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1.
Biochim Biophys Acta Proteins Proteom ; 1869(2): 140562, 2021 02.
Article in English | MEDLINE | ID: mdl-33221341

ABSTRACT

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.


Subject(s)
Recombinant Proteins/ultrastructure , Serine Proteases/chemistry , Serine Proteinase Inhibitors/chemistry , Urokinase-Type Plasminogen Activator/chemistry , Amino Acid Sequence/genetics , Humans , Quinuclidines/chemistry , Quinuclidines/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Serine Endopeptidases/chemistry , Serine Endopeptidases/ultrastructure , Serine Proteases/genetics , Serine Proteinase Inhibitors/pharmacology , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Urokinase-Type Plasminogen Activator/genetics
2.
Sci Rep ; 6: 31362, 2016 08 10.
Article in English | MEDLINE | ID: mdl-27506494

ABSTRACT

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.


Subject(s)
Bacteria, Anaerobic/enzymology , Carboxy-Lyases/metabolism , Toluene/chemistry , Amides/chemistry , Anaerobiosis , Catalysis , Cell-Free System , Clostridium , Industrial Microbiology , Oxygen/chemistry , Polymerase Chain Reaction , Proteomics , RNA, Ribosomal, 16S , Sewage
3.
J Ind Microbiol Biotechnol ; 41(11): 1687-96, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25223615

ABSTRACT

Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, ß-xylosidase GH43, ß-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.


Subject(s)
Ascomycota/enzymology , Xylosidases/biosynthesis , Ascomycota/growth & development , Ascomycota/metabolism , Glycoside Hydrolases/metabolism , Industrial Microbiology , Lignin/metabolism , Polysaccharides/metabolism , Proteome/metabolism , Zea mays
4.
Antiviral Res ; 99(3): 292-300, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23769894

ABSTRACT

Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA74, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA74 as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA74 in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development.


Subject(s)
Dengue Virus/enzymology , Dengue/virology , Drug Evaluation, Preclinical/methods , Enzyme Assays/methods , Methyltransferases/analysis , Viral Nonstructural Proteins/analysis , Antiviral Agents/pharmacology , Dengue/drug therapy , Dengue Virus/drug effects , Dengue Virus/genetics , Dengue Virus/metabolism , Enzyme Inhibitors/pharmacology , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/genetics , Methyltransferases/metabolism , RNA Caps/genetics , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
5.
Acta Biomater ; 9(3): 5759-70, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23201015

ABSTRACT

Vitamin D plays a central role in bone regeneration, and its insufficiency has been reported to have profound negative effects on implant osseointegration. The present study aimed to test the in vitro biological effect of titanium (Ti) implants coated with UV-activated 7-dehydrocholesterol (7-DHC), the precursor of vitamin D, on cytotoxicity and osteoblast differentiation. Fourier transform infrared spectroscopy confirmed the changes in chemical structure of 7-DHC after UV exposure. High-pressure liquid chromatography analysis determined a 16.5±0.9% conversion of 7-DHC to previtamin D(3) after 15min of UV exposure, and a 34.2±4.8% of the preD(3) produced was finally converted to 25-hydroxyvitamin D(3) (25-D(3)) by the osteoblastic cells. No cytotoxic effect was found for Ti implants treated with 7-DHC and UV-irradiated. Moreover, Ti implants treated with 7-DHC and UV-irradiated for 15min showed increased 25-D(3) production, together with increased ALP activity and calcium content. Interestingly, Rankl gene expression was significantly reduced in osteoblasts cultured on 7-DHC-coated Ti surfaces when UV-irradiated for 15 and 30min to 33.56±15.28% and 28.21±4.40%, respectively, compared with the control. In conclusion, these findings demonstrate that UV-activated 7-DHC is a biocompatible coating of Ti implants, which allows the osteoblastic cells to produce themselves active vitamin D, with demonstrated positive effects on osteoblast differentiation in vitro.


