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Biomedical subjects

D M Gibson

Publications and source records attributed to D M Gibson.

At least 55 records · Page 3Linked to original sources
PubMed1989

Phosphorylation and degradation of HMG CoA reductase.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the limiting enzyme step in cholesterol formation in mammalian liver and other tissues. It is a glycoprotein of 97,000 daltons embedded in the endoplasmic reticulum with a long cytoplasmic extension that is the site of catalytic conversion of HMG CoA to mevalonate. The enzyme is subject to both long-term (induction/repression; degradation) and short-term control (reversible phosphorylation) mediated by endocrine signaling (insulin, glucagon) and through negative feedback by metabolic products of mevalonate (e.g., cholesterol). The catalytic capacity of microsomal reductase falls rapidly in the presence of several protein kinases (reductase kinase, protein kinase-C, calmodulin-dependent protein kinase). Activity is restored with various protein phosphatases. Increased phosphorylation of reductase in intact cells after addition of glucagon or mevalonate is followed by enhanced degradation of the enzyme. In an in vitro model system, phosphorylated, native microsomal reductase is more rapidly cleaved by the calcium-dependent, neutral protease calpain than the dephosphorylated from of reductase. Our present research which centers on the mechanism of the in vitro model system is reviewed. Calpain in the presence of Ca2+ cleaves the cytosolic domain of phosphorylated 97 kDa reductase at two points giving rise to two fragments of nearly the same size that appear as a 52-56,000 dalton doublet by electrophoresis and immunoblotting. In the same system native reductase labeled with [gamma-32P]ATP generates a doublet with 32P solely in the upper (heavier) band. This indicates that serine phosphorylation sites lie between the two calpain cleavage loci. These are positioned in the "linker" region of the long carboxy-terminal cytosolic domain near the membrane. This segment possesses five invariant serine residues and two PEST sequences (constellations of proline, glutamate, serine and threonine) that are characteristic of proteins with short half-lives. If phosphorylation of HMG CoA reductase is confined to the linker region, we must look to this domain in order to interpret the resulting conformational changes that markedly influence reductase catalytic activity and prepare the enzyme for degradation.

Adenosine Triphosphate↗
PubMed1988

Evidence of functional lymphocytes in some (leaky) scid mice.

Although the majority of severe combined immune deficiency (scid) mice lack functional lymphocytes, some (2-23%) appear to develop a limited number of B and T cells between 3 and 9 mo old. Most of these leaky scid mice were shown to contain very few clones (less than or equal to 3) of Ig-producing plasmacytes. Clonal progeny were distributed unevenly in the lymphatic tissues and appeared as discrete plasmacytic foci. In many cases, individual clones persisted for several months and produced abnormally high concentrations of Ig that included multiple isotypes. Functional T cells were inferred from the ability of leaky mice to reject allogeneic skin grafts, a T cell-dependent reaction. Interestingly, approximately 40% of leaky mice developed thymic lymphomas. In other respects, leaky mice resembled regular scid mice; e.g., their splenic cells failed to express common lymphocyte antigens (Ly-5[B220], Ly-1) and to proliferate in response to lymphocyte mitogens. Histologically, their lymphoid tissues retained the same general pattern of severe lymphocytic deficiency as scid mice.

Animals↗
PubMed1988

Purification and characterization of phytase from cotyledons of germinating soybean seeds.

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.

6-Phytase↗
PubMed1988

Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds.

Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min.

6-Phytase↗
PubMed1988

Microbiological quality and shelf life prediction of chilled fish.

A number of storage experiments have been carried out with whole cod and vacuum-packed cod fillets stored in ice. The microbiological quality of the fish was determined on the basis of detection time estimated rapidly by conductance assays in a TMAO-containing medium at 25 degrees C. Detection time and sensory data have been incorporated into a predictive linear model to estimate the remaining shelf life of the products. It is concluded that the shelf life of iced whole cod can be predicted using this model but not that of vacuum-packed fillets because of the greater variability of bacterial activity in packaged fish.

Animals↗
PubMed1987

Word and feature identification by profoundly deaf teenagers using the Queen's University tactile vocoder.

