RESUMO
Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
Assuntos
Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Sítios de Ligação , Geobacillus stearothermophilus/genética , Cinética , L-Lactato Desidrogenase/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
In 1990, two selection lines of Merino sheep were established for low and high behavioural reactivity (calm and nervous temperament) at the University of Western Australia. Breeding records consistently showed that calm ewes weaned 10% to 19% more lambs than the nervous ewes. We hypothesise that calm ewes could have a higher ovulation rate than nervous ewes and/or calm ewes could have a lower rate of embryo mortality than nervous ewes. We tested these hypotheses by comparing the ovulation rate and the rate of embryo mortality between the calm and nervous lines before and after synchronisation and artificial insemination. Merino ewes from the temperament selection lines (calm, n=100; nervous, n=100) were synchronised (early breeding season) for artificial insemination (day 0) (intravaginal sponges containing fluogestone acetate and eCG immediately after sponge withdrawal). On day-17 and 11 ovarian cyclicity and corpora lutea, and on days 30 and 74 pregnancies and embryos/foetuses were determined by ultrasound. Progesterone, insulin and leptin concentrations were determined in blood plasma samples from days 5, 12 and 17. Ovarian cyclicity before and after oestrus synchronisation did not differ between the lines, but ovulation rate did (day-17: calm 1.63; nervous 1.26; P<0.01; day 11: calm 1.83; nervous 1.57; P<0.05). Ovulation rate on day 11 in nervous ewes was higher than on day-17. Loss of embryos by day 30 was high (calm: 71/150; nervous: 68/130); but nervous ewes had a lower proportion (15/47) of multiple pregnancies compared with calm ewes (30/46; P<0.01). Reproductive loss between days 30 and 74 represented 7.3% of the overall loss. Temperament did not affect concentrations of progesterone, but nervous ewes had higher insulin (32.0 pmol/l±1.17 SEM; P=0.013) and lower leptin (1.18 µg/l±0.04 SEM; P=0.002) concentrations than calm ewes (insulin: 27.8 pmol/l±1.17 SEM; leptin: 1.35 µg/l±0.04 SEM). The differences in reproductive outcomes between the calm and nervous ewes were mainly due to a higher ovulation rate in calm ewes. We suggest that reproduction in nervous ewes is compromised by factors leading up to ovulation and conception, or the uterine environment during early pregnancy, that reflect differences in energy utilisation.
Assuntos
Comportamento Animal , Ovulação/fisiologia , Resultado da Gravidez/veterinária , Reprodução/fisiologia , Ovinos/fisiologia , Animais , Austrália , Cruzamento , Corpo Lúteo/fisiologia , Sincronização do Estro/fisiologia , Feminino , Fertilização , Inseminação Artificial/veterinária , Gravidez , Gravidez Múltipla , Progesterona/sangue , Estações do AnoRESUMO
Rapid mixing, kinetic experiments were performed on native and modified [Tyr(3NO2)237)] porcine H4 lactate dehydrogenase at low temperatures in a medium containing 30% dimethyl sulphoxide. In the temperature range -16 to +8 degrees C, the modified enzyme-NADH complex, when mixed with 1 mM pyruvate, is converted to enzyme, NAD+ and lactate at two distinctly different rates. At -16 degrees C the more rapid process occurs at a rate of 40 s-1 and the slower at 3 s-1. The slower rate is identical to that assigned to the steady-state turnover of the enzyme in these conditions and therefore reflects the slow, rate-limiting rearrangement of protein structure which has been inferred from previous kinetic experiments. The fast phase of NADH oxidation, however, proceeds at a rate which coincides with that of the closure of a loop of polypeptide over the active site of the enzyme (sensed by the nitrotyrosine group, which protonates in response to the approach of glutamate 107, a residue situated on this mobile loop). We explain these results by proposing that: (i) both the slow and fast changes in protein structure must occur before the enzyme can accomplish the redox step, (ii) the enzyme-NADH (binary) complex exists in two, slowly interconverting forms, (iii) the structural change giving rise to this slow conformational equilibrium can also occur in the ternary (enzyme-NADH-pyruvate) complex and (iv) it is this step which limits the rate of the steady-state reaction. Both of the binary forms are able to bind pyruvate, but the rate of NADH oxidation in one of the forms is rapid, since it has already undergone this slow rearrangement. In this rapidly reacting form, it is the closure of the loop (not transfer of the hydride ion) which limits the rate at which the coenzyme is oxidized, while the slowly reacting form must undergo both loop-closure and the slow structural conversion before the redox reaction can occur.