Subject(s)
Cell Differentiation/drug effects , Dehydrocholesterols/radiation effects , Osteoblasts/cytology , Prostheses and Implants , RANK Ligand/genetics , Titanium/pharmacology , Ultraviolet Rays , Alkaline Phosphatase/metabolism , Animals , Calcification, Physiologic/drug effects , Calcification, Physiologic/genetics , Cell Count , Cell Differentiation/genetics , Cell Line , Cell Shape/drug effects , Cell Survival/drug effects , Cholecalciferol/pharmacology , Coated Materials, Biocompatible/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/radiation effects , Mice , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteoblasts/radiation effects , RANK Ligand/metabolism , Spectroscopy, Fourier Transform Infrared , Time Factors
7.
Antimicrob Agents Chemother ; 47(2): 739-46, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12543686

ABSTRACT

We evaluated the activities of clarithromycin and azithromycin against 19 isolates of Streptococcus pneumoniae using a neutropenic lung infection model. The isolates included five susceptible isolates (clarithromycin and azithromycin MICs, /=64 micro g/ml). Infected mice were administered either saline (control), clarithromycin (4, 40, or 200 mg/kg of body weight twice daily or 200 mg/kg once daily), or azithromycin (4, 40, or 200 mg/kg once daily or 40 mg/kg twice daily) by oral gavage for 72 h. Mortality was assessed at regular intervals for 10 days, and survival in each group was compared to that of untreated controls. Animals infected with susceptible isolates demonstrated significant improvement in survival compared to the controls following treatment with either agent at doses of >/=40 mg/kg. In contrast, none of the regimens improved the survival of animals infected with isolates exhibiting high-level macrolide resistance. Among mice infected with strains expressing low-level resistance, significant improvement in survival compared to the controls was noted among isolates treated with clarithromycin at 40 (seven of nine isolates) and 200 (nine of nine isolates) mg/kg twice a day and with azithromycin at 40 (one of nine isolates) and 200 (three of nine isolates) mg/kg once a day. Animals infected with isolates of S. pneumoniae exhibiting low-level, mefA-mediated macrolide resistance responded to treatment with clarithromycin at rates similar to those observed among mice infected with fully susceptible isolates.


Subject(s)
Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Clarithromycin/pharmacology , Streptococcal Infections/drug therapy , Streptococcus pneumoniae/drug effects , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/therapeutic use , Area Under Curve , Azithromycin/pharmacokinetics , Azithromycin/therapeutic use , Clarithromycin/pharmacokinetics , Clarithromycin/therapeutic use , Disease Models, Animal , Drug Resistance, Multiple, Bacterial , Half-Life , Humans , Lung Diseases/drug therapy , Lung Diseases/microbiology , Mice , Microbial Sensitivity Tests , Serotyping , Streptococcus pneumoniae/classification
8.
Antimicrob Agents Chemother ; 46(12): 3846-53, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12435687

ABSTRACT

We evaluated the in vitro activity of the new echinocandin antifungal micafungin against Candida spp. using microdilution and time-kill methods. Additionally, we examined the postantifungal effect (PAFE) of micafungin. Finally, we evaluated the effect of the addition of serum and plasma on the MIC of micafungin. Four Candida albicans isolates and two isolates of each Candida glabrata, Candida krusei, and Candida tropicalis were selected for testing. The MICs of micafungin were determined in RPMI 1640 medium buffered with morpholinepropanesulfonic acid alone and with the addition of 10, 20, and 50% human serum and plasma. MICs were determined by using two endpoints: a prominent reduction in growth (the MIC at which 80% of isolates are inhibited [MIC(80)]) and complete visual inhibition of growth (MIC(100)). The minimum fungicidal concentration (MFC) of micafungin for each isolate was also determined. Time-kill curves were determined for each isolate in RPMI 1640 medium with micafungin at concentrations ranging from 0.125 to 16 times the MIC(80) to assess the correlation between MIC(80) and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC(80). The MIC(80)s for the test isolates ranged from 0.0039 to 0.25 micro g/ml. Overall, the addition of serum or plasma increased the MIC 6 to 7 doubling dilutions for C. albicans and 3 to 4 doubling dilutions for C. krusei and C. tropicalis. Micafungin time-kill studies demonstrated fungicidal activity at concentrations ranging from 4 to 16 times the MIC(80). Micafungin is very potent agent against a variety of Candida spp., producing fungicidal activity against 7 of 10 isolates tested. A PAFE was observed against all isolates. The PAFE was influenced by the drug concentration, with the highest concentration resulting in the longest observed PAFE in each case. The highest concentration tested, four times the MIC, resulted in a PAFE of more than 9.8 h for 5 of 10 isolates tested (range, 0.9 to > or =20.1 h).


