Biomedical subjects

R A Holt

Publications and source records attributed to R A Holt.

At least 19 recordsLinked to original sources

PubMed2005

Flavocytochrome P450 BM3: an update on structure and mechanism of a biotechnologically important enzyme.

Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner--a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved.

Animals↗

PubMed2001

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Algorithms↗

PubMed2000

The genome sequence of Drosophila melanogaster.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Animals↗

PubMed2000

A morphometric evaluation of allograft matrix combinations in the treatment of osseous defects in a baboon model.

Recent and ongoing research efforts have been made to increase the efficacy of biomaterials as structural fillers during in vivo bony reconstructions. Although the selection of the possible material choices has grown, a biomaterial that can be physically molded to the defect/void space as well as offer biomimetic tissue regeneration has yet to be made available. With the potential success of demineralized freeze-dried bone allografts (DFDBA) combined with tendonous collagen as an effective filling material, further research should help to elucidate its use. The purpose of this study was to evaluate the regenerative healing response of five allograft mixtures via the morphology of filled, periodontal defects. Critical size mandibular and maxillary osseous defects were surgically created in six adult baboons. The filling response of four combinations of DFDBA and tendon collagen was compared with an all-collagen graft after 3 months of implantation. The overall results indicate that all combinations of DFDBA and collagen provided a better fill response than the all-collagen matrix (P < 0. 05). Statistically, however, all of the combinations were similar (P > 0.05) with a 60:40 collagen to DFDBA mass ratio resulting in the largest defect fill response.

Alveolar Ridge Augmentation↗

PubMed1999

Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam.

The GABA and benzodiazepine binding sites on GABA(A) receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug-naive rats was potentiated approximately 1.6-fold by 100 microM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA potentiation were observed. Diazepam-induced changes in GABA(A) receptor gamma2-subunit gene transcription and alpha1-, beta2-, and gamma2-subunit steady-state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Animals↗

PubMed1998

Gynecologic and reproductive services for women with developmental disabilities.

A two-phase study using mail-out questionnaires examined the availability and accessibility of gynecologic and reproductive services for women with developmental disabilities. First, 127 women with developmental disabilities responded to queries about the accessibility and type of available services, the providers of necessary care, and satisfaction with the services. The age range of the women was 18-80 years, with a mean age of 40 years. Of the women queried, 40% indicated that they had not received health education regarding gynecologic and reproductive needs, and 27% indicated that they disliked health education about such matters. In the second phase of the study, agencies that provide services to these women were queried about patterns of the providers, available services, and identified barriers, including financial problems, difficulty in finding a provider, family perceptions, and fear of and distaste for examinations. A surprising finding was that more than one-third of the reporting agencies indicated that questions about the identification and treatment of sexual abuse were not applicable to their agencies. The findings are discussed with an emphasis on identified barriers to care, available services, and the implications for nurse practitioner practice.

Adult↗

PubMed1997

Redox control of the catalytic cycle of flavocytochrome P-450 BM3.

Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.

Bacillus megaterium↗

PubMed1997

Chronic zolpidem treatment alters GABA(A) receptor mRNA levels in the rat cortex.

The effect of chronic zolpidem treatment on the steady-state levels of gamma-aminobutyric acidA alpha1-6, beta1-3 and gamma1-3 subunit mRNAs in rat cortex has been investigated. Male Sprague-Dawley rats were injected once daily, for 7 or 14 days, with 15 mg/kg of zolpidem in sesame oil vehicle. The levels of the alpha4 and beta1 subunit mRNAs were significantly increased after 7 days of treatment and the level of alpha1 subunit mRNA was significantly decreased after 14 days of treatment, as determined by solution hybridization. These results are compared to the previously determined effects of an equivalent schedule of treatment with diazepam.

Animals↗

PubMed1997

Lased and sandblasted denture base surface preparations affecting resilient liner bonding.

STATEMENT OF PROBLEM: Adhesive failure between the liner and the denture base creates an environment for potential bacterial growth and accelerated breakdown of the soft liner resulting in a deteriorating prosthesis. PURPOSE: This study evaluated the effects of a specific sandblasted or lased preparation on the interfacial bonding of polymethyl methacrylate and silicone and polyethyl methacrylate resilient liners. MATERIAL AND METHODS: Polymethyl methacrylate test specimens were fabricated and received one of three surface treatments: untreated (control), sandblasted (250 microns aluminum oxide particles), and lased (carbon dioxide). Polyethyl methacrylate and silicone resilient lining materials were applied to these surfaces and the peel strengths were determined with the American Society for Testing and Materials peelin-adhesion test. RESULTS: Altering the polymethyl methacrylate surface by sandblasting significantly reduced the peel strengths for the polymethyl methacrylate/polyethyl methacrylate and polymethyl methacrylate/silicone specimens. Altering the polymethyl methacrylate surface by delivering carbon dioxide laser energy to form a grid pattern produced lower peel strengths that were statistically significant from the controls for the polymethyl methacrylate/polyethyl methacrylate specimens, but not so for the polymethyl methacrylate/silicone specimens. Untreated polymethyl methacrylate/polyethyl methacrylate peel strengths were significantly higher than polymethyl methacrylate/silicone. CONCLUSIONS: Results of this study imply that mechanical surface preparation of denture bases before application of a resilient liner may not be warranted.