Assuntos
L-Lactato Desidrogenase/metabolismo , Animais , Fenômenos Químicos , Química , Isoenzimas , Cinética , Lactatos/metabolismo , Ácido Láctico , NAD/metabolismo , Oxirredução , Conformação Proteica , Piruvatos/metabolismo , Ácido Pirúvico , SuínosRESUMO
Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines. Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme. Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce. By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (less than 0.3 ns). Trp-203 is responsible for 75-80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%.
Assuntos
Proteínas de Bactérias/genética , Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/genética , Espectrometria de Fluorescência , Triptofano , Animais , Proteínas de Bactérias/metabolismo , Geobacillus stearothermophilus/genética , L-Lactato Desidrogenase/metabolismo , Mutação , SuínosRESUMO
Using site-directed mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine. In the closely homologous eukaryotic lactate dehydrogenase, this residue binds the carboxylate group of the substrate by forming a planar bifurcated bond. The mutation diminishes the binding energy of pyruvate, alpha-ketobutyrate and alpha-ketovalerate (measured by kcat/Km) by the same amount (about 6 kcal/mol). For each additional methylene group on the substrate, there is a loss of about 1.5 kcal/mol of binding energy in both mutant and wild-type enzymes. From these parallel trends in the two forms of enzyme, we infer that the mode of productive substrate binding is identical in each, the only difference being the loss of a strong carboxylate-guanidinium interaction in the mutant. In contrast to this simple pattern in kcat/Km, the Km alone increases with substrate-size in the wild-type enzyme, but decreases in the mutant. These results can be most simply explained by the occurrence of relatively tight unproductive enzyme-substrate complexes in the mutant enzyme as the substrate alkyl chain is extended. This does not occur in the wild-type enzyme, because the strong orienting effect of Arg-171 maximizes the frequency of substrates binding in the correct alignment.
Assuntos
Arginina , Ácidos Carboxílicos/metabolismo , Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/metabolismo , Lisina , Mutação , Sítios de Ligação , Calorimetria , Geobacillus stearothermophilus/genética , Cinética , L-Lactato Desidrogenase/genética , Ligação Proteica , Especificidade por SubstratoRESUMO
OBJECTIVES: Unwarranted variance in healthcare has been associated with prolonged length of stay, diminished health and increased cost. Practice variance in the management of asthma can be significant and few investigators have evaluated strategies to reduce this variance. We hypothesized that selective redesign of order sets using different ways to frame the order and physician decision-making in a computerized provider order entry system could increase adherence to evidence-based care and reduce population-specific variance. PATIENTS AND METHODS: The study focused on the use of an evidence-based asthma exacerbation order set in the electronic health record (EHR) before and after order set redesign. In the Baseline period, the EHR was queried for frequency of use of an asthma exacerbation order set and its individual orders. Important individual orders with suboptimal use were targeted for redesign. Data from a Post-Intervention period were then analyzed. RESULTS: In the Baseline period there were 245 patient visits in which the acute asthma exacerbation order set was selected. The utilization frequency of most orders in the order set during this period exceeded 90%. Three care items were targeted for intervention due to suboptimal utilization: admission weight, activity center use and peak flow measurements. In the Post-Intervention period there were 213 patient visits. Order set redesign using different default order content resulted in significant improvement in the utilization of orders for all 3 items: admission weight (79.2% to 94.8% utilization, p<0.001), activity center (84.1% to 95.3% utilization, p<0.001) and peak flow (18.8% to 55.9% utilization, p<0.001). Utilization of peak flow orders for children ≥8 years of age increased from 42.7% to 94.1% (p<0.001). CONCLUSIONS: Details of order set design greatly influence clinician prescribing behavior. Queries of the EHR reveal variance associated with ordering frequencies. Targeting and changing order set design elements in a CPOE system results in improved selection of evidence-based care.
RESUMO
The objective of this study was to investigate variation in human papillomavirus (HPV) type-16 load within histologically defined grades of cervical intraepithelial neoplasia. Two hundred and thirty-seven liquid based cytology samples were collected from women attending colposcopy clinics, DNA was extracted, and presence of virus determined by PCR-enzyme immunoassay. Quantitative real-time PCR was used to determine viral load for 70 HPV-16 positive single infections. Viral load was expressed as the ratio of copies of the viral L1 gene to copies of the human beta-globin gene. Measurements varied from 0.019 to 4,194 HPV genomes per cell. Our data demonstrate that in cervical neoplasia, HPV load tends to correlate with disease severity, but that the number of viral genomes/cell varies considerably within histological grades. This variation within disease grades currently limits the clinical utility of viral load measurement.