Subject(s)
Candida/drug effects , Lipoproteins/pharmacology , Peptides, Cyclic/pharmacology , Echinocandins , Humans , Lipopeptides , Lipoproteins/blood , Micafungin , Microbial Sensitivity Tests , Peptides, Cyclic/blood , Time Factors
9.
Pharmacotherapy ; 22(4): 463-70, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11939681

ABSTRACT

STUDY OBJECTIVE: To compare the antistreptococcal activity of five fluoroquinolone antibiotics, using a neutropenic murine model of pneumococcal pulmonary infection. DESIGN: Animal experiment. SETTING: University-affiliated research center. ANIMALS: Neutropenic and control mice weighing 24-29 g. INTERVENTION: After induction of neutropenia, renal failure, and infection with Streptococcus pneumoniae, the mice received one of five fluoroquinolones twice/day for 72 hours beginning 12 hours after infection. Dosages were selected to approximate 0.1 x AUC0-24 (area under the concentration-time curve from 0-24 hours) and AUC0-24 achieved in humans. Control mice received normal saline. Survival was assessed at regular intervals for up to 10 days. At least 10 mice were included in each cohort (range 10-34). MEASUREMENTS AND MAIN RESULTS: Ciprofloxacin, clinafloxacin, grepafloxacin, levofloxacin, and moxifloxacin were studied at subtherapeutic and therapeutic dosages against three quinolone-susceptible isolates of S. pneumoniae that lacked mutations in parC, parE, and gyrA. Pharmacokinetic profile of each agent and dosing regimen was determined. A composite survival curve for all fluoroquinolones and isolates was constructed. Relationships between survival rate at 72 hours and AUC:MIC (minimum inhibitory concentration), peak:MIC, time above the MIC (percentage of dosing interval) for total and free drug concentrations were fit by using a sigmoid maximal effect (Emax) model. Survival was significantly better in the higher dosage group than in the lower dosage group. Time above MIC did not display a correlation with outcome. The AUC:MIC showed a greater correlation with outcome (R2 = 0.56 total, 0.54 free) than did peak:MIC (R2 = 0.52 total, 0.51 free). With use of composite data, total AUC:MIC ratios associated with 50%, 90%, and 99% of Emax were 34:1, 56:1, and 95:1, respectively CONCLUSIONS: In this model, efficacy was achieved with the fluoroquinolone antibiotics at dosages yielding AUC0-24 comparable to those obtained in humans. One pharmacodynamic parameter (i.e., AUC:MIC) may be applied to various fluoroquinolones and isolates of S. pneumoniae. The AUC:MIC was more predictive of outcome than was time above the MIC or peak:MIC.


Subject(s)
Anti-Infective Agents/pharmacokinetics , Disease Models, Animal , Neutropenia/blood , Pneumonia, Pneumococcal/blood , Animals , Anti-Infective Agents/therapeutic use , Area Under Curve , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/statistics & numerical data , Fluoroquinolones , Mice , Microbial Sensitivity Tests/statistics & numerical data , Neutropenia/chemically induced , Neutropenia/drug therapy , Pneumonia, Pneumococcal/drug therapy , Renal Insufficiency/blood , Renal Insufficiency/chemically induced , Renal Insufficiency/drug therapy , Survival Analysis
10.
Antimicrob Agents Chemother ; 45(3): 673-8, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11181341

