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Biomedical subjects

A Camacho

Publications and source records attributed to A Camacho.

At least 37 records · Page 2Linked to original sources

Pleiotropic effect of protein P6 on the viral cycle of bacteriophage phi29.

The product of bacteriophage phi29 early gene 6, protein p6, is a double-stranded-DNA binding protein and one of the more abundant proteins during viral infection. We have studied the role of protein p6 in vivo through the infection of suppressor and nonsuppressor Bacillus subtilis strains with a phage carrying a nonsense mutation in gene 6, sus6(626). In the absence of functional protein p6, the two major processes of the viral cycle, transcription and DNA replication, were affected. Viral DNA synthesis was practically abolished, and early transcription was remarkably delayed and, in addition, underregulated at late times of the infection. The amount of protein p6 synthesized after infection with mutant phage sus6(626) under suppressor conditions was sixfold lower than that produced after wild-type infection. Nonetheless, phage production was as high as that obtained after wild-type infection. These results indicate that p6 is synthesized in amounts higher than those needed for most of its functions. However, the concentration of protein p6 appeared to be important for repression of the early promoter C2.

Bacillus Phages↗

Effect of mutations in the "extended -10" motif of three Bacillus subtilis sigmaA-RNA polymerase-dependent promoters.

The "extended -10" motif described originally in Escherichia coli promoters occurs frequently in other bacterial promoters. Most Bacillus subtilis bacteriophage o29 promoters contain this motif. To analyse the influence of the motif on sigmaA-RNA polymerase transcription, the 5'-TG-3' dinucleotide was changed to 5'-AC-3' in three o29 promoters. This change impaired RNA polymerase binding to the promoters; the yields of closed and open complexes were reduced independently of other differences inherent to each promoter. The mutation abolished transcription in vitro from a promoter lacking the consensus sequence at the -35 hexamer. In contrast, at other promoters with a -35 consensus sequence, the yield of run off transcription was not reduced by the mutation. Indeed an apparent interference phenomenon at high polymerase/DNA ratios was relieved. These results indicate that the extended -10 motif provides contact points for sigmaA-RNA polymerase with a role restricted to the first steps of transcription.

Bacillus subtilis↗

Apurinic/apyrimidinic endonuclease genes from the trypanosomatidae leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli.

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E. coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing.

Amino Acid Sequence↗

Conversion of US3-encoded protein kinase gene from pseudorabies virus in a diploid gene located within inverted repeats by genetic recombination between the viral genome isomers.

The pseudorabies virus (PRV) genome consists of two components, long (U(L)) and short (U(S)) regions. The U(S) region is the only one capable of inverting itself relative to the U(L) region during productive infection, generating two equimolecular isomeric forms of viral DNA. Here we describe a recombinant virus (gIp2) generated by genetic recombination between pseudorabies viral isomers. This recombination event was observed in the parental virus gIS8, which was obtained by insertion of the alpha4-TK herpes simplex virus type 1 (HSV1) gene. The growth of gIS8 virus in the presence of 5-bromodeoxyuridine (BrdU) yielded gIp2. This was generated by nonhomologous recombination either between the two viral genomic isomers of gIS8, P and I(U/S), or between the same P isomer using nonhomologous and homologous recombination, with loss of the HSV1 sequences and duplication of the PRV US3-encoded protein kinase gene. Virus gIp2 is negative for TK, gI, gE, 11K and 28K and shows an in vitro replication capacity in neuronal cells approximately 22 times lower than that of parental virus gIS8, and similar to that of the Bartha vaccine virus strain in monkey kidney and human neuronal cells.

Animals↗

Crystallization and preliminary X-ray diffraction of Trypanosoma cruzi dUTPase.

Crystals of Trypanosoma cruzi dUTPase have been grown. Two different morphologies are observed, depending on the molecular weight of the PEG used as precipitating agent in the mother liquor, both having a hexagonal unit cell with similar dimensions. Complete X-ray diffraction data have been collected to low resolution for one of the forms. The space group is P6322, with unit-cell dimensions a = 134.15, c = 147.05 A. Peaks in the self-rotation function and the solvent content are consistent with two molecules of dUTPase per asymmetric unit.

