Biomedical subjects

C R Machado

Publications and source records attributed to C R Machado.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of promoter regulatory elements involved in downexpression of the DNA polymerase kappa in colorectal cancer.

The low-fidelity DNA polymerases thought to be specialized in DNA damage processing are frequently misregulated in cancers. We show here that DNA polymerase kappa (polkappa), prone to replicate across oxidative and aromatic adducts and known to function in nucleotide excision repair (NER), is downregulated in colorectal tumour biopsies. Contrary to the replicative poldelta and polalpha, for which only activating domains were described, we identified an upstream 465-bp-long repressor region in the promoter of POLK. We also found an activating 237-bp region that includes stimulating protein-1 (SP1) and cyclic AMP-responsive element (CRE)-binding sites. Mutations at one CRE-binding site led to a dramatic 80% decrease in promoter activity. Alterations of the SP1-binding site also affected, to a lesser extent, the transcription. Gel shift assays confirmed the role played by CRE/SP1 recognition sequences. Moreover, ectopic expression of SP1 or CRE-binding protein (CREB) protein favoured polkappa transcription. Finally, we found that polkappa downexpression in colorectal biopsies correlated with a decreased level of CREB and SP1 transcripts. This work shows that the promoter of POLK is cis-controlled and suggests that silencing of CREB and SP1 proteins could contribute to downregulation of this repair polymerase in colorectal tumours.

Acetylation↗

PubMed2003

Beta2-agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle.

1. The role of beta2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective hbetaagonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10(-5)M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5)M ICI 118.551, a selective fbeta2antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4)M dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by fbeta2adrenoceptors, with the participation of a cAMP-dependent pathway.

Adrenergic beta-Agonists↗

PubMed2001

Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi.

Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment was used as a probe to select the corresponding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T. cruzi MSH2 interferes with the bacterial mismatch system. Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.

Amino Acid Sequence↗

PubMed2001

Cardiac autonomic denervation and functional response to neurotoxins during acute experimental Chagas' disease in rats.

Severe cardiac autonomic denervation occurs in the acute Chagas' disease in rats. The present study aims at verifying whether this denervation was accompanied by impairment of heart function. Scorpionic (Tityus serrulatus) crude venom was used for neurotransmitter release in isolated hearts (Langendorff's preparation). In control hearts, the venom induced significant bradycardia followed by tachycardia. In infected animals, despite the severe (sympathetic) or moderate (parasympathetic) cardiac denervation, the venom provoked similar bradycardia but the tachycardia was higher. The hearts of infected animals beat at significantly lower rate. Atropine prevented this lower rate. Our results demonstrated sympathetic dysfunction during the acute phase of Trypanosoma cruzi infection in rats, the parasympathetic function being spared.

Acetylcholine↗

PubMed2001

Trypanosoma cruzi: peripheral blood monocytes and heart macrophages in the resistance to acute experimental infection in rats.

The contribution of mononuclear phagocytes to host resistance against acute Chagas disease has been studied in vivo in a rat model inoculated with Trypanosoma cruzi, Y strain. Acute T. cruzi infection triggered a dramatic increase (937%) in peripheral blood monocyte number at day 12 of infection. At this point, histological analysis of the heart showed high parasitism and diffuse and a moderate to intense mononuclear inflammatory process. Ultrastructural study revealed a large number of macrophages, in addition to lymphocytes and undiffer entiated cells. Clusters of macrophages exhibited different morphologi cal phenotypes, with evident signs of activation (increase in size, surface rufflings, and amount of cytoplasmic organelles). Cell-to-cell contacts involving macrophages and lymphocytes or macrophages and mono cytes were observed. Depletion of macrophages by treatment with silica, a selective cytotoxic agent for these cells, caused a significant increase in the number of amastigote nests in cardiomyocytes. The present findings indicate that the early phase of infection with T. cruzi induces rapid production, maturation, and activation of the monocyte/macrophage system so as to control T. cruzi replication, emphasizing the crucial role for macrophages in the rat resistance to Chagas disease.

Animals↗

PubMed2001

Evidence for a direct action of Tityus serrulatus scorpion venom on the cardiac muscle.