ABSTRACT

Several new quinolones that exhibit enhanced in vitro activity against Streptococcus pneumoniae have been developed. Using a dynamic in vitro model, we generated time-kill data for ciprofloxacin, clinafloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin against three isolates of quinolone-susceptible S. pneumoniae. Three pharmacokinetic profiles were simulated for each of the study agents (0.1, 1, and 10 times the area under the concentration-time curve [AUC]). Target 24-h AUCs were based upon human pharmacokinetic data resulting from the maximal daily doses of each agent. Ciprofloxacin was the least active agent against all three isolates. With regimens that simulated the human 24-h AUC, ciprofloxacin resulted in an initial, modest decline in the numbers of CFU per milliliter; however, by 48 h the numbers of CFU per milliliter returned to or exceeded the starting inoculum. At the AUC, levofloxacin resulted in variable bacteriostatic and bactericidal activities against the isolates. The remaining agents yielded bactericidal (99.9% reduction) activity by 48 h with regimens that simulated the AUC. At 0.1 time the AUC ciprofloxacin and levofloxacin produced no inhibitory effect, grepafloxacin exhibited bacteriostatic activity, trovafloxacin had mixed static and cidal activities, and clinafloxacin and moxifloxacin caused significant reductions in the numbers of CFU per milliliter by 48 h. All six agents produced cidal activity at 10 times the AUC. In this dynamic in vitro model of infection, the quinolones demonstrated various degrees of activity against S. pneumoniae. The rank order of activity, with respect to bactericidal effect, was ciprofloxacin (least active) << levofloxacin < grepafloxacin, trovafloxacin < clinafloxacin and moxifloxacin (most active). The rank order of the agents with respect to the selection of resistance was ciprofloxacin (most likely) > grepafloxacin, moxifloxacin, and trovafloxacin > levofloxacin > clinafloxacin.


Subject(s)
Anti-Infective Agents/pharmacology , Aza Compounds , Fluoroquinolones , Quinolines , Streptococcus pneumoniae/drug effects , Ciprofloxacin/pharmacology , Colony Count, Microbial , Humans , Levofloxacin , Microbial Sensitivity Tests , Models, Biological , Moxifloxacin , Naphthyridines/pharmacology , Ofloxacin/pharmacology , Piperazines/pharmacology , Time Factors
11.
J Am Soc Mass Spectrom ; 10(11): 1105-10, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10536817

ABSTRACT

We report here the first application of laser desorption (LD) in transmission geometry (backside irradiation of the sample through a transparent support) inside a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR). A probe-mounted fiber optic assembly was used to simplify the implementation of this LD technique. This setup requires little or no instrument modifications, has minimum maintenance requirements, and is relatively inexpensive to build. The performance of the probe was tested by determining the molecular weight of a commercial polystyrene standard from its matrix-assisted laser desorption/ionization (MALDI) spectrum. The measured average molecular weight is comparable to that obtained for the same sample by MALDI in the conventional top-illumination arrangement (reflection geometry) and by the manufacturer of the sample by gel permeation chromatography. The average velocities measured for ions evaporated by transmission mode LD of several neat samples are about half the velocity of those obtained by using the reflection geometry. Therefore, transmission mode irradiation of the sample holds promise to desorb ions that are easier to trap in an ICR cell. An oscillating capillary nebulizer was adapted for the deposition of analytes to improve sampling reproducibility.


Subject(s)
Cyclotrons , Fourier Analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Chromatography, Gel , Crystallization , Fiber Optic Technology , Molecular Weight , Optical Fibers , Polystyrenes/chemistry
12.
Z Geburtshilfe Neonatol ; 202(5): 187-91, 1998 Sep.
Article in German | MEDLINE | ID: mdl-9857443

ABSTRACT

OBJECTIVE: The purpose of this study was to determine the significance of fetal acidemia for newborn encephalopathy (NEP) and for the combination of NEP and multiorgan system dysfunction (MOS). In particular the influence of acidemia will be contrasted with criteria of pregnancy and delivery. STUDY DESIGN: 248 infants delivered with cord umbilical arterial pH < 7.15 (UApH) were retrospectively studied. Infants with similar or identical neonatal characteristics formed different groups: unaffected neonatal development, NEP, NEMO (NEP combined with MOS), other diseases, infection, exitus letalis. Statistics and calculations were done by means of factor analysis, univariate analysis, Student-Newman-Keuls-test, and Chi 2-test (p < 0.05). RESULTS: Twenty seven infants with UApH < 7.15 suffered from NEP. NEP in combination with MOS occurred in 11 cases. There was no relationship between the degree of acidemia and a single NEP. Infants with NEMO differ from all other groups in their mean UApH (p < 0.05). The Apgar scores 1 min and 5 min (A1, A5) separated newborns with unaffected neonatal development from all other groups (p < 0.05). UApH-differences in the group: NEMO resulted from a combination of acute intranatal fetal distress and operative delivery and a combination of A1, A5, preterm delivery, and pathologic CTG (p < 0.01). CONCLUSIONS: The degree of acidemia impacts the occurrence of NEMO. However, UApH alone does not predict this combination of characteristics. The Apgar score should be taken into account to evaluate an acidemic UApH. In case of acidemia complex factors of pregnancy and delivery are associated with an increased risk of NEMO.