Adhesiveness↗

PubMed1997

Chronic diazepam exposure decreases transcription of the rat GABA(A) receptor gamma2-subunit gene.

The rate of transcription of the GABA(A) receptor gamma2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABA(A) receptor gamma2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABA(A) receptor gene.

Animals↗

PubMed1997

Effect of resin/fluoride and holmium:YAG laser irradiation on the resistance to the formation of caries-like lesions.

PURPOSE: This study compared the effects of a combination of a topical application of resin/NaF mixture and holmium-doped yttrium aluminum garnet (HO:YAG) laser energy with untreated control sites for their resistance to acid destruction/mechanical challenges of root surfaces adjacent to restorative margins. MATERIALS AND METHODS: Forty-eight extracted human teeth were prepared for restorations with coronal margins in enamel and the apical margins on root surfaces. Four types of restorative materials were used. One half of the restorative/root surface margins were treated with an application of nonfilled resin/fluoride mixture and holmium-doped yttrium aluminum garnet laser irradiation; the remaining portion served as the untreated control for each tooth. Acid (10% formic) and mechanical (air sandblaster) challenges were used to test the effects of these treated sites to their controls and to other types of restorative/root surface margins. The depth of root surface loss within 0 to 2 mm apical from the restorative margins was measured and evaluated after 24, 48, and 72 hours of acid exposures. RESULTS: The measurements of tooth loss for the 24- and 48-hour acid-exposure cycles were subjected to statistical analysis (ANOVA) using a paired t test for the variables. The variables, depth of tooth surface loss for the control sites was compared with the depth of loss of the treated sites on opposing sides of each tooth. The depth of tooth surface loss for the control sites had an average mean of 0.35 (0.25) mm for the 24-hour acid exposures, with the opposing treated sides having a mean of 0.053 (0.05) mm (significant at the 0.05 level). The 48-hour acid-exposure results showed significant increased tooth loss for the control sites, but minimum additional loss on the treated sites. The control sites showed an average mean of 1.16 (0.24) mm and a mean of 0.095 (0.103) mm for the treated areas, significant at the 0.05 level. No measurements were made for the 72-hour cycle groups of composite and amalgam alloy restorations because of the loss of most of the filling materials in the control sites. The control groups of crowns showed an average mean loss of 2.06 (0.37) mm with only 0.20 (0.19) mm surface loss for the treated sites. The paired t test for variables indicated no significant differences of tooth surface loss between various types of restorative margins used in this study. CONCLUSIONS: HO:YAG laser energy irradiation after application of resin/NaF to restorative margins and adjacent areas showed a significant increased resistance to acid/mechanical destruction on cementum-dentin root surfaces. The integrity of the restorative/dentin margins were maintained after extended exposures to formic acid and mechanical challenges.

Composite Resins↗

PubMed1996

Dimethylsulfide:acceptor oxidoreductase from Rhodobacter sulfidophilus. The purified enzyme contains b-type haem and a pterin molybdenum cofactor.

Dimethylsulfide:receptor oxidoreductase was purified from the purple non-sulfur phototrophic bacterium Rhodobacter sulfidophilus. The native form of the enzyme had a molecular mass of 152 kDa and was composed of three distinct subunits of 94, 38 and 32 kDa. Dimethylsulfide:acceptor oxidoreductase did not oxidise other thioethers which were tested. The enzyme was able to reduce a variety of N-oxides using reduced methylviologen as electron donor but it reduced dimethylsulfoxide at a very low rate. The resting form of dimethylsulfide:acceptor oxidoreductase exhibited a spectrum which was characteristic of a reduced cytochrome with absorbance maxima at 562 nm, 533 nm and 428 nm. Pyridine haemochrome analysis established that the cytochrome contained a b-type haem and a content of 0.65 mol protohaem/mol enzyme was determined. After oxidation of the haem with ferricyanide, the absorbance spectrum of the reduced cytochrome was restored by reduction with dimethylsulfide. Metal analysis revealed that dimethylsulfide:acceptor oxidoreductase contained 0.5 mol Mo and 3.5 mol Fe/mol enzyme. Heat treatment of the enzyme released material with fluorescence excitation and emission spectra which were characteristic of form B of the pterin component of the pterin molybdenum cofactor. From this analysis it is concluded that dimethylsulfide:acceptor oxidoreductase is a molybdenum oxotransferase which may also contain a iron-sulfur cluster. It is suggested that the haem and pterin molybdenum cofactor are associated with the 94-kDa subunit.

Chromatography↗

PubMed1996

Chronic treatment with diazepam or abecarnil differently affects the expression of GABAA receptor subunit mRNAs in the rat cortex.

