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1.
Indian J Med Res ; 133: 431-3, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21537098

ABSTRACT

BACKGROUND & OBJECTIVES: Almost round-the-year occurrence of Salmonella Typhi and Salmonella Paratyphi A has been noticed in Rourkela since last 13 and five years respectively. The incidence of infection along with the antibiogram of these two serotypes in this area were carried out. METHODS: The study was carried out at Ispat General Hospital, Rourkela, India, between January 2005 and December 2008 with 5340 blood samples collected from patients with suspected enteric fever and pyrexia of unknown origin. Isolation, identification and antibiogram of the causative organisms were performed according to standard bacteriological procedures. RESULTS: A total of 298 Salmonella isolates showed an overall per cent positivity of 5.58. Multidrug resistance was found in 11.96 per cent and 15.62 per cent isolates of S. Typhi and S. Paratyphi A respectively. Less than 2 per cent isolates of Salmonella showed resistance to ciprofloxacin. A resistance of 3.0 to 6.25 per cent against third generation cephalosporins was observed among the salmonella isolates. INTERPRETATION & CONCLUSION: A round-the-year occurrence of Salmonella spp. in Rourkela might have been due to the presence of a considerable number of carriers in the locality, poor sanitation in nearby slum areas, and inadequate and contaminated community water supply at times. Higher degree of susceptibility among S. Typhi isolates against various antibiotics was encouraging, but increasing trend of resistance observed among S. Paratyphi A isolates was a matter of concern.


Subject(s)
Paratyphoid Fever/epidemiology , Salmonella paratyphi A/drug effects , Salmonella paratyphi A/isolation & purification , Salmonella typhi/drug effects , Salmonella typhi/isolation & purification , Typhoid Fever/epidemiology , Anti-Bacterial Agents/pharmacology , Cephalosporins/pharmacology , Ciprofloxacin/pharmacology , Community-Acquired Infections , Drug Resistance, Multiple/drug effects , Fever/drug therapy , Fever/epidemiology , Fever/microbiology , Humans , Incidence , India/epidemiology , Microbial Sensitivity Tests/methods , Paratyphoid Fever/drug therapy , Paratyphoid Fever/microbiology , Salmonella paratyphi A/metabolism , Salmonella typhi/metabolism , Sanitation , Typhoid Fever/drug therapy , Typhoid Fever/microbiology , Water Pollutants
2.
Clin Exp Allergy ; 32(4): 627-32, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11972612

ABSTRACT

BACKGROUND: Intrapulmonary administration of IL-12 has been shown to inhibit the number of eosinophils in lung murine models of asthma, but the precise mechanism of this inhibition has not been reported. The purpose of this study was to examine whether IL-12 treatment inhibits bone marrow eosinophilopoiesis, and to elucidate the role of IFN-gamma in this process. OBJECTIVE: To elucidate the in vivo and in vitro effects of IL-12 on eosinophil differentiation from murine bone marrow (BM) stem cells, and to examine the mechanistic role of IFN-gamma in this process. METHODS: Allergen-sensitized BALB/c mice were administered low doses of intranasal IL-12 at the time of allergen challenge, and the number of eosinophils in BM was determined 3 days later. The direct actions of IL-12 on eosinophil differentiation from BM cells were determined in vitro. The mechanistic role of IFN-gamma was assessed by measuring IFN-gamma induction by IL-12 in BM cell cultures, and through the use of IFN-gamma KO mice. RESULTS: Treatment of allergic mice with intrapulmonary IL-12 (1 ng or 10 ng) reduced eosinophils in BM by 43%. Culture of BM cells from allergen-sensitized mice with IL-3 + IL-5 induced eosinophil differentiation in vitro. Addition of IL-12 to these cultures inhibited eosinophil differentiation, with maximal inhibition (45%) occurring at 10 ng/mL IL-12 concentration. IL-12 induced IFN-gamma production from BM cultures, and failed to inhibit eosinophil differentiation in IFN-gamma-knockout mice, indicating a critical mechanistic role for IFN-gamma. CONCLUSION: This study demonstrates that IL-12 selectively inhibits BM eosinophilopoiesis, and that this effect is mediated by IFN-gamma. Intrapulmonary IL-12 has suppressive effects on BM eosinophilopoiesis that may represent a novel mechanism contributing to the anti-eosinophilic effects of IL-12 in allergic airway disease.


