986

Thyroid hormone effects on lactase expression by rat enterocytes.

Thyroxine (T4) and triiodothyronine (T3) injected into adult rats causes first an increase and then a decrease in lactase activity measured subsequently in intestinal homogenates of rat jejunum. These changes are not associated with any alteration in intestinal structure or enterocyte migration rate. Quantitative cytochemistry shows T4 stimulation and inhibition of lactase activity to take place in upper villus and crypt cells respectively (O- and C-enterocytes). T3 injected into thyroidectomized rats produces identical stimulatory effects on lactase development to T4 injected into control animals. Radioactive T3 is distributed in all cell types following intraperitoneal injection into thyroidectomized rats. Highest amounts of recovered T3 are found in C- rather than O- enterocytes. Quantitative autoradiography shows intracellular T3 to be located in nuclear and cytoplasmic compartments following intraperitoneal injection. Simultaneous injection of non-radioactive hormone displaces 50-75% of radioactive T3. These results are discussed in relation to what is already known concerning the ability of thyroid hormones to affect intestinal development. The future need to study the physiological effects of T3 at the cellular level in the intestine is also emphasized.

Animals↗
PubMed1986

Binding of triiodothyronine by fully differentiated rat enterocytes.

The cellular binding characteristics of triiodothyronine (T3) have been measured in a preparation of isolated fully differentiated enterocytes prepared from thyroidectomized rats. Specific binding of [125I]T3, defined as that portion displaced by the presence of 20 microM-non-radioactive hormone, was shown to become maximal after 100 min incubation with cells at a temperature of 15 degrees C. The cellular release of lactic dehydrogenase measured under these conditions was found to be 18%. Thin layer chromatography of radioactive compounds recovered from enterocytes and from incubation medium showed less than 2% radioactivity to be as free iodide. The amount of [125I]T3 specifically bound to enterocytes showed a curvilinear dependence on the concentration of T3 present in the incubation medium. Scatchard plot analysis of this data revealed the presence of both high (KD 1.2 +/- 1.1 X 10(-9) M) and low (KD 1.9 +/- 0.8 X 10(-7) M) affinity binding sites in these cells. Previous injection of non-radioactive T3 into thyroidectomized rats, at a time chosen to ensure negligible levels of circulating T3 at the time of the experiment, caused a 3-fold reduction in the maximal binding capacity of the low-affinity site (83 compared with 28 pmol/mg protein). Neither the affinity nor the number of high-affinity binding sites for T3 were significantly affected by this treatment. These results are compared with those obtained using other types of cell responsive to T3. The possible physiological importance of these findings is discussed.

Animals↗
PubMedDate not supplied

Molecular genetics of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder. The disease affects specific muscles of the face, shoulder-girdle and upper arm. The biochemical defect underlying FSHD is unknown and there are no specific tests that are diagnostic of FSHD. Genetic linkage studies have mapped the FSHD gene to chromosome 4q35. A DNA marker (p13E-11; D4F104S1, formerly D4S810) has been isolated which recognizes two highly polymorphic loci detectable by EcoRI or HindIII; one locus maps to chromosome 4q35 and shows fragments between about 50 and 320 kb. In FSHD patients deletions occur within this EcoRI/HindIII fragment, yielding fragments that are usually smaller than 28 kb. Characterization of the polymorphic fragments demonstrates that they consist of a 3.2 kb tandem repeat; their number can range between approximately 12 and 96 within the 4q35-specific fragments. In FSHD patients, an integral number of these tandem repeats are deleted, leaving at maximum eight copies.

Chromosome Mapping↗