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Biomedical subjects

M John

Publications and source records attributed to M John.

At least 163 records · Page 9Linked to original sources
PubMed1988

Identification of Mycobacterium bovis isolates using a monoclonal antibody.

A rapid immunoperoxidase slide assay for the identification of Mycobacterium bovis culture isolates is described. The monoclonal antibody used in this assay is specific for the M. tuberculosis complex of organisms. All M. bovis isolates tested, including 151 separate field isolates of M. bovis were positive as were 11 out of 12 M. tuberculosis strains and 4 out of 6 Bacillus Calmette Guerin (BCG) strains. One strain each of M. africanum and M. microti was negative. This assay provides a considerable improvement in both time and expense over the conventional methods of biochemical typing of M. bovis.

Antibodies, Monoclonal↗
PubMed1988

Rhizobium meliloti nodA and nodB genes are involved in generating compounds that stimulate mitosis of plant cells.

The nodB gene of Rhizobium meliloti encodes a 23.8-kDa protein that is conserved in several Rhizobium species. Monospecific polyclonal antibodies against NodB were used to localize this protein in the cytosol of R. meliloti and Escherichia coli cells containing nodABC genes. In comparison to the NodA and NodC proteins, NodB is synthesized in a disproportionately low amount. The NodA and NodB proteins are involved in generating small, heat-stable compounds that stimulate the mitosis of various plant protoplasts. Our experiments suggest that NodC is not involved in the synthesis of the factors. On the basis of their properties, we speculate that the factors are cytokinin-like substances.

Journal Article↗
PubMed1988

Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning.

The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15.1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxyl-terminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.

Amino Acid Sequence↗
PubMed1988

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis.

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.

Animals↗
PubMed1988

Glucose homeostasis in the newborn. Effects of oral feeding on response to fasting and intravenous glucose infusion in neonatal piglets.

The effect of oral feeding on the response to fasting and to an intravenous glucose infusion was investigated in neonatal piglets. Piglets, which had received no food at birth, reduced both their rates of glucose appearance and glucose utilisation (at 24 h: 43 +/- 3.5 mumol/min in previously fed piglets and 28 +/- 6 mumol/min in fasted piglets; at 48 h: 45 +/- 3.5 mumol/min in previously fed piglets and 25 +/- 5 mumol/min in fasted piglets). This reduction in glucose turnover was not associated with a fall in plasma glucose concentration, and all groups of piglets were normoglycaemic at the start of the study. An intravenous infusion of glucose (100 mumol/kg/min) resulted in suppression of hepatic glucose output and an increase in the glucose utilisation rate. Piglets, which had received no food at birth, had lower absolute rates of glucose utilisation throughout the period of infusion. The present study indicates that major changes in glucose turnover rates may occur prior to these being reflected in changes in blood glucose concentration. The results also indicate that oral feeding affects both steady-state glucose turnover and the response to an intravenous glucose infusion.

Administration, Oral↗
PubMed1987

Preliminary results of concomitant radiotherapy and chemotherapy in advanced cervical carcinoma.

Ten patients with advanced and previously untreated squamous cell carcinoma of the cervix were treated with a synchronous course of radiotherapy (RT) and chemotherapy (CT). RT consisted of 3600 to 4500 cGy external pelvic treatments on a 6-MeV linear accelerator followed by two intracavitary applications administering a total of 4000 mg hr of radium equivalent cesium. CT consisted of a course of mitomycin C (10 mg/m2 iv bolus) and 5-fluorouracil (5FU; 1000 mg/m2/24 hr for 96 hr) during the second week of external RT and another course of cis-platinum (CDDP; 75 mg/m2, 1-6 hr infusion) and 5FU (1000 mg/m2/24 hr for 96 hr) during the first intracavitary cesium application. Toxicity was acceptable and complete clinical response was obtained in all patients at the end of the regimen. Nine patients are alive (eight without disease) 6 to 37 months following initiation of treatment (median 20 months). One patient has developed lumbar spine bone metastases and another died of local and pulmonary disease at 28 months. This combination of 5FU/mitomycin C/CDDP and RT appears to be a practical, well-tolerated, and highly effective regimen for advanced cervical carcinoma.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1987

Cloning of cDNA encoding steroid 11 beta-hydroxylase (P450c11).

We have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11 beta-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11 beta-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.

Adrenal Cortex↗
PubMed1987

Glucose homeostasis in the newborn: effects of an intravenous glucose infusion in normal and intra-uterine growth-retarded neonatal piglets.

Disturbances of glucose homeostasis are a common clinical problem in low birth weight human infants. Previous studies have demonstrated that the intra-uterine growth-retarded piglet is a suitable animal model for the study of such problems. The effects of an intravenous glucose infusion on glucose homeostasis have been studied in normal and intrauterine growth-retarded neonatal piglets using a non-steady state tracer technique. Despite suppression of hepatic glucose output, hyperglycaemia (plasma glucose greater than 10.0 mmol/l) developed in the majority of animals. The hyperglycaemia resulted from a failure to increase glucose utilisation adequately in response to the glucose infusion. Growth-retarded animals had an impaired ability to increase their rate of glucose utilisation, although, in common with the normally grown animals, they demonstrated a graded response to different infusion rates of glucose.

Animals↗
PubMed1987

Inoperable esophageal carcinoma: results of aggressive synchronous radiotherapy and chemotherapy. A pilot study.