Subject(s)
Asthma/immunology , Eosinophils/immunology , Hematopoietic Stem Cells/immunology , Interferon-gamma/physiology , Interleukin-12/pharmacology , Animals , Cell Differentiation/drug effects , Cells, Cultured , Eosinophils/drug effects , Female , Hematopoietic Stem Cells/drug effects , Interferon-gamma/genetics , Interleukin-5/antagonists & inhibitors , Lung/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Models, Immunological
3.
Exp Lung Res ; 26(6): 457-76, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11033768

ABSTRACT

The relative efficacy of mucosal (intratracheal) and systemic (intraperitoneal) delivery of interleukin (IL)-12 was evaluated in a mouse model of allergic lung eosinophilia. Mucosal administration of IL-12 achieved 100- to 600-fold higher bronchoalveolar lavage (BAL) levels of IL-12, but 2- to 10-fold lower serum levels compared to systemic administration. Whereas both mucosal and systemic IL-12 inhibited BAL eosinophil recruitment at high doses (100-1000 ng), only mucosal IL-12 was effective at low doses (1-10 ng). Mucosal, but not systemic, administration of 1000 ng of IL-12 increased interferon (IFN)-gamma expression in BAL cells. In a model of ongoing eosinophilic inflammation, when mucosal or systemic IL-12 doses were initiated prior to peak eosinophilia, further eosinophil recruitment was inhibited. However, when IL-12 treatment was initiated after peak eosinophil recruitment occurred, recovery from eosinophilic inflammation was not facilitated. Our findings are the first to demonstrate that locally administered IL-12 inhibits eosinophil recruitment at 100-fold lower doses than systemic IL-12. The most likely mechanism of this enhanced inhibitory activity is a sustained increase in lung levels of IL-12 that augments IFN-gamma production from BAL cells. We suggest that future studies should evaluate the efficacy of low doses of nebulized IL-12 in inhibiting eosinophilic lung inflammation in asthma.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Interferon-gamma/biosynthesis , Interleukin-12/administration & dosage , Lung/drug effects , Pulmonary Eosinophilia/prevention & control , Respiratory Mucosa/drug effects , Adjuvants, Immunologic/blood , Allergens/immunology , Animals , Asthma , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Disease Models, Animal , Injections, Intraperitoneal , Interferon-gamma/genetics , Interleukin-12/blood , Intubation, Intratracheal , Lung/pathology , Mice , Mice, Inbred BALB C , Pollen/immunology , Pulmonary Eosinophilia/immunology , Pulmonary Eosinophilia/pathology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
4.
J Immunol ; 165(4): 2198-204, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10925307

ABSTRACT

The activation of eosinophils by cytokines is a major event in the pathogenesis of allergic diseases. We have investigated the activation of mitogen-activated protein (MAP) kinases and their functional relevance in eosinophil differentiation, survival, degranulation, and cytokine production. IL-5 induced phosphorylation and activation of extracellular signal-regulated kinases (ERK) and p38 MAP kinases in eosinophils. PD98059, a MAP/ERK kinase inhibitor, blocked phosphorylation of ERK1/2 in a dose-dependent manner. SB202190, a p38 inhibitor, blocked p38-dependent phosphorylation of activating transcription factor-2. To study the importance of the MAP kinases on eosinophil differentiation, we cultured mouse bone marrow cells with IL-3 and IL-5 in the presence of the inhibitors. SB202190 dramatically inhibited eosinophil differentiation by 71%. PD98059 was less potent and reduced eosinophil differentiation by 28%. Both inhibitors marginally inhibited eosinophil survival only at the highest doses. Prolonged incubation of eosinophils with IL-5 induced significant eosinophil-derived neurotoxin release. Both PD98059 and SB202190 nearly completely inhibited (87% and 100% inhibition, respectively) IL-5-stimulated eosinophil-derived neurotoxin release in a dose-dependent manner. Next, we examined the effect of the MAP kinase inhibitors on eosinophil production of the cytokine macrophage-inflammatory protein (MIP)-1alpha. PD98059 blocked C5a- but not ionomycin-induced MIP-1alpha production (59% inhibition at 50 microM concentration). In contrast, SB202190 nearly completely inhibited (99%) C5a-induced MIP-1alpha production. Further, it blocked ionomycin-stimulated production by 66%. Our results suggest that both p38 and ERK1/2 MAP kinases play an important role in eosinophil differentiation, cytokine production, and degranulation. The p38 MAP kinase plays a greater role than ERK1/2 in eosinophil differentiation and cytokine production.