Diazepam and abecarnil produce their overt effects by interaction with the GABAA receptor. Chronic treatment with abecarnil, however, does not induce diazepam-like tolerance. This study investigates the effects of chronic diazepam and abecarnil treatment on expression of GABAA receptor alpha 1-6 beta 1-3 and gamma 1-3 subunit isoform mRNAs in rat cortex. Male Sprague-Dawley rats were injected subcutaneously once daily for 7 or 14 days with 15 mg/kg diazepam or 6 mg/kg abecarnil in sesame-oil vehicle, and steady-state levels of GABAA receptor subunit mRNAs were quantified by solution hybridization. The levels of alpha 4- and alpha-, beta 1- and gamma 3-subunit mRNAs were significantly increased after 7 days of diazepam treatment, and this effect was maintained at 14 days. A significant increase in alpha 3-subunit mRNA was apparent only after 14 days of diazepam treatment and a significant decrease in beta 2-subunit mRNA was seen only after 14 days of abecarnil treatment. Gamma 2-Subunit mRNA was significantly decreased after 14 days of either diazepam or abecarnil exposure. A degree of association between a particular drug treatment and changes in the levels of mRNAs arising from a given gene cluster was noted. Our results are consistent with a model of diazepam dependence based on GABAA receptor subunit isoform switching.

Animals↗

PubMed1995

Evaluation of the potential of a polylactic acid barrier for correction of periodontal defects in baboons: a clinical and histologic study.

Created periodontal defects in baboons were treated with one of four possible treatment modes: (1) root preparation and Epi-Guide biodegradable polylactic acid barrier, (2) root preparation and Gore-Tex e-PTFE membrane, (3) root preparation only (no barrier), and (4) no root preparation and no barrier (control). Root preparation consisted of hand instrumentation and use of finishing burs. Measurements of gingival recession were recorded from color photographic slides taken weekly for 6 weeks following barrier placement. Block sections were removed from one animal 6 weeks after barrier placement and prepared for histologic evaluation. Significantly more gingival recession was observed at the Gore-Tex sites than at the Epi-Guide sites. There were no significant differences in gingival recession between the Epi-Guide sites and root preparation-only sites or control sites. Both types of barriers were histologically acceptable. At 6 weeks, the Epi-Guide material was present histologically in a partially resorbed state. There was a mild inflammatory reaction in the surrounding connective tissues.

Alveolar Bone Loss↗

PubMed1994

Metabolism of styrene by Rhodococcus rhodochrous NCIMB 13259.

Rhodococcus rhodochrous NCIMB 13259 grows on styrene, toluene, ethylbenzene, and benzene as sole carbon sources. Simultaneous induction tests with cells grown on styrene or toluene showed high rates of oxygen consumption with toluene cis-glycol and 3-methylcatechol, suggesting the involvement of a cis-glycol pathway. 3-Vinylcatechol accumulated when intact cells were incubated with styrene in the presence of 3-fluorocatechol to inhibit catechol dioxygenase activity. Experiments with 18O2 showed that 3-vinylcatechol was produced following a dioxygenase ring attack. Extracts contained a NAD-dependent cis-glycol dehydrogenase, which converted styrene cis-glycol to 3-vinylcatechol. Both catechol 1,2- and 2,3-dioxygenase activities were present, and these were separated from each other and from the activities of cis-glycol dehydrogenase and 2-hydroxymuconic acid semialdehyde hydrolase by ion-exchange chromatography of extracts. 2-Vinylmuconate accumulated in the growth medium when cells were grown on styrene, apparently as a dead-end product, and extracts contained no detectable muconate cycloisomerase activity. 3-Vinylcatechol was cleaved by catechol 2,3-dioxygenase to give a yellow compound, tentatively identified as 2-hydroxy-6-oxoocta-2,4,7-trienoic acid, and the action of 2-hydroxymuconic acid semialdehyde hydrolase on this produced acrylic acid. A compound with the spectral characteristics of 2-hydroxypenta-2,4-dienoate was produced by the action of 2-hydroxymuconic acid semialdehyde hydrolase on the 2,3-cleavage product of 3-methylcatechol. Extracts were able to transform 2-hydroxypenta-2,4-dienoate and 4-hydroxy-2-oxopentanoate into acetaldehyde and pyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylates↗

PubMed1986

Occurrence of two distinct succinate thiokinases in animal tissues.

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.

Adenosine Diphosphate↗

PubMed1986

Instructions for patients who receive immediate dentures.

Adverse physiologic and psychologic effects may occur in patients who receive immediate dentures. These effects may be caused by ill-fitting dentures that result from oral changes that occur after insertion. To improve the comfort of and acceptance by patients with immediate dentures, it is suggested that written instructions be given to each patient before insertion of the dentures.

Consumer Behavior↗

PubMed1986

Contours of the edentulous palate.

Diagnostic casts were made of the palates of 123 patients who had complete dentures. Categorization of all cross-arch palatal forms yielded a distribution in which 93% showed some variation of a U-shaped palatal form, be it angular or mildly curved. Along the midline, 12% had steep anterior inclines, curved midpalatal regions, and curved posterior palates; 69% had moderate anterior inclinations, curved midpalates, and curved posterior palates; 12% had palates that were moderately inclined anteriorly, flat in the midpalatal section, and flat posteriorly. The remaining palates were predominantly flat in two of the three regions measured.

Cephalometry↗