The experiments described are part of an ongoing evaluation of the Queen's University Tactile Vocoder, a device that allows the acoustic waveform to be felt as a vibrational pattern on the skin. Two prelingually profoundly deaf teenagers reached criterion on a 50-word vocabulary (live voice, single speaker) using information obtained solely from the tactile vocoder with 28.5 and 24.0 hours of training. Immediately following word-learning experiments, subjects were asked to place 16 CVs into five phonemic categories (voiced & unvoiced stops, voiced & unvoiced fricatives, approximants). Average accuracy was 84.5%. Similar performance (89.6%) was obtained for placement of 12 VCs into four phonemic categories. Subjects were able to acquire some general rules about voicing and manner of articulation cues.

Adolescent↗
PubMed1987

Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization.

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.

6-Phytase↗
PubMed1987

Some modification to the media for rapid automated detection of salmonellas by conductance measurement.

A selenite medium for the automated detection of salmonellas by conductance measurements has been modified to eliminate the negative results given by some dulcitol-negative strains. The dulcitol is replaced with mannitol and pre-enrichment is best done in buffered peptone water containing mannitol and dimethylsulphoxide. It is suggested that both versions of the selenite medium be used initially.

Culture Media↗
PubMed1986

The organization of immunoglobulin variable kappa chain genes on mouse chromosome 6.

One mouse with a known recombination (NAK) at the Igk locus on chromosome 6 and two new recombinants [B6.PL (75NS) and B6.PL (85NS)] were examined using a series of probes, each of which is specific for a set of immunoglobulin (Ig) Vk genes. Under high stringency conditions, each probe detects from 1 to 19 Bam HI restriction endonuclease fragments (REFs) in genomic DNA by Southern transfer hybridization techniques. Analysis of the REF patterns indicate that the NAK recombination event occurred within the variable region of Igk. The REF patterns of the two B6.PL congenic mice provided two additional recombination events which could be examined. Although some of the REFs had shared mobility among the parental strains, at least 1 and up to 13 polymorphic REFs were present for a given probe among the NZB and AKR parental strains. The results from the NAK mouse indicate that at least some members of Vk4, Vk8, Vk10, and Vk21 were on one side of the recombination event linked to the Lyt-2 alpha and Igk-Ef1 alpha alleles of AKR, while the Vk9, Vk11, and Vk24 REF patterns came from the NZB parental strain linked to the Igk-Ef2 beta (Vk1) allele. The two B6.PL congenics produced a refined map on the Lyt-2, Lyt-3 side of the Vk region. The B6.PL (85NS) mice retained the Vk21 REF pattern of the Lyt-2 alpha, Lyt-3 alpha donor strain PL/J, while displaying the C57BL/6 REF pattern for the other Vk gene groups tested. The B6.PL (75NS) mice retained the REF patterns of PL/J for Vk21 and Ef-1, indicating a third recombination. This indicates the Vk gene order is (Lyt-2; Vk21); Ef-1; (Vk4; Vk8; Vk10); and (Vk9; Vk11; Vk24; Ef-2).

Alleles↗
PubMed1986

Phosphorylation state of HMG CoA reductase affects its catalytic activity and degradation.

The expressed catalytic activity of liver microsomal HMG CoA reductase, the limiting enzyme in cholesterol synthesis, is reversibly diminished by phosphorylation in vitro. In intact hepatocytes the expressed activity of HMG CoA reductase is enhanced by incubation of cells with insulin, and diminished by treatment with glucagon or with mevalonate. In the latter situations the level of total reductase activity falls following initial inactivation (phosphorylation) of the enzyme. This observation suggested that the phosphorylated form of HMG CoA reductase is more sensitive to proteolysis. HMG CoA reductase is a 97,000 dalton (97 K) integral protein of the endoplasmic reticulum with a cytosolic domain that includes the catalytic site and serine residues that may be reversibly phosphorylated. In vitro the Ca2+-activated proteolytic enzyme, calpain, generates two catalytically-active fragments: a membrane bound 62 K and a soluble 53 K form of the enzyme which are quantified by specific immunoblot procedures. Cleavage of the native 97 K HMG CoA reductase is enhanced by pretreatment (inactivation) of microsomes with ATP (Mg2+) and liver reductase kinase compared to microsomes pretreated with protein phosphatase. This is reflected in a loss of 97 K reductase and an increase in the soluble 53 K form of the enzyme. Degradation of HMG CoA reductase in hepatocytes is partially blocked by lysosomotropic agents and insulin. A steady state model for the turnover of proteins subject to reversible phosphorylation has been developed which recognizes fractional degradative rate constants for the phosphorylated and dephosphorylated species.