Assuntos
Papillomavirus Humano 16/isolamento & purificação , Infecções por Papillomavirus/virologia , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Carga Viral , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/patologiaRESUMO
The development of effective strategies against cervical cancer in Africa requires accurate type specific data on human papillomavirus (HPV) prevalence, including determination of DNA sequences in order to maximise local vaccine efficacy. We have investigated cervical HPV infection and squamous intraepithelial lesions (SIL) in an unselected cohort of 1061 women in a rural Gambian community. Squamous intraepithelial lesions was diagnosed using cytology and histology, HPV was typed by PCR-ELISA of DNA extracts, which were also DNA sequenced. The prevalence of cervical HPV infection was 13% and SIL were observed in 7% of subjects. Human papillomavirus-16 was most prevalent and most strongly associated with SIL. Also common were HPV-18, -33, -58 and, notably, -35. Human papillomavirus DNA sequencing revealed HPV-16 samples to be exclusively African type 1 (Af1). Subjects of the Wolof ethnic group had a lower prevalence of HPV infection while subjects aged 25-44 years had a higher prevalence of cervical precancer than older or younger subjects. This first report of HPV prevalence in an unselected, unscreened rural population confirms high rates of SIL and HPV infection in West Africa. This study has implications for the vaccination of Gambian and other African populations in the prevention of cervical cancer.
Assuntos
Carcinoma de Células Escamosas/epidemiologia , DNA Viral/análise , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Displasia do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/epidemiologia , Adolescente , Adulto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Gâmbia/epidemiologia , Humanos , Pessoa de Meia-Idade , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Prevalência , Fatores de Risco , População Rural/estatística & dados numéricos , Análise de Sequência de DNA , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologiaRESUMO
The National Center for Biotechnology Information (NCBI) has created a database collection that includes several protein and nucleic acid sequence databases, a biosequence-specific subset of MEDLINE, as well as value-added information such as links between similar sequences. Information in the NCBI database is modeled in Abstract Syntax Notation 1 (ASN.1) an Open Systems Interconnection protocol designed for the purpose of exchanging structured data between software applications rather than as a data model for database systems. While the NCBI database is distributed with an easy-to-use information retrieval system, ENTREZ, the ASN.1 data model currently lacks an ad hoc query language for general-purpose data access. For that reason, we have developed a software package, SORTEZ, that transforms the ASN.1 database (or other databases with nested data structures) to a relational data model and subsequently to a relational database management system (Sybase) where information can be accessed through the relational query language, SQL. Because the need to transform data from one data model and schema to another arises naturally in several important contexts, including efficient execution of specific applications, access to multiple databases and adaptation to database evolution this work also serves as a practical study of the issues involved in the various stages of database transformation. We show that transformation from the ASN.1 data model to a relational data model can be largely automated, but that schema transformation and data conversion require considerable domain expertise and would greatly benefit from additional support tools.
Assuntos
Bases de Dados Factuais , Software , Algoritmos , Sequência de Aminoácidos , Sequência de Bases , Sistemas de Gerenciamento de Base de Dados , Humanos , National Library of Medicine (U.S.) , Design de Software , Estados UnidosRESUMO
The rational design of enzyme catalysts for chiral chemistry and of drugs which bind to proteins would be facilitated if rules for the recognition of one partner by the other could be formulated. This communication suggests and tests one generalization: arginine forms a tighter ion pair with a carboxylate group than does lysine and is always used for ion-pairs which are not broken during turnover in naturally-occurring enzymes.
Assuntos
Arginina , L-Lactato Desidrogenase , Sequência de Aminoácidos , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Íons , Lisina , Ligação ProteicaRESUMO
A general technique for monitoring the intramolecular motion of a protein is described. Genetic engineering is used to replace all the natural tryptophan residues with tyrosine. A single tryptophan residue is then inserted at a specific site within the protein where motion is then detected from the fluorescence characteristics of this fluorophore. This technique has been used in B. stearothermophilus lactate dehydrogenase mutant (W80Y, W150Y, W203Y, G106W) to correlate the slow closure of a surface loop of polypeptide (residues 98-110) with the maximum catalytic velocity of the enzyme.
