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PubMed1982

The effect of atropine and duct decompression on the evolution of Diazinon-induced acute canine pancreatitis.

Three groups of eight dogs each were studied to evaluate the early evolution of the hyperamylasemia, hyperlipasemia, and acinar cell pathology at the light and electron microscopic levels during acute Diazinon-induced pancreatitis. Two more groups of five dogs each were evaluated for the effects of cholinergic receptor blockade with atropine and ductal decompression on the evolution of serum enzyme changes and acinar cell pathology. Group I dogs received a secretin infusion of 2 units/kg/hr, and a Diazinon infusion of 75 mg/kg, and demonstrated significant increases in serum amylase and lipase at one, two and three hours. Light microscopy revealed acinar cell vacuolization and progressive interstitial edema. Electron microscopy revealed the formation of large intracytoplasmic vacuoles filled with flocculent material, the fusion of these vacuoles with basolateral membrane, and the formation of interstitial edema. In both group II dogs (that received secretin alone) and Group III dogs (that received atropine, 200 micrograms/kg IV prior to secretin and Diazinon), the serum enzyme levels and histologic results were normal. In group IV dogs, pancreatic duct cannulation to prevent hypertension prevented the hyperamylasemia and hyperlipasemia, but not the acinar cell vacuolization and interstitial edema. This model for acute interstitial pancreatitis is apparently cholinergic-receptor mediated, the serum enzyme elevations are due primarily to ductal hypertension, and the acinar cell pathology is primarily due to cholinergic stimulation and occurs independent of ductal hypertension.

Acute Disease↗

PubMed1999

A new GTP-cyclohydrolase I mutation in an unusual dopa-responsive dystonia, familial form.

We found a new mutation in the GTP cyclohydrolase gene involved in dopa-responsive dystonia. We sequenced the GTP cyclohydrolase gene in a family with four siblings affected by this disorder and identified an A-T mutation in exon 2, leading to a non conservative amino acid substitution at codon 135 of the protein (Ile135Lys), which may change the conformation of the binding site of this enzyme. The clinical evolution was heterogeneous among carriers of the same mutation, underlining the involvement of other determinants modulating the occurrence of the disease such as genetic or environmental susceptibility factors.

Adolescent↗

PubMed1988

Spinal cord trauma: in search of the meaning of granular axoplasm and vesicular myelin.

The rapid appearance of selective axonal calcification following experimentally induced spinal cord impact injury has suggested that there is a sudden influx of intracellular calcium which mediates many of the pathological changes subsequent to the trauma. Granular dissolution of axoplasm and vesicular disruption of myelin are the most characteristic alterations in the affected white matter. These changes, which appear early and progress, are identical to those that can be induced by exposing myelinated axons to increased extracellular calcium or to calcium ionophores. These observations have led us to develop the hypothesis that calcium mediates the destruction of traumatized axons in the spinal cord by stimulating calcium-activated neutral proteinases (CANP), which in turn degrade axonal and myelin proteins. A rapid increase in CANP activity has been demonstrated in our experimental rat trauma model and a CANP has been isolated from rat spinal cord myelin which degrades both neurofilament and myelin proteins. However, the interpretation of the role of calcium has been complicated by the fact that granular axoplasm and vesicular myelin can be induced under conditions other than those favorable for CANP enhancement. The ultimate proof of the calcium hypothesis will depend on analyzing early intracellular ionic fluxes and correlating these with the localization of calcium-mediated enzymes and the evolution of pathological events.

Animals↗

PubMed1995

Hepatic toxicity associated with 2'-3' dideoxyinosine in children with AIDS.