Subject(s)
Eosinophils/enzymology , Eosinophils/immunology , Mitogen-Activated Protein Kinases/physiology , Animals , Cell Degranulation/immunology , Cell Differentiation/immunology , Cell Survival/immunology , Chemokine CCL3 , Chemokine CCL4 , Enzyme Activation/drug effects , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Eosinophils/cytology , Eosinophils/metabolism , Flavonoids/pharmacology , Humans , Imidazoles/pharmacology , Interleukin-5/physiology , Macrophage Inflammatory Proteins/biosynthesis , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Pyridines/pharmacology , p38 Mitogen-Activated Protein Kinases
6.
J Immunol ; 162(10): 6284-93, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10229876

ABSTRACT

Asthma is an inflammatory disease of the airways that is induced by Th2 cytokines and inhibited by Th1 cytokines. Despite a steady increase in the incidence, morbidity, and mortality from asthma, no current treatment can reduce or prevent asthma for a prolonged period. We examined the ability of unmethylated CpG oligodeoxynucleotides (ODN), which are potent inducers of Th1 cytokines, to prevent the inflammatory and physiological manifestations of asthma in mice sensitized to ragweed allergen. Administration of CpG ODN 48 h before allergen challenge increased the ratio of IFN-gamma to IL-4 secreting cells, diminished allergen-induced eosinophil recruitment, and decreased the number of ragweed allergen-specific IgE-producing cells. These effects of CpG ODN were sustained for at least 6 wk after its administration. Furthermore, there was a vigorous Th1 memory response to the recall Ag, inhibition of peribronchial and perivascular lung inflammation, and inhibition of bronchial hyperresponsiveness 6 wk after administration of CpG ODN. Administration of CpG ODN in IFN-gamma -/- mice failed to inhibit eosinophil recruitment, indicating a critical role of IFN-gamma in mediating these effects. This is the first report of a treatment that inhibits allergic lung inflammation in presensitized animals for a prolonged period and thus has relevance to the development of an effective long term treatment for asthma.


Subject(s)
Anti-Allergic Agents/therapeutic use , Asthma/prevention & control , Oligodeoxyribonucleotides/therapeutic use , Allergens/immunology , Animals , Asthma/immunology , Asthma/pathology , Chemotaxis, Leukocyte/drug effects , Cytidine/therapeutic use , Dinucleoside Phosphates/therapeutic use , Disease Models, Animal , Female , Guanosine/therapeutic use , Immunoglobulin E/biosynthesis , Interferon-gamma/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Nebulizers and Vaporizers , Plant Proteins/immunology , Pollen/immunology , Pulmonary Eosinophilia/prevention & control , Th1 Cells/immunology , Th2 Cells/immunology , Time Factors , Trachea/drug effects
7.
Gene ; 195(1): 41-8, 1997 Aug 11.
Article in English | MEDLINE | ID: mdl-9300818

ABSTRACT

The two full-length cDNAs encoding ESG1 (Enhancer of split groucho) and related AES (Amino Enhancer of split) proteins of 767 and 197 amino acids, respectively, were cloned and sequenced from the African frog Xenopus laevis. The amino acid sequence of Xenopus ESG1 protein had 61% identity to the full-length Drosophila groucho. Xenopus AES protein exhibited 91%, 58% and 48% identity to the mouse AES, amino-terminal regions of Xenopus ESG1 and Drosophila groucho, respectively. Northern blot analysis showed that widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts were seen in adult tissues, whereas ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. The overall structural relationships of ESG and AES proteins among human, mouse, rat, Xenopus and Drosophila were analysed.


Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Gene Expression Regulation, Developmental , Insect Proteins/genetics , Proteins/genetics , Repressor Proteins , Xenopus Proteins , Xenopus/genetics , Amino Acid Sequence , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Cloning, Molecular , DNA-Binding Proteins/metabolism , Drosophila/genetics , Embryo, Nonmammalian/physiology , Female , Humans , Insect Proteins/metabolism , Mice , Molecular Sequence Data , Oocytes/physiology , Ovum/physiology , Proteins/metabolism , Rats , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue Distribution , Xenopus/growth & development
8.
Biochem Biophys Res Commun ; 234(3): 788-91, 1997 May 29.
Article in English | MEDLINE | ID: mdl-9175795

ABSTRACT

SMT3 gene from nematode, Caenorhabditis elegans, was identified and sequenced. The nematode SMT3 gene codes for a homolog to the yeast SMT3, suppressor of MIF2 mutation in a centromere protein gene. Further, nematode SMT3 cDNA was amplified by polymerase chain reaction from a cDNA library and its nucleotide sequence determined. A comparison of the SMT3 genomic and cDNA sequences established that the protein-encoding sequence is interrupted by two introns of 56 and 50 bp at codon Nos. 22-23 and 56, respectively. The sequence of 91 amino acids deduced from the nematode SMT3 nucleotide sequence exhibited 42 and 47% identity to the yeast and human SMT3 protein sequences, respectively. The evolutionary relationships of SMT3 proteins from human, nematode, Arabidopsis, rice, and yeast were analyzed.


Subject(s)
Caenorhabditis elegans/genetics , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , Evolution, Molecular , Humans , Molecular Sequence Data , Oryza/genetics , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Small Ubiquitin-Related Modifier Proteins
9.
J Biol Chem ; 269(9): 6900-7, 1994 Mar 04.
Article in English | MEDLINE | ID: mdl-8120052

ABSTRACT

We introduced a promoter trap vector carrying a neo gene as a selectable marker into F9 cells and established several cell lines in which the expression of neo gene is under the control of an endogenous host gene that is active only in the undifferentiated F9 cells. Using one of these cell lines, G19, we isolated the integrated neo construct and its flanking host sequences by the plasmid rescue method, identified the host gene which contributes to the expression of neo gene, and named it the Zfp-57 gene. Two different Zfp-57 transcripts (1.8 and 3.2 kilobases) were identified in the undifferentiated F9 cells, and the levels of these transcripts were decreased significantly within a short time after induction of differentiation. We examined mouse organs for the presence of the Zfp-57 RNAs and found that the 1.8-kilobase RNA was detected only in the testis. The Zfp-57 cDNAs corresponding to the two different RNAs were isolated, and a comparison of the nucleotide sequences revealed that their coding regions were completely identical, but they differed both in length and in sequence of the 3'-untranslated region. The Zfp-57 cDNA encoded a protein consisting of 421 amino acids with an extremely high content of basic amino acid residues and multiple zinc finger motifs. Immunocytochemical analysis revealed that this protein is localized in the nucleus. These findings suggest that the Zfp-57 protein is a DNA-binding protein.


Subject(s)
Cell Differentiation/physiology , DNA-Binding Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Promoter Regions, Genetic , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , DNA-Binding Proteins/genetics , Genetic Vectors , Kanamycin Kinase , Kinetics , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Restriction Mapping , Teratoma , Transcription, Genetic , Transfection , Tumor Cells, Cultured
10.
Biochem Mol Biol Int ; 32(1): 131-8, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8012278

ABSTRACT

We previously reported that the promoter region of the mouse mitochondrial malate dehydrogenase gene is located within the 160 base-pairs (bp) region upstream from the initiation codon, named P1, and that the -160/-131 bp region contains sequence(s) essential for the promoter activity of mitochondrial malate dehydrogenase gene. To define more precisely the regulatory mechanism of this gene, a 3' deletion analysis was performed using constructs with a constant 3' boundary at -130 bp. We obtained evidence of an additional positive regulatory element located between -393/-249 bp, named P2, the function of which can be separated from that of the P1. DNase I footprinting and gel-sift mobility experiments showed that nuclear protein binding to the P2 was not inhibited in the presence of the P1 sequences.


