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Biomedical subjects

M Garret

Publications and source records attributed to M Garret.

At least 19 recordsLinked to original sources
PubMed1992

Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness. High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types. Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR). Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure. Among the sorted transfectants were cells producing approx. 150,000 receptors per cell (Kd of approx. 1 nM). These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids. These cells also overproduced the mRNA for TNFR. Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli. One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene. Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR. Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF. The rescued clone contains approx. 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene. Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb. The mechanism by which this clone induces TNFR expression has not been determined.

Base Sequence↗
PubMed1991

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor.

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).

Amino Acid Sequence↗
PubMed1989

Determination of total and segmental colonic transit time in constipated patients. Results in 91 patients with a new simplified method.

Ninety-one patients with idiopathic constipation had segmental colonic transit studied with radiopaque markers using a new simplified technique to determine frequency and type of colonic transit time (CTT) abnormalities and to determine the utility of this test in planning therapy. Colonic transit studies defined four groups: normal CTT (N = 49), right colonic stasis (N = 16), outlet obstruction (N = 12), and isolated left colonic stasis (N = 14). Right colonic stasis and outlet obstruction were associated with frequent use of digital pressure to assist defecation. Right colonic stasis was characterized by a low stool frequency (less than 3 per week) in 93% of cases and failure to respond to bran therapy. Outlet obstruction also showed a poor response to bran therapy but weekly stool frequency was higher than 3 in 46% of cases. Normal colonic transit time and isolated left colonic stasis were characterized by a normal stool frequency (5.8 +/- 0.05 and 4.2 +/- 0.1, respectively) and clinical help with the use of bran treatment (72 and 64%, respectively). Our study suggested that patients who complain of idiopathic constipation represent a heterogenous group of disorders. Segmental CTT determination is a simple, useful, and noninvasive test of patients with constipation.

Adolescent↗
PubMed1989

Specific amplification of a DNA sequence common to all Chlamydia trachomatis serovars using the polymerase chain reaction.

Enzymatic DNA amplification was applied to DNA and elementary bodies of C. trachomatis. Oligonucleotide primers were chosen in a sequence of a conserved domain of the major outer membrane protein to generate the amplification of a 129-base pair fragment. This sequence was amplified in the 15 serovars of C. trachomatis; however, serovar J gave a weaker signal than the others. The specificity was controlled by EcoRI restriction enzyme digestion and Southern analysis using an internal probe of the amplified sequence. No cross-reaction was shown with DNA of 11 other bacteria. Thus, enzymatic DNA amplification by the polymerase chain reaction appears to be a potential tool for the specific detection of C. trachomatis.

Base Sequence↗
PubMed1989

Detection of Mycoplasma pneumoniae by using the polymerase chain reaction.

The polymerase chain reaction (PCR) technique was used to detect Mycoplasma pneumoniae. A specific DNA sequence for M. pneumoniae was selected from a genomic library, and two oligonucleotides were chosen in this sequence to give an amplified fragment of 144 base pairs. We show that DNA from different M. pneumoniae strains can be detected by PCR, with DNA from other Mycoplasma species giving negative results. Analysis of biological samples (throat swabs) obtained from hamsters that were experimentally infected with M. pneumoniae showed that PCR was more sensitive and reliable than conventional culture techniques for the detection of M. pneumoniae. Initial experiments on artificially seeded human bronchoalveolar lavages showed that PCR can be used to detect 10(2) to 10(3) organisms.

Animals↗
PubMed1988

[Hypergastrin and hyperprolactin syndrome caused by mucinous cystadenoma of the ovary].

We report a case of a clinically and biologically typical hypergastrinemia syndrome due to ovarian mucinous cystadenoma. When examined under histoimmunofluorescence, this "border-line" tumor was shown to contain a polymorphous endocrine cell proliferation, composed primarily of G cells and, to some degree, of D cells. Electron microscopic study further evidenced a small amount of prolactin containing cells, which coincided with elevated prolactin-levels in blood. Ovariectomy combined with vagotomy and pyloroplasty resulted in a prompt disappearance of both clinical and biological abnormalities. Ovarian mucinous cystadenomas have been known for a long time to contain endocrine cells, but only five cases, including the present one, have been described with fully developed endocrine expression. We suggest that this particular condition could be more frequent than generally admitted, and could justify systematic screening for mucinous cystadenoma in the case of peptide hormone dysfunction.

Adult↗
PubMed1986

Inhibition of human pancreatic elastase II activity on human aortic elastin by human alpha 2-macroglobulin.

Human alpha 2-macroglobulin-human pancreatic elastase II binding were investigated using a homologous substrate, human aortic elastin, in order to test the enzymatic activity. We demonstrated that two moles of alpha 2-M are required to inhibit one mole of HPEII when the enzyme is added to a mixture of elastin and alpha 2-M. In addition, when the elastase-alpha 2-M complex is prepared under some circumstances, it exhibits an elastinolytic activity.

Aorta↗
PubMed1986

Tryptophanyl-tRNA synthetase is a major soluble protein species in bovine pancreas.

Besides their central role in protein synthesis, aminoacyl-tRNA synthetases have been found or thought to be involved in other processes. We present here a study showing that tryptophanyl-tRNA synthetase has a surprising tissular distribution. Indeed, immunochemical determinations showed that in several bovine organs such as liver, kidney and heart, tryptophanyl-tRNA synthetase constitutes, as expected, about 0.02% of soluble proteins. In spleen, brain cortex, stomach, cerebellum or duodenum, this amount is about 10-times higher, and in pancreas it is 100-fold. There is no correlation between these amounts and the RNA content of the organs. Moreover, the concentration of another aminoacyl-tRNA synthetase (methionyl-tRNA synthetase) is higher in liver than in pancreas, while the amount of tRNATrp is not higher in pancreas than in liver as compared to other tRNAs. Among several interpretations, it is possible that tryptophanyl-tRNA synthetase is involved in a function other than tRNA aminoacylation. This unknown function would be specific to the differentiated organs, since fetal cerebellum and fetal pancreas contain the same amount of tryptophanyl-tRNA synthetase as adult liver.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Formycin 3' end modified tRNATrp. Recognition by avian myeloblastosis virus reverse transcriptase and primer function.

Primer tRNATrp has been modified at the 3' end by adenosine analogues: 2'deoxyadenosine, 3'deoxyadenosine, 3' amino-3' deoxyadenosine and formycin. Aminoacylation of modified tRNATrp with cognate aminoacyl-tRNA synthetase and primer function for DNA synthesis catalyzed by AMV reverse transcriptase have been studied. The tRNATrp was able to accept tryptophan but did not initiate the DNA synthesis directed by 35S AMV RNA. Recognition of modified tRNATrp by AMV reverse transcriptase was not affected as followed by enzyme-tRNA complex formation. The functional consequences of these effects are discussed.

Adenosine↗