Animals↗

Hb D-Los Angeles associated with Hb S or beta-thalassemia in four Mexican Mestizo families.

Twenty-five individuals were studied from four unrelated Mexican Mestizo families with Hb D-Los Angeles. We observed five compound heterozygotes: four for Hb S and Hb D, and one for Hb D and beta-thalassemia (beta(0) 39 nonsense mutation); 16 heterozygotes: four for Hb S, seven for Hb D, and five for beta-thalassemia, while the remaining four were normal. The four Hb S/Hb D patients had severe hemolytic anemia, while in the Hb D/beta-thalassemia patient, the anemia was similar to that of a beta-thalassemia heterozygote; therefore, Hb D is clinically harmful when it is associated with Hb S. The beta(S) chromosomes were associated with the Benin haplotype in two families and Bantu in one family, while the beta(D) and beta(0) 39 mutations were associated with haplotype 1 [+ - - - - + +]. The Bantu and Benin haplotypes have been found with high frequency in Hb S individuals from the East Coast and Northwestern Mexico. The beta(D) chromosomes from Italy were also shown to be associated with haplotype 1, the most frequently observed haplotype in the world; there are no haplotype studies on beta(D) chromosomes from India or China where Hb D-Los Angeles is most common. Thus, the true origin of this mutation observed in these Mestizo families remains to be elucidated.

Adolescent↗

5-hydroxytryptamine1A receptor-mediated effects on adenylate cyclase and nitric oxide synthase activities in rat ventral prostate.

The rat ventral prostate possesses specific 5-hydroxytryptamine (5-HT1A) receptors coupled to adenylate cyclase. In vivo treatment of rats or in vitro preincubation of minced prostatic tissue with the 5-HT1A receptor agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) in different experimental conditions shows the possibility of desensitisation mechanisms with switching from inhibitory to stimulatory pattern on adenylate cyclase activity. As in the majority of systems, we observed the inhibition of forskolin-stimulated adenylate cyclase activity as a functional correlate of 5-HT1A receptor activation. A similar feature occurred when the direct stimulation of the enzyme by the diterpene was replaced by a receptor-mediated activation with the neuropeptide vasoactive intestinal peptide. Furthermore, 8-OH-DPAT stimulated nitric oxide synthase (NOS) activity in a dose-dependent manner. Thus, serotonin appears to be able to act in the rat prostate gland through specific 5-HT1A receptors coupled to a complex system of signal transduction involving an inhibitory response of adenylate cyclase that can become stimulatory, as well as an enhancement of NOS activity.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major.

A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichia coli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

Amino Acid Sequence↗

A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi.

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

Amino Acid Sequence↗

Characterization of the genes, including that encoding the viral proteinase, contained in BamHI restriction fragment 9 of the pseudorabies virus genome.

We describe the nucleotide sequence, transcription pattern and open reading frames (ORFs) located on BamHI restriction fragment 9 (0.406-0.435 map units) in the unique long segment of the pseudorabies virus (PRV) genome. The fragment contains three nested genes with a common 3' end. The 5' ends of the corresponding 0.9, 1.7 and 3.3 kb mRNAs have been mapped. Fragment BamHI-9 contains three complete ORFs, ORF1, ORF2 and ORF2.5. ORF1, which is within the 3.3 kb transcript, encodes a protein with an apparent molecular mass of 60 kDa which is homologous to the product of the herpes simplex virus type 1 UL25 gene. The 1.7 kb mRNA contains ORF2, whose product is homologous to the herpesvirus proteinases, while the 0.9 kb transcript contains ORF2.5, which probably encodes the assembly protein precursor. ORF2 was identified as the PRV proteinase gene following expression in E. coli, using the product of ORF2.5 as the substrate protein.

Amino Acid Sequence↗

Kinetic effects of trimethoprim-sulfamethoxazole in children with biliary atresia: a new dosing regimen.