Subject(s)
Malate Dehydrogenase/genetics , Mitochondria/enzymology , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , 3T3 Cells , Animals , Base Composition , Deoxyribonuclease I/metabolism , Electrophoresis, Polyacrylamide Gel , Malate Dehydrogenase/metabolism , Mice , Protein Binding
11.
Eur J Biochem ; 216(1): 343-52, 1993 Aug 15.
Article in English | MEDLINE | ID: mdl-8365415

ABSTRACT

Mouse and human cDNA encoding AES (amino-terminal enhancer of split) and ESG (enhancer of split groucho) proteins with strong similarity to Drosophila enhancer of split groucho protein were isolated and sequenced. Mouse AES-1 and AES-2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins were found to exhibit approximately 50% identity to the amino-terminal region of Drosophila groucho, mouse ESG and human transducin-like enhancer of split (TLE) proteins. Mouse AES transcripts of 1.5 kb and 1.2 kb were abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 kb and 1.4 kb were predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3 kb and 4.0 kb were found only in testis, while human TLE 1 transcripts of 4.5 kb was more abundant in muscle and placenta compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosomes 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines.


Subject(s)
Cloning, Molecular , DNA-Binding Proteins/genetics , Drosophila/chemistry , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Chromosome Mapping , Chromosomes, Human, Pair 15 , Chromosomes, Human, Pair 19 , Chromosomes, Human, Pair 9 , DNA/chemistry , DNA/genetics , DNA-Binding Proteins/chemistry , Female , Humans , Hybrid Cells , Male , Mice , Molecular Sequence Data , Organ Specificity , Poly A/metabolism , Sequence Homology, Amino Acid , Transcription Factors/chemistry
12.
Trans R Soc Trop Med Hyg ; 86(6): 615-6, 1992.
Article in English | MEDLINE | ID: mdl-1337634

ABSTRACT

A 20 d drug regimen of aminosidine (= paromomycin) at 12 mg/kg/d in combination with sodium stibogluconate at 20 mg/kg/d proved efficacious and well-tolerated in patients with visceral leishmaniasis in the State of Bihar, India. Eighteen of 22 evaluable patients achieved an ultimate cure. The remaining 4 patients, although not cleared of parasites, had their parasite grade reduced and also improved clinically. This confirms prior findings in Kenyan patients with kala-azar, and indicates that this regimen is a valid alternative to antimonial compounds alone in the State of Bihar, where cases of kala-azar not responding to antimonial drugs and intolerant of pentamidine are increasingly recorded.


Subject(s)
Antimony Sodium Gluconate/therapeutic use , Leishmaniasis, Visceral/drug therapy , Paromomycin/therapeutic use , Adolescent , Adult , Aged , Child , Child, Preschool , Drug Therapy, Combination , Female , Humans , Infant , Male , Middle Aged , Pilot Projects
13.
J Indian Med Assoc ; 89(10): 294-6, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1815014

ABSTRACT

Forty patients suffering from intractable unilateral trigeminal neuralgia involving more than one division of the trigeminal nerve were treated by percutaneous radiofrequency thermocoagulation of the trigeminal sensory root. The aim of the operation was to relieve the pain without producing dense sensory deficit in the face. This goal was achieved by making selective lesions in the sensory root with gradually increasing temperature 60 degrees C to 90 degrees C. Three to four consecutive lesions each for 60 seconds have been found to produce excellent pain relief in 77.7% with good and fair results in the rest. The recurrence rate has been found to be 15% during 2 years of follow-up. Considerable dysaesthesia was observed in 5% of cases. Corneal anaesthesia was found in 5% cases while one patient developed neuroparalytic keratitis. Transient trigeminal motor weakness was observed in 10% of patients.


Subject(s)
Electrocoagulation , Trigeminal Nerve/surgery , Trigeminal Neuralgia/surgery , Aged , Anti-Bacterial Agents/therapeutic use , Electrocoagulation/adverse effects , Female , Humans , Male , Middle Aged , Recurrence
14.
Biochem Int ; 22(3): 583-91, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2076113

ABSTRACT

Structural organization of the human cytosolic aspartate aminotransferase gene was determined by analyzing the phage clones obtained from two kinds of genomic DNA libraries, using mouse cytosolic aspartate aminotransferase cDNA as a probe. The gene is more than 32 kb long and is split into 9 exons by 8 introns of various sizes. The 5' and 3'-flanking regions and the exact sizes and boundaries of the exon blocks were determined. The 5' end of the gene lacks the TATA and CAAT boxes, but contains G+C rich sequences and one potential binding site for the transcription factor, Sp1. Comparison of the nucleotide sequence of 250 bp upstream from the translation-initiation site revealed that the sequences of binding sites for the nuclear proteins, previously identified in the mouse, are highly conserved between human and mouse cytosolic aspartate aminotransferase genes.