Twenty-one consecutive patients with squamous cell carcinoma of the esophagus (EC) were referred, with advanced Stage III disease in nine patients, severe pulmonary/cardiac disease in five patients, and postsurgical recurrences or metastatic disease in seven patients. They were treated with one to four courses of 5-fluorouracil (5-FU) + mitomycin C, alternating with 5-FU + cisplatin (5-FU: 1,000 mg/m2/24 X 96-h infusion; Mitomycin C: 10 mg/m2 i.v.; cisplatin: 75 mg/m2 i.v.) simultaneously with 3,000-5,000 cGy of local radiotherapy (RT) in 3.5-5.5 weeks. These doses of chemotherapy (CT) and RT were generally well tolerated except for prolonged thrombocytopenia in two patients, pancytopenia in one, pulmonary fibrosis in one, and acute renal failure in one. Six patients were alive and free of disease 8-40 months (median, 16 months; mean, 21.5 months) after initiation of treatment. Two additional patients died of unrelated causes without evidence of viable disease at autopsy. Our experience confirms the rapid and sustained palliation of dysphagia and pain obtained within 7-14 days after initiation of treatment. Mean survival of patients receiving one to two courses of CT and less than 4,000 cGy RT was 3.4 months compared to 16 months in patients receiving more than 2 courses of CT with RT. Five of six patients who are alive and free of disease received 4,000 cGy or greater. This experience suggests that escalated and concurrent doses of RT (greater than 4,000 cGy) and CT (three to four cycles) are tolerated with acceptable morbidity and could provide good palliation and sometimes prolonged disease-free status in those patients with EC who are considered inoperable because of advanced disease or medical reasons.

Aged↗
PubMed1986

Organization, structure and symbiotic function of Rhizobium meliloti nodulation genes determining host specificity for alfalfa.

In R. meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D. The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units. Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range. These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R. leguminosarum. The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

Bacterial Proteins↗
PubMed1986

Purification and characterization of ovine angiotensinogen.

The two major forms of ovine angiotensinogen (renin substrate) have been purified to homogeneity from plasma and a third form has been partially purified. The purification procedure involved ammonium sulphate fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The pure proteins have apparent relative molecular masses of 56 000 as determined by sodium dodecyl sulphate gel electrophoresis. The amino acid compositions of the two major forms appear to be the same; however, they do have different carbohydrate compositions. Antibodies raised against the a form showed complete cross-reactivity with the b and c forms. Both major forms have the same amino-terminal sequence, which includes that of ovine angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu. Thus ovine angiotensin is the Ile5 form and not the Val5 form as had previously been suggested.

Amino Acid Sequence↗
PubMed1986

Expression of the nodulation gene nodA in Rhizobium meliloti and localization of the gene product in the cytosol.

The nodA gene of Rhizobium meliloti encodes a 21.8-kDa protein, which is conserved in several Rhizobium species. We overproduced the nodA protein as a fusion product with a portion of the lambda cI repressor in Escherichia coli. This fusion protein was purified from inclusion bodies by gel and hydroxyapatite chromatography in the presence of NaDodSO(4). Monospecific polyclonal antibodies against the hybrid protein were used to detect the nodA protein in the cytosol of E. coli and R. meliloti by immunoblotting. In contrast to experiments with antibodies against the R. meliloti nodC membrane protein, the alfalfa-R. meliloti nodulation was not affected by the addition of anti-nodA antibodies to medium and inoculum. This suggests that the nodA protein is located within the cell and is therefore not accessible to antibodies. The expression of the nodA gene is induced in R. meliloti by various compounds present in the exudate of leguminous plants, particularly by the flavone luteolin. We show that the plant hormone trigonelline also has some inducing activity. The nodC protein was further localized in the membrane fraction of R. meliloti. Our experiments demonstrate that the nodC transmembrane protein is not necessary for the uptake of the compounds inducing the synthesis of the nodA protein. The nodA and the nodC proteins were also detected in mature nodules. During nodule development, the nodC protein may be processed to a 34-kDa protein.

Journal Article↗
PubMed1985

Expression of the nodulation gene nod C of Rhizobium meliloti in Escherichia coli: role of the nod C gene product in nodulation.

The nod C gene of Rhizobium meliloti encodes a protein of mol. wt. 44 000 which is highly conserved in at least three Rhizobium species. In order to overproduce this protein, a gene fusion of lambda cI repressor sequences to a large fragment of nod C was constructed. The fusion was placed under control of the tac promoter on plasmid pEA305 to yield pJS1035. IPTG-induced Escherichia coli cells harbouring pJS1035 accumulated the cI-nod C hybrid protein up to 19% of total cellular protein. The synthesis of the hybrid protein drastically inhibits the growth rate of the bacterium. The fusion protein was purified by gel and hydroxyapatite chromatography in the presence of SDS. Antibodies raised against the purified fusion protein precipitated the mol. wt. 44 000 nod C proteins of R. meliloti and of the broad-host range Rhizobium strain NGR234, which were both expressed in E. coli mini-cells. The hybrid protein is associated with the outer membrane of E. coli cells, and the cI-nod C fusion protein appears to be an integral membrane protein. Nodulation of alfalfa by R. meliloti and of clover by R. trifolii was markedly inhibited (approximately 50%) by the addition of antibodies against the hybrid protein to plant growth medium and inoculum.

Bacterial Proteins↗