Among 34 children with AIDS enrolled in a trial with 2'-3' dideoxyinosine (ddI), six (aged 1-6 years) developed liver abnormalities within 2-18 months after institution of ddI. Two children died of fulminant hepatic failure (one had also an adenovirus infection), and four had a striking elevation of alkaline phosphatases (AP: 1120-7000 IU/L). All of them received sulfa drugs and antifungic treatment with ketoconazole or fluconazole. Three had a serology positive for hepatitis C virus. The evolution of liver enzymes following withdrawal and rechallenge with ddI in the children with elevated AP was an indication of drug-induced toxicity in two patients. In both of the others, readministration of ddI did not cause any subsequent problems. Liver histology in the patients with fulminant hepatitis showed an extensive hepatic necrosis. Liver biopsy done in two other patients revealed a mild granulomatous hepatitis in one and nonspecific changes (i.e., steatosis and a mild inflammation) in the other. Hepatic toxicity has been described with ddI and other nucleoside analogs in adult patients. These six children had many potential causes of liver disease. It may be that ddI is not hepatotoxic per se but that it precipitates liver disease in predisposed patients. We recommend that liver functions be carefully monitored when using this drug.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Length of hospital stay after acute myocardial infarction.

A number of trials show that long stay in hospital after an acute myocardial infarction (AMI) is not necessary for many patients and that stays of three-ten days may be adequate. All patients aged under 70 years with a diagnosis of AMI admitted to the seven public hospitals in the Lower Hunter Region of New South Wales are monitored as part of the WHO MONICA Study. Between August 1984 and December 1985 of 438 hospitalised patients with a 'definite' AMI according to MONICA criteria and a clinical discharge diagnosis of AMI, 386 (88%) patients were discharged alive from hospital. Four patients had lengths of stay between 46 and 77 days and have been omitted from further analysis. The mean length of hospital stay was 13.6 days (95% confidence intervals 12.9 to 14.3 days); 74% of all patients stayed in hospital for more than ten days. The mean length of stay in the Coronary Care Unit (CCU) was 4.5 days (95% confidence intervals 4.2 to 4.8 days) with 60% staying longer than three days. Mean hospital stay varied from 10.5 to 17.4 days among the seven hospitals, although most of this variation was accounted for by three hospitals with few patients. Restricting analysis to the four hospitals with 90% of all the patients, multiple regression analysis showed that the CK enzyme levels, the evolution of Q waves on ECG, the presence of an anterior AMI and the use of nitrates and digoxin during hospitalisation were all associated with increased length of stay in hospital.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1986

Cytochrome-free mitochondria of an anaerobic protozoan--Blastocystis hominis.

Isolated mitochondria of the anaerobic protozoan Blastocystis hominis were subjected to spectral analysis, color, catalase, and peroxidase tests and found to be completely negative for cytochrome enzymes, catalase, and peroxide. Based on the absence of cytochrome enzymes, the possible evolution of B. hominis mitochondria from anaerobic bacteria is postulated.

Aerobiosis↗

PubMed2005

In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase.

The (beta(alpha))8-barrel is the most common enzyme fold and it is capable of catalyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (beta(alpha))8-barrels with in vitro selection methods remains largely untested. We have investigated plasmid display as a suitable in vitro format by engineering a variant of phosphoribosylanthranilate isomerase (PRAI) that carried the FLAG epitope in active-site-forming loop 6. Trial enrichments for binding of mAb M2 (a mAb to FLAG) demonstrated that FLAG-PRAI could be identified from a 10(6)-fold excess of a FLAG-negative competitor in three rounds of in vitro selection. These results suggest PRAI as a useful scaffold for epitope and peptide grafting experiments. Further, we constructed a FLAG-PRAI loop library of approximately 10(7) clones, in which the epitope residues most critical for binding mAb M2 were randomized. Four rounds of selection for antibody binding identified and enriched for a variant in which a single nucleotide insertion produced a truncated (beta(alpha))8-barrel consisting of (beta(alpha))1-5beta6. Biophysical characterization of this clone, trPRAI, demonstrated that it was selected because of a 21-fold increase in mAb M2 affinity compared with full-length FLAG-PRAI. Remarkably, this truncated barrel was found to be soluble, structured, thermostable and monomeric, implying that it represents a genuine subdomain of PRAI and providing further evidence that such subdomains have played an important role in the evolution of the (beta(alpha))8-barrel fold.

Aldose-Ketose Isomerases↗

PubMed1976

Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli.

Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.

Alcohol Oxidoreductases↗

PubMed1978

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.

Arylsulfatases↗

PubMed2004

Palm mutants in DNA polymerases alpha and eta alter DNA replication fidelity and translesion activity.