Pharmacokinetics for combination trimethoprim/ sulfamethoxazole (TMP/SMX) was studied in only four patients with biliary atresia (BA): three girls, 6.2,8.0 and 8.2 years of age and one boy 8.4 years of age, as this is an uncommon obstructive anomaly of the extrahepatic biliary system and has been described as having a poor prognosis. These four patients are the survivors of 27 initial children who were operated on previously. They have been receiving 2.3 +/- 0.5 mg/kg TMP, and 11.5 +/- 2.6 mg/kg SMX every 12 h since 2 weeks after surgical treatment for biliary atresia performed at 2-2.5 months of age. The patients have suffered some episodes of cholangitis during their short lives, most of them after interrupting temporally the chemotherapy. Nevertheless, they have achieved a favorable quality of life. TMP/SMX disposition was well characterized by a one compartment open pharmacokinetic model. Wide interpatient variability was observed for all pharmacokinetic parameters with coefficients of variation for t1/2 el, ClT, and Vd of 33.2, 49.6, and 26.3%, respectively, for SMX and 108.9, 52.1, and 71.0%, respectively, for TMP. A marked difference in the pharmacokinetics of TMP and SMX was observed, for example; (ClT: mean +/- SD; 90.3 +/- 47.0 ml/kg/g for TMP and 13.7 +/- 6.8 ml/kg/h for SMX), (t1/2 el with 7.93 +/- 8.64 h for TMP and 10.51 +/- 3.49 h for SMX). In order to develop dosage schedules that would reliably achieve peak serum concentrations of TMP/SMX in the therapeutic range, we found that established dose leads to high fluctuations at steady state between C(max), ss and C(min), ss, without maintaining therapeutic levels. Recommended maintenance dose varied from 8 to 30 mg/kg for SMX with a mean of 21.9 +/- 10.89 mg/kg/12 h, and from 0.8 to 4.5 mg/kg/12 h with a mean of 3.2 +/- 1.7 mg/kg/12 h. The present study illustrates the need for pharmacokinetic studies for the individualization of drug dosing in patients with BA.

Biliary Atresia↗

Isolation and characterization of TK-deficient mutants of African swine fever virus.

African swine fever virus induces the synthesis of thymidine kinase (TK) in BHK TK-negative cells as an immediate early protein. The TK gene is not essential for growth of ASFV in cell culture and a stable viral strain deficient in TK has been isolated (E70NTKp). The genetic lesion of this ASFV TK- strain was identified by TK gene nucleotide sequencing, showing a nucleotide deletion leading to a -1 frameshift and a nonsense codon residue downstream of the deletion. The availability of this viable ASFV variant deficient in TK activity allows the insertion of foreign genes in the ASFV genome for genetic and biochemical studies.

African Swine Fever Virus↗

Direct enantiomeric separation of cis-(+/-)diltiazem in plasma by high-performance liquid chromatography with ovomucoid column.

A method is proposed for the direct enantiomeric separation of cis-(+/-)diltiazem in plasma. The method is based on a commercial Ultron ES-OVM column with a mobile phase of different mixtures of ethanol-phosphate buffer. The degree of extraction of the enantiomers was more than 86%. The detection limit of the method used was 5 ng of racemic mixture on column (coefficient of variation = 13%), corresponding to 3.2 ng for cis-(+/-)diltiazem and 1.8 ng for cis-(+/-)diltiazem.

Chromatography, High Pressure Liquid↗

Chronic ear pathology in a model of neonatal amniotic fluid ear inoculation.

Human histopathologic studies have demonstrated that amniotic fluid cellular contents, keratinized squamous epithelial cells and lanugo hair, induce an intense inflammatory reaction including granulation tissue in the neonatal temporal bone. To investigate this reaction over a prolonged period, an animal model was studied. An aliquot of sterilized autologous hair and keratinized epithelial cells was placed into 14 gerbil bullae; saline was used as a contralateral control. The animals were sacrificed at intervals up to 6 months and the temporal bones were studied by light microscopy. All animals demonstrated nonpurulent inflammatory changes on the experimental side including granulation tissue, osteoneogenesis, tympanosclerosis, and cholesteatoma; the control side demonstrated minimal middle ear changes. We conclude that in this model autologous keratinized tissue provokes a foreign body response similar to the granulation tissue observed in human infants and, further, that over a prolonged period the middle ear demonstrates more severe pathologic consequences.

Amniotic Fluid↗