Assuntos
Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/genética , Triptofano , Tirosina , Engenharia Genética , Geobacillus stearothermophilus/genética , Cinética , L-Lactato Desidrogenase/metabolismo , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
A variant of lactate dehydrogenase from Bacillus stearothermophilus has been engineered by site-directed mutagenesis in which an active-site arginine residue at position 171 in the protein sequence is replaced by lysine. Replacement of this arginine by lysine has no effect on co-enzyme binding, a relatively small effect on the rate of turnover of the enzyme, but causes a 2000-fold increase in the Michaelis constant for pyruvate, a 6000-fold increase in the dissociation constant for oxamate and results in a Michaelis constant for lactate which is too high to measure. The decrease in binding energy for these carboxylate-containing substrates caused by this mutation is very large, around 5.5 kcal.mol-1 and in part, is explained by the small increase in the distance of a lysine-substrate carboxylate interaction at this site and the absence of the additional hydrogen bond from a two-point arginine-carboxylate interaction. Consistent with this last observation, the ability of this mutant enzyme to stabilize an NAD+-sulphite compound in its active site (an alternative enzyme-substrate complex which does not involve bifurcated bonding to arginine) is only reduced 14-fold.
Assuntos
Arginina , Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Lactatos/metabolismo , Ácido Láctico , Mutação , Piruvatos/metabolismo , Ácido Pirúvico , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Restriction sites were introduced into the gene for Bacillus stearothermophilus lactate dehydrogenase which enabled a region of the gene to be excised which coded for a mobile surface loop of polypeptide (residues 98-110) which normally seals the active site vacuole from bulk solvent and is a major determinant of substrate specificity. Oligonucleotide-overlap extension (using the polymerase chain reaction) was used to obtain double-stranded DNA regions which coded for different length and sequence loops and which also contained the same restriction sites. The variable length and sequence loops were inserted into the cut gene and used to synthesize hydroxyacid dehydrogenases with altered substrate specificities. Loops which were longer and shorter than the original were made. The substrate specificities of enzymes with these new loops were considerably altered. For many poor enzyme-substrate pairs, the effect of fructose 1,6-bisphosphate on the steady-state kinetic parameters suggested that the substrate was mainly bound in a nonproductive mode. With one longer loop construction (BL1), activity with pyruvate was reduced one-million-fold but activity with phenylpyruvate was largely unaltered. A switch in specificity (kcat/KM) of 390,000-fold was achieved. The 1700:1 selectivity of enzyme BL1 for phenylpyruvate over pyruvate is that required in a phenyllactate dehydrogenase to be used in monitoring phenylpyruvate in the urine of patients with phenylketonuria consuming an apparently phenylalanine-free diet.
Assuntos
Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Enzimas de Restrição do DNA , DNA Bacteriano/química , Frutosedifosfatos/metabolismo , Geobacillus stearothermophilus/genética , Cinética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Ácidos Fenilpirúvicos/metabolismo , Especificidade por SubstratoRESUMO
We report the development of a novel detection and typing methodology for human papillomaviruses (HPV) based on real-time PCR with the self-probing fluorescent primers known as Scorpions. This technique is quick, simple, specific, sensitive, and capable of estimating viral load per cell. We report the results of over 100 typing reactions performed on cell lines, biopsies, and cervical cytobrush samples which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. We further report preliminary data suggesting a relationship between viral load per cell and grade of cervical disease.
Assuntos
Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Infecções Tumorais por Vírus/virologia , Colo do Útero/virologia , Primers do DNA , DNA Viral/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Papillomaviridae/genética , Papillomaviridae/fisiologia , Células Tumorais Cultivadas , Carga ViralRESUMO
The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.
Assuntos
Genes Bacterianos , Geobacillus stearothermophilus/enzimologia , Glicina/genética , L-Lactato Desidrogenase/genética , Mutação , Treonina/genética , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Geobacillus stearothermophilus/genética , Cinética , Especificidade por SubstratoRESUMO
Polymerase chain reaction using sequence-specific primers was utilized to ascertain the prevalence of 3 polymorphisms of the vitamin D receptor (VDR) gene (FokI F/f, ApaI A/a, and TaqI T/t) in 56 patients with Mycobacterium malmoense pulmonary disease. When compared with 101 controls, M. malmoense patients displayed an increased prevalence of Apa1 A (P=.03; Fisher's exact test), TaqI t (P=.04), and the At VDR haplotype (P=.04), and they displayed a decreased prevalence of FokI f (P=.04). Only 4 (7%) of 56 patients (vs. 29 [28%] of 101 controls) were both positive for FokI f and negative for At (P=.001). This indicates that polymorphisms in the VDR (or in closely linked genes) modulate the susceptibility to M. malmoense and that susceptibility involves multiple genetic and environmental factors.
Assuntos
Pneumopatias/etiologia , Infecções por Mycobacterium/etiologia , Infecções Oportunistas/etiologia , Polimorfismo Genético , Receptores de Calcitriol/genética , Suscetibilidade a Doenças , Haplótipos , Humanos , FenótipoRESUMO
Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.