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase alpha that were associated with a defect in error discrimination. Among them, L868F DNA polymerase alpha has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase alpha. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase alpha-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase alpha catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3' T 26000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase eta, and the F34L mutant of S. cerevisiae DNA polymerase eta has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase alpha is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

Amino Acid Sequence↗

PubMed2006

Vitamin synthesis in plants: tocopherols and carotenoids.

Carotenoids and tocopherols are the two most abundant groups of lipid-soluble antioxidants in chloroplasts. In addition to their many functional roles in photosynthetic organisms, these compounds are also essential components of animal diets, including humans. During the past decade, a near complete set of genes required for the synthesis of both classes of compounds in photosynthetic tissues has been identified, primarily as a result of molecular genetic and biochemical genomics-based approaches in the model organisms Arabidopsis thaliana and Synechocystis sp. PCC6803. Mutant analysis and transgenic studies in these and other systems have provided important insight into the regulation, activities, integration, and evolution of individual enzymes and are already providing a knowledge base for breeding and transgenic approaches to modify the types and levels of these important compounds in agricultural crops.

Carotenoids↗

PubMedDate not supplied

Differential induction of glutathione transferases and glucosyltransferases in wheat, maize and Arabidopsis thaliana by herbicide safeners.

By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: (1) The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. (2) The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. (3) The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.

Arabidopsis↗

PubMed2004

Global dissemination of beta-lactamases mediating resistance to cephalosporins and carbapenems.

While the main era of beta-lactam discovery programs is over, these agents continue to be the most widely prescribed antimicrobials in both community and hospital settings. This has led to considerable beta-lactam pressure on pathogens, resulting in a literal explosion of new beta-lactamase variants of existing enzyme classes. Recent advances in the molecular tools used to detect and characterize beta-lactamases and their genes has, in part, fueled the large increase in communications identifying novel beta-lactamases, particularly in Gram-negative bacilli. It now seems clear that the beta-lactams themselves have shaped the field of new enzymes, and the evolution of key amino acid substitutions around the active sites of beta-lactamases continues to drive resistance. Over 130 variants of TEM beta-lactamase now exist, and more are reported in the scientific literature each month. The most disturbing current trend is that many bla structural genes normally limited to the chromosome are now mobilized on plasmids and integrons, broadening the spread of resistance to include carbapenems and cephamycins. Furthermore, in some Enterobacteriaceae, concomitant loss of outer membrane porins act in concert with these transmissible beta-lactamase genes to confer resistance to the most potent beta-lactams and inhibitor combinations available. Continued reviews of the literature are necessary in order to keep abreast of the ingenuity with which bacteria are changing the current genetic landscape to confer resistance to this important class of antimicrobials.

Anti-Bacterial Agents↗

PubMed1997

Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

Bacillus subtilis↗

PubMed2006

High-throughput selection system for assessing the activity of epoxide hydrolases.

Crucial to the success of directed evolution of enantioselective enzymes for use as catalysts in synthetic organic chemistry is the availability of high-throughput assays for determining the enantiopurity of thousands of samples. Although several such ee-assays are available, they entail time and effort, which means that pre-tests for activity have been developed to eliminate non-active mutants prior to ee-screening. Pre-selection systems may be even more efficient and simple to perform. In the present paper an efficient pre-selection test for assessing the activity of epoxide hydrolases has been developed. The bacterial (E. coli) growth on agar plates is shown to be directly related to the presence of active epoxide hydrolases which catalyze the detoxicating hydrolysis of the epoxide substrates. Visual inspection of agar plates is all that is necessary to identify positive (active) hits in large libraries of mutant epoxide hydrolases.

Combinatorial Chemistry Techniques↗

PubMed2004

[Genomic, molecular biology and malaria: new medical perspectives?].

