Biomedical subjects
Publications and source records attributed to J Bartley.
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[2-(13)C]glycine metabolism was studied in freshly isolated rat renal proximal tubules. Mitochondrial coupling of the glycine cleavage complex (GC) and serine hydroxymethyltransferase (SHMT) was confirmed by the formation of three serine isotopomers, [2-(13)C]-, [3-(13)C]- and [2,3-(13)C]serine, detected by 13C-NMR. Incubation with different fractions of 13C-labelled glycine altered the labelling pattern of the serine isotopomers predictably and allowed calculation of the 13C-labelled fractions of total glycine and methylene in N5,N10-methylenetetrahydrofolate (m-THF) available for serine metabolism. Within 20 min there was a fall in labelled glycine (to 42 +/- 3, 68 +/- 3 and 93 +/- 2%, (n = 4, mean +/- S.D.) from 50%, 75% and 100% 13C-labelled added glycine respectively), followed by a slow rate of endogenous glycine formation for up to 80 min incubation. The C2 of glycine was the source of more than 90% of the methylene group of m-THF formed. Gas chromatography-mass spectroscopy (GC-MS) showed that greater than 50% of serine formed was unlabelled. GC and SHMT proceeded in the direction of serine formation. Serine isotopomer analysis by NMR and GC-MS allowed the actions of GC and SHMT and de novo contributions to glycine, serine and m-THF to be monitored in situ in fresh renal proximal tubules.
A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity of this domain. Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infected with homologous or heterologous meningococci or other neisserial species. These results have important implications for the understanding of PDHC and the design of future outer membrane vesicle-based vaccines.
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A subtle mutation in the type II collagen gene COL2A1 was detected in a case of human hypochondrogenesis by using a chondrocyte culture system and PCR-cDNA scanning analysis. Chondrocytes obtained from cartilage biopsies were dedifferentiated and expanded in monolayer culture and then redifferentiated by culture over agarose. Single-strand conformation polymorphism and direct sequencing analysis identified a G----A transition, resulting in a glycine substitution at amino acid 574 of the pro alpha 1(II) collagen triple-helical domain. Morphologic assessment of cartilage-like structures produced in culture and electrophoretic analysis of collagens synthesized by the cultured chondrocytes suggested that the glycine substitution interferes with conversion of type II procollagen to collagen, impairs intracellular transport and secretion of the molecule, and disrupts collagen fibril assembly. This experimental approach has broad implications for the investigation of human chondrodysplasias as well as human chondrocyte biology.
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The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.
In 1987 the Centers for Disease Control published a Universal Precautions Policy establishing blood and body fluid procedures to be used consistently with all patients. An important and unequivocal Universal Precautions Policy recommendation with regard to avoidance of needlestick injuries is that needles should never be recapped. We examined the recapping-related attitudes and behaviors of physicians and nurses at four large teaching hospitals with patients with acquired immunodeficiency syndrome and with Universal Precautions Policy in-service training programs. Compliance was found to be considerably less than optimal. According to unannounced needle counts in disposal boxes, the percentage of recapped needles was always greater than 25% and exceeded 50% in four instances. Recapping was related to inadequate knowledge, concerns about personal risk, forgetfulness, being "too busy" to follow the Universal Precautions Policy, and the misperception that recapping is a way to avoid needlestick injury. Strategies are suggested to improve and supplement traditional in-service education.
The pediatric literature has documented a growing attention to defining the nature and quality of residency training. The critical incident technique, a method widely accepted in industrial settings, was used in this study to determine attitudes and behaviors deemed critical for successful performance of residents in a pediatric training program. Structured interviews with 17 senior teaching faculty produced descriptions of resident behavior that were classified into the following mutually exclusive categories: commitment to learning, clinical judgment, communicating medical information, recognition of limits, professional behavior, interpersonal skills with patients, and dealing with emergency situations. Only 30% of the critical incidents obtained from the faculty were related to criteria traditionally used to select and evaluate residents, such as knowledge and technical skills, while the remaining incidents were noncognitive in nature. The results of this study have implications for the evaluation and selection of residents and suggest that pediatric program directors and faculty must give attention to the means by which noncognitive skills are fostered in residents.
We have studied the generation of reactive oxygen species during the metabolism of a carcinogen, benzo[a]pyrene, by human mammary epithelial cells. We have quantitated the production of one type of oxidative DNA damage, thymine glycols, by using a monoclonal antibody specific to this base modification. Thymine glycols were produced in DNA in a dose-dependent manner after exposure of human mammary epithelial cells to benzo[a]pyrene. The number of thymine glycols formed in the DNA was similar to that of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene covalently bound to the DNA. Exposure of cells to the carcinogen in the presence of superoxide dismutase, which reduces superoxide anions, inhibited the production of thymine glycols and increased cell survival but had little effect on adduct formation. At equitoxic doses, approximately equal to 10-fold more thymine glycols were formed after exposure to benzo[a]pyrene than to gamma-irradiation. Thymine glycols, produced by either agent, were efficiently removed from the DNA of the cells. Since thymine glycols represent only a portion of the oxidative damage possibly produced, our results indicate that the total amount of oxidative damage induced during the exposure of human mammary epithelial cells to benzo[a]pyrene greatly exceeds the amount produced by direct adduct formation and that this indirect damage plays an important role in the cytotoxicity of benzo[a]pyrene.
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The murine mammary gland is a prime target organ for 7,12-dimethylbenz[a]anthracene, (DMBA)-induced carcinogenesis. We analyzed the metabolism of 3H-DMBA in a cell-free microsomal activation system derived from mouse mammary microsomes and in a whole mammary organs in culture. The in vitro microsomal activation system failed to show the more polar diol derivative of DMBA after HPLC. The metabolites obtained directly from the 3H-DMBA-treated whole mammary organs, however, revealed the presence of both the diol as well as the phenolic derivatives of DMBA. Analysis of the glands and the culture medium further showed that nearly 95% of the radioactivity added to the culture medium was associated with the adipose tissue and complete solubilization of the fat pad released substantial amounts of DMBA and its metabolites. It appears that a large portion of DMBA and its metabolites remain entrapped in the adipose tissue surrounding the parenchyma. Formic acid digestion of the gland releases the DMBA and the metabolites allowing their ethylacetate extraction, and HPLC characterization.
By cloning the endpoints of a DMD-associated deletion, we have "jumped" 1100 kb from pERT87-1 (DSX164) to a new locus designated J66 (DXS268), mapping distally within the Duchenne muscular dystrophy (DMD) gene. Both J66 and JBir are mapped by field-inversion gel electrophoresis and detect abnormal SfiI fragments in DMD patients and distal DMD-associated X; autosome translocations. Our long-range map extends the physical map of the DMD gene from 800 to 2000 kb (2 Mb) and increases the mapped portion of Xp21 to approximately 8 Mb. The position of the glycerol kinase gene and the adrenal hypoplasia locus are further confined to the region between J66 and the nearest distal probe L1-4. This region spans at least 1.5 Mb. The multiallelic J66 polymorphism has immediate application in the diagnosis of DMD and generally appears to be distal to DMD mutations.