Subject(s)
Acidosis/congenital , Brain Damage, Chronic/congenital , Acidosis/diagnosis , Apgar Score , Brain Damage, Chronic/diagnosis , Diagnosis, Differential , Female , Fetal Distress/diagnosis , Gestational Age , Humans , Hydrogen-Ion Concentration , Infant, Newborn , Multiple Organ Failure/congenital , Multiple Organ Failure/diagnosis , Pregnancy , Prognosis , Retrospective Studies , Risk Factors
13.
Toxicology ; 128(2): 151-67, 1998 Jul 03.
Article in English | MEDLINE | ID: mdl-9710156

ABSTRACT

The polycyclic aromatic hydrocarbon (PAH), methylcholanthrene (MCA), is a well studied carcinogen and a teratogen. MCA and other PAH cause immune suppression of B cell and T cell responses in mice and MCA had been reported to induce thymus atrophy. Here we show that MCA treatment causes thymus atrophy in adrenalectomized mice and in C57BL/6 and DBA/2 mice which differ in aryl hydrocarbon receptor (AhR) expression. This indicates that MCA-mediated thymus atrophy is mediated, at least in part, by glucocorticoid hormone receptor- and aryl hydrocarbon receptor-independent mechanisms. Assay of thymocytes, both in situ and ex vivo, demonstrate that MCA induces thymocyte apoptosis. Apoptotic thymocytes can be found within or adjacent to thymic Mphi, suggesting rapid phagocytosis. Mice that are deficient in tumor necrosis factor-alpha receptor-1 or p53, or that overexpress bcl-2 are susceptible to MCA-mediated thymus atrophy.


Subject(s)
Apoptosis/drug effects , B-Lymphocytes/drug effects , Carcinogens/toxicity , DNA/metabolism , Methylcholanthrene/toxicity , Receptors, Aryl Hydrocarbon/biosynthesis , T-Lymphocytes/drug effects , Thymus Gland/drug effects , Animals , B-Lymphocytes/pathology , B-Lymphocytes/ultrastructure , DNA/analysis , Female , Flow Cytometry , Genetic Techniques , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Microscopy, Electron , Phagocytosis/drug effects , Receptors, Aryl Hydrocarbon/drug effects , Species Specificity , T-Lymphocytes/pathology , T-Lymphocytes/ultrastructure , Thymus Gland/pathology , Thymus Gland/ultrastructure
14.
J Clin Invest ; 100(6): 1400-5, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9294105

ABSTRACT

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic disorder characterized by autosomal dominant inheritance, onset usually before 25 yr of age, and abnormal pancreatic beta-cell function. Mutations in the hepatocyte nuclear factor(HNF)-4alpha/MODY1, glucokinase/MODY2, and HNF-1alpha/MODY3 genes can cause this form of diabetes. In contrast to the glucokinase and HNF-1alpha genes, mutations in the HNF-4alpha gene are a relatively uncommon cause of MODY, and our understanding of the MODY1 form of diabetes is based on studies of only a single family, the R-W pedigree. Here we report the identification of a second family with MODY1 and the first in which there has been a detailed characterization of hepatic function. The affected members of this family, Dresden-11, have inherited a nonsense mutation, R154X, in the HNF-4alpha gene, and are predicted to have reduced levels of this transcription factor in the tissues in which it is expressed, including pancreatic islets, liver, kidney, and intestine. Subjects with the R154X mutation exhibited a diminished insulin secretory response to oral glucose. HNF-4alpha plays a central role in tissue-specific regulation of gene expression in the liver, including the control of synthesis of proteins involved in cholesterol and lipoprotein metabolism and the coagulation cascade. Subjects with the R154X mutation, however, showed no abnormalities in lipid metabolism or coagulation except for a paradoxical 3.3-fold increase in serum lipoprotein(a) levels, nor was there any evidence of renal dysfunction in these subjects. The results suggest that MODY1 is primarily a disorder of beta-cell function.