The knowledge of genomic structure of man, of Plasmodium falciparum and of its main vector Anopheles gambiae has led to great progress in the understanding of malaria pathophysiology. It may also offer new perspectives for malaria therapy vaccines or control of mosquito-borne transmission. In pathophysiology, genes encoding adhesion plasmodial proteins of their receptors were identified, as well as other genes controlling the patient immune response or the antigenic parasite variability. From a therapeutic point of view, new targets for future antimalarial drugs were identified, mainly in apicoplast (a vestige of vegetal structure incorporated by the parasite during its phylogenic evolution) and several enzymes, particularly proteases. It will be now necessary among these "promising new molecules" to select a few ones (probably no more than 5 or 6) for a pre clinical and clinical pharmaceutical development. Indeed, the industrial possibilities for developing new antimalarial drugs are evidently limited and several other antimalarial drugs are already under development. For future malaria vaccines, several new targets and antigenic proteins were also identified. As for new drugs, a complete evaluation of these antigens is absolutely necessary to select few of them for clinical development. Particularly for malaria, ADN vaccines may offer very promising perspectives with the possibility to obtain both humoral and cellular immunity and to use at the same time a panel of plasmodial antigens. It could be thus possible to obtain a simultaneous immunization against different stages of Plasmodium falciparum (sporozoites, merozoites, gametocytes) and to use, as an adjuvant, a gene encoding a viral protein or a cytokine (GMCSF). In Anopheles gambiae genome, several genes encoding for key-proteins, particularly odorant receptors necessary for blood meals, were identified. Non biting-non transmitting mosquitoes were obtained by genetic manipulation and, from an academic viewpoint, offer a very attractive new perspective for the interruption of malaria transmission. Unfortunately several practical problems remain unsolved and genetically modified mosquitoes do not survive long enough among "wild" strains. On the whole genomic and proteomic gave very exciting scientific results in malaria and, very probably the post-genomic phase will even give more new data. From a practical, medical viewpoint, it is still too early and speculative to imagine their possible applications for malaria control.

Animals↗

PubMed1990

Identification of the reverse transcriptase encoded by the Mauriceville and Varkud mitochondrial plasmids of Neurospora.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) that have characteristics of mtDNA introns and retroid elements. The plasmids contain a single long open reading frame (710 amino acids), whose amino-terminal half has structural similarity to reverse transcriptases. Using antibodies against synthetic peptides and trpE fusion proteins, we detected an 81-kDa protein encoded by this open reading frame in mitochondrial preparations from the plasmid-containing strains. This 81-kDa protein cosegregates with reverse transcriptase activity in sexual crosses and comigrates with reverse transcriptase activity in sodium dodecyl sulfate-polyacrylamide gels, where it can be assayed after renaturation of the protein. In glycerol gradients under nondenaturing conditions, the reverse transcriptase activity sediments at approximately 145 kDa, close to the value expected for a dimer of the 81-kDa protein. The 81-kDa protein represents most of the 710-amino acid open reading frame, but may be missing some amino acids at the amino terminus. The regions upstream and downstream of the putative reverse transcriptase domain lack sequences characteristic of gag, protease, RNase H, or integrase domains found in other retroid elements. The plasmid-encoded 81-kDa protein seems to be a novel type of reverse transcriptase that may provide insight into the evolution of these enzymes.

Cloning, Molecular↗

PubMed1989

Recent findings and future perspectives of digestive physiology in rabbits: a review.

The rabbit is one of the most common laboratory animals. Consequently, there is an enormous number of publications concerning its physiology. The present review is restricted to a special field and deals with the new findings of the rabbit's digestive physiology. For the sake of better intelligibility the most important antecedents are also given. First the particularities of feed intake regulation are demonstrated. The strong control mechanism can be disturbed by feed antinutritives, mycotoxins and high environmental temperature. Caecotrophy depends on three main factors: stimulation of rectal mechanoreceptors, perception of the specific odour of the soft faeces, and the inner motive determined by the blood level of metabolites and hormones. The species characteristics of proportions, pH conditions, microflora and -fauna of the rabbit's digestive tract are given. The digestion and absorption of dietary nutrients are discussed. Special sections deal with the sorption of electrolytes and VFAs in the gut, the caecal digestive process, the formation of hard and soft faeces and the role of the caecotrophy in stomachal carbohydrate degradation. A relatively new area, i.e. the development and maturation of the gastrointestinal tract, is also shown. The postnatal evolution of digestive enzyme activity is also summarized. The main endogenic factors (breed, sex, age, stress, caecotrophy, presence of hairball in the stomach) influencing the digestion of dietary nutrients are also described. The probable future trends are given, too.

Animals↗