Animals↗
PubMed1986

An exploratory study of poor adjustment styles among retirees.

This paper focuses on the one third of retirees who report difficulty in making the transition from work to retirement. On the basis of Levy's [16] research, it is hypothesized that there are different styles of poor adjustment that have different consequences for the retirees experiencing them. Data from 487 male and female retirees identify four poor adjustment styles--poor health, negativism, change adaptation and retirement reluctance. While the latter two responses to retirement tend to be problems only in the short term, the former two may have far reaching consequences. Health related retirement and negativism about retiring were linked with low activity and involvement, poor physical and mental health, inadequate income and low life satisfaction in the years following retirement.

Adaptation, Psychological↗
PubMed1986

Continuing evaluation of the Queen's University tactile vocoder. I: Identification of open set words.

Identification of open set words, by an experienced normal hearing subject using the tactile vocoder developed at Queen's University, was examined. The tactile vocoder filters and processes the acoustic waveform into 16 filter channels, each of which controls a vibrator on the skin surface. After acquiring a 250-word vocabulary through the tactile vocoder, the subject was presented with three sets of 1000 different open set words in three reception conditions. The percentages of words correctly identified in the tactile vocoder (TV), lipreading (L), and lipreading plus tactile vocoder (L + TV) conditions were 8.8, 39.4, and 68.7 percent respectively. Phonemic analysis of stimulus/response pairs revealed that 36.7, 64.9 and 85.5 percent of the phonemes were correctly identified in TV, L, and L + TV conditions, respectively, indicating that incorrect-response words often contained many correct phonemes. Also, syllabic stress of stimulus and response words was identical 88 percent of the time in the TV condition. Important information about speech was transmitted through the tactile vocoder.

Deafness↗
PubMed1986

Continuing evaluation of the Queen's University tactile vocoder II: Identification of open set sentences and tracking narrative.

Identification of open set words by an experienced normal hearing subject using the tactile vocoder developed at Queen's University was examined. The tactile vocoder filters and processes the acoustic waveform into 16 filter channels each of which activates a vibrator on the skin surface. After acquiring a 250-word vocabulary through the tactile vocoder, the subject was presented with 1000 different open set sentences in two reception conditions. The percentages of words correctly identified within the sentences in the lipreading (L) and lipreading plus tactile vocoder (L + TV) conditions were 57.8 percent and 79.6 percent, respectively. After two presentations of the stimulus sentences, the number of sentences repeated verbatim in the L + TV condition was 58.4 percent, which was 27.8 percent higher than scores obtained in the L condition. Performance on the tactile vocoder was also evaluated using the "tracking procedure," in which phrases of text are read to the subject who attempts to repeat the material verbatim. The final tracking rate obtained using lipreading was 15.3 words per minute, which was considerably lower than the L + TV tracking rate of 49.3 words per minute. The tactile vocoder provided valuable information which improved the subject's lipreading ability.

Evaluation Studies as Topic↗
PubMed1985

Monoclonal hybridoma screening by analysis of immunoglobulin light chains.

A technique is described for the characterization of immunoglobulin light chains of hybridomas in culture. Immunoglobulins biosynthetically labelled with 14C are obtained from culture supernatants. Following complete reduction and alkylation light chains are separated from heavy chains and most other labelled contaminants by urea-formate gel electrophoresis. They are subsequently analyzed by isoelectric focusing using a simple transfer procedure. The method can be used to analyze up to 30 samples at a time and has a potential for the distinction of 750 light chains. The technique is especially useful (1) to determine the monoclonality of antibodies at an early stage in production, (2) to identify and classify antibodies having different structures but similar specificities, (3) to identify any alterations which may occur in quantity or quality of antibodies in long term culture, (4) to identify different hybridomas which produce antibodies of identical light chain subgroup.

Animals↗
PubMed1985

Linkage of a 7S RNA sequence and kappa light chain genes in the mouse.

A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Ef1. Two mouse stocks with previously identified crossovers within the Ly-2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3)-(Rn7s-6, Igk-Ef1)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3a-Igk-Ef1a haplotype.

Animals↗