Subject(s)
DNA-Binding Proteins , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/physiopathology , Liver/physiology , Mutation , Phosphoproteins/genetics , Transcription Factors/genetics , Adult , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Blood Glucose/analysis , C-Peptide/blood , Female , Germany/ethnology , Glucose Tolerance Test , Hepatocyte Nuclear Factor 4 , Humans , Insulin/blood , Islets of Langerhans/physiology , Kidney/physiology , Male , Polymerase Chain Reaction , Proinsulin/blood , Time Factors
16.
Diabetes ; 46(3): 528-35, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9032114

ABSTRACT

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1alpha gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1alpha gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyC tract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1alpha gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1alpha gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T-->G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1alpha gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal beta-cell functions.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Exons , Mutation , Nuclear Proteins , Transcription Factors/genetics , Adolescent , Adult , Age of Onset , Amino Acid Sequence , Animals , Base Sequence , Child , Child, Preschool , Codon , DNA-Binding Proteins/genetics , Female , Frameshift Mutation , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Humans , Male , Mice , Molecular Sequence Data , Nuclear Family , Pedigree , Point Mutation , Polymerase Chain Reaction , Polymorphism, Genetic , Promoter Regions, Genetic , Rats , Sequence Deletion , Sequence Homology, Nucleic Acid
17.
Nat Genet ; 13(2): 161-6, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8640221

ABSTRACT

Non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is a common disorder of middle-aged individuals characterized by high blood glucose levels which, if untreated, can cause serious medical complications and lead to early death. Genetic factors play an important role in determining susceptibility to this disorder. However, the number of genes involved, their chromosomal location and the magnitude of their effect on NIDDM susceptibility are unknown. We have screened the human genome for susceptibility genes for NIDDM using non-and quasi-parametric linkage analysis methods in a group of Mexican American affected sib pairs. One marker, D2S125, showed significant evidence of linkage to NIDDM and appears to be a major factor affecting the development of diabetes mellitus in Mexican Americans. We propose that this locus be designated NIDDM1.


Subject(s)
Chromosomes, Human, Pair 2 , Diabetes Mellitus, Type 2/genetics , Mexican Americans/genetics , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/ethnology , Genetic Linkage , Genetic Markers , Genetic Predisposition to Disease , Humans , Japan , White People
18.
Zentralbl Hyg Umweltmed ; 193(2): 123-9, 1992 Aug.
Article in German | MEDLINE | ID: mdl-1388614

ABSTRACT

Based on a impurity of industrial made pizza products with Salmonella it was checked to what extent the preparation instructions given by the producer are connected with a hygienic risk for the consumers. It can be declared that the producer didn't deal with its duty for exactness and that the made controls were insufficient.


Subject(s)
Food Microbiology , Salmonella/growth & development , Cryopreservation , Hot Temperature
19.
Article in German | MEDLINE | ID: mdl-2144084

ABSTRACT

The posteruptive maturation of enamel is an important factor in caries disposition. In an experimental study the demineralization of enamel as well as its permeability and its morphological features in dependence of eruption stage were examined on extracted premolars. Chemical analysis showed variations in the Ca/P molar ratio. It is possible, that the instability of Ca-ions in crystalline configuration decreased during the time of eruption. The permeability of enamel decreased between 5 and 11.8%. It appears to be useful to concentrate preventive measure on this phase of dentition development.


Subject(s)
Dental Enamel Permeability , Tooth Calcification , Tooth Eruption , Tooth Permeability , Tooth/anatomy & histology , Bicuspid , Calcium/analysis , Dental Caries Susceptibility , Humans , In Vitro Techniques , Phosphorus/analysis , Surface Properties , Tooth/analysis
20.
J Antimicrob Chemother ; 23 Suppl D: 105-8, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2656623

ABSTRACT

The efficacies of a single 2 g intra-operative iv dose of cefmetazole or cefotetan in women undergoing non-elective Caesarean section were compared. Thirty-six women (18 in each group) completed the double-blind protocol. Both antibiotics were equally effective as agents for prophylaxis of post-Caesarean infections. Combined data suggest that routine multi-dose antibiotic prophylaxis regimens could be replaced by a single dose regimen.


Subject(s)
Cefmetazole/therapeutic use , Cefotetan/therapeutic use , Cesarean Section , Premedication , Surgical Wound Infection/prevention & control , Adolescent , Adult , Clinical Trials as Topic , Double-Blind Method , Female , Humans , Pregnancy , Random Allocation , Surgical Wound Infection/microbiology
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