Subject(s)
Aspartate Aminotransferases/genetics , Cytosol/enzymology , Amino Acid Sequence , Animals , Bacteriophages/genetics , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA Probes , Humans , Isoenzymes/genetics , Mice , Molecular Sequence Data , Nucleic Acid Hybridization , Restriction Mapping , Sequence Homology, Nucleic Acid
15.
J Biol Chem ; 265(3): 1293-9, 1990 Jan 25.
Article in English | MEDLINE | ID: mdl-2295630

ABSTRACT

The malate-aspartate shuttle, consisting of mitochondrial and cytosolic aspartate aminotransferase and mitochondrial and cytosolic malate dehydrogenase, is a major pathway for the transport of reducing equivalents from cytosol to mitochondria in mammals. To elucidate molecular mechanisms regulating metabolic coordination between the mitochondria and the cytosol, we analyzed the 5'-flanking regulatory regions of the complete set of mouse isoenzyme genes playing a pivotal role in the shuttle. Deletion analysis and an in vivo transfection assay, using NIH3T3 cells, revealed that all the promoter regions are located within the 300-base pair regions upstream from the initiation codon. Subsequently, DNase I footprinting analyses using NIH3T3 cell nuclear extracts led to identification of several protein binding sites within these promoter regions. A synthetic oligomer containing the consensus binding site sequence for CTF/NFI, a transcription factor for RNA polymerase II, competed for the binding of proteins to the promoter regions of cytosolic aspartate aminotransferase and mitochondrial and cytosolic malate dehydrogenase genes, but not for that of the mitochondrial aspartate amino-transferase gene. On the other hand, a synthetic oligomer containing the consensus binding site sequence for Sp1, which activates transcription from promoters containing properly positioned GC boxes, competed for protein(s) binding to the promoter region of the mitochondrial aspartate aminotransferase gene.


Subject(s)
Aspartate Aminotransferases/genetics , Malate Dehydrogenase/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Chromosome Deletion , Cytosol/enzymology , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , Mice , Mitochondria/enzymology , Molecular Sequence Data , Nuclear Proteins/metabolism , Transcription, Genetic , Transfection
16.
Prog Clin Biol Res ; 344: 139-58, 1990.
Article in English | MEDLINE | ID: mdl-2203048

ABSTRACT

To examine molecular mechanisms of transcription of mammalian isozyme genes functioning in the malate-aspartate shuttle and to observe structural and evolutionary relationships, we investigated gene organizations of cAspAT and mAspAT, and cMDH and mMDH, and isolated and characterized cDNAs and genomic DNAs for these isozymes in mice. The deduced amino acid sequences of mouse cAspAT and mAspAT showed about 47%, and those of mouse cMDH and mMDH, about 23% overall homology. Surprisingly, the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and E. coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice. The first duplication of a common ancestral MDH gene should thus have occurred long before the emergence of the eukaryotic cells, and subsequently, the mammalian mMDH and E. coli MDH genes have evolved from one of the duplicates. The mammalian cMDH and Thermus flavus MDH genes have no doubt evolved from one of the other duplicates. Moreover, structural organizations of the two-pairs of isozyme genes indicated that introns antedate the divergence of these mitochondrial and cytosolic isozyme genes. The 5' ends of all four isozyme genes lacked the TATA and CAAT boxes characteristic of eukaryotic promoters but did contain G + C-rich sequences and multiple transcription-initiation sites. We found several highly conserved regions in the 5' flanking sequences between mAspAT and cAspAT, between mMDH and mAspAT, and between cMDH and cAspAT genes.


Subject(s)
Aspartate Aminotransferases/genetics , Genes , Isoenzymes/genetics , Malate Dehydrogenase/genetics , Animals , Aspartic Acid/metabolism , Biological Evolution , Escherichia coli/enzymology , Escherichia coli/genetics , Exons , Introns , Malates/metabolism , Mice , Models, Molecular , Protein Conformation , Thermus/enzymology , Thermus/genetics
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