The ability of toxins to activate the cardiovascular system plays an important role in the morbidity and lethality of the Tityus serrulatus scorpion envenoming. Most of the actions of the scorpion toxins are indirect and due to the release of adrenergic and cholinergic neurotransmitters. Accordingly, treatment following envenoming is targeted towards inhibition of adrenergic and cholinergic receptors. Here, we have sought evidence for a direct action of T. serrulatus venom on the isolated rat heart (Langendorff's method). We show that the bradycardia induced by T. serrulatus venom was completely blocked by atropine, a muscarinic receptor antagonist. Similarly, the increase in heart rate that follows the venom-induced bradycardia was totally inhibited by a beta(1)-adrenoceptor antagonist or by chemical sympathetic denervation with 6-hydroxydopamine. In contrast to these findings, the venom-induced increase in contractile force was not modified by beta(1)-adrenoceptor blockade or by chemical sympathetic denervation. The results clearly demonstrate that the chronotropic effects of T. serrulatus are dependent on neurotransmitter release, but the inotropic effects are not. The neurotransmitter-independent increase in contractility seems to be a direct action of the venom on cardiomyocytes. We suggest that this direct effect on cardiac fibers may play a role in the development of cardiac arrhythmias and contractility defects following envenoming with T. serrulatus scorpion.

Acetylcholine↗

PubMed2000

Trypanosoma cruzi infection and the rat central nervous system: proliferation of parasites in astrocytes and the brain reaction to parasitism.

Chagas' disease, caused by the protozoan Trypanosoma cruzi, is characterized by an acute phase in which parasites circulate in the blood and proliferate in several cell types, especially muscle cells. A life-long chronic phase follows the acute phase. In young patients, the acute phase is more severe, and meningoencephalitis frequently occurs in children before 2 years of age. Parasites have been rarely observed in neurons but their presence inside glial cells has been reported without characterization of the glial cell type. The cells involved in the brain reaction to the parasites and the time course of this reaction remain to be studied. Therefore, using suckling and juvenile rats and different T. cruzi populations, we aimed at determining the brain target for parasite proliferation and the cells involved in the brain reaction. Around the middle of the acute phase, histological and ultrastructural findings indicated that T. cruzi proliferates in astrocytes, forming nests devoid of enclosing membrane as described for non-glial cells. The brain nodular reaction comprised astrocytes, microglia, macrophages and neutrophils. Resting microglia was devoid of parasites in contrast to macrophages and neutrophils that probably participate in parasite removal. Suckling animals were significantly more affected, the numbers of nests and nodules varying with inoculum size. Histoquantitative analysis showed higher number of nests at the parasitemic peak (day 13) and drastic fall at day 20 post-inoculation. The highest number of nodules occurred at day 20 with drastic reduction at day 30. Recovery from histopathological alterations occurred even in surviving younger animals.

Age Factors↗

PubMed2000

Early damage of sympathetic neurons after co-culture with macrophages: a model of neuronal injury in vitro.

Since activated immune cells may damage peripheral nerves during inflammation, we developed a co-culture model that permits the direct study of macrophage-induced neuronal damage. Sympathetic neurons were enzymatically isolated from neonatal mice and co-cultured with increasing numbers of peritoneal macrophages for 24 h. This caused rapid neuronal cell death, reducing neuronal number by 24.1 +/- 4% with the addition of 11.5 x 10(3) macrophages, representing a ratio of 8 macrophages per neuron. Nuclear analysis showed that cell death occurred by both apoptosis and necrosis. These effects were not mimicked by addition of macrophage-conditioned medium, and were prevented by 10 microM dexamethasone. Although no appreciable neuronal death occurred beyond 24 h, the density of neurites was decreased between 1 and 2 days of co-culture (p < 0.05). There is, therefore, a rapid induction of cytotoxicity by macrophages after their addition to the neuronal cultures, followed by axonal damage without neuronal cell death.

Animals↗

PubMed2000

Mast cell types and cell-to-cell interactions in lymph nodes of the opossum Didelphis albiventris.

Previous light-microscopic studies have shown a unique population of mast cells in lymphatic sinuses of lymph nodes located in the head, neck, axillary fossa and inguinal region of the opossum. In the present work, scanning and transmission electron-microscopic studies in the opossum mandibular and superficial axillary lymph nodes have strengthened the differences between connective-tissue mast cells (CTMC) and the lymphatic-sinus mast cells (LSMC). Further, close appositions of mast cells to other cells were described. At the nodal capsule, CTMC contacted fibroblast and granulocytes. In the lymphatic sinuses a few CTMC contacted LSMC, macrophages and reticular cells. The LSMC contacted macrophages, reticular cells and other LSMC. A few LSMC could be located in the medullary cord in close contact with plasma cells or other lymphoid cells, keeping the same ultrastructural features of those found in the lymphatic sinuses. An important new finding was provided by light-microscopic studies in nine abdominal lymph nodes. Most of them (para-aortic, common iliac, cardial, cecocolic and those of the body and tail of the pancreas) displayed numerous LSMC with the same distribution and histological features described herein. However, the mesenteric, pyloric and head-of-pancreas lymph nodes were virtually devoid of LSMC. Instead, their mast cells occurred mainly at the medullary cords and were very similar to the CTMC. Ultrastructural studies at the mesenteric lymph nodes confirmed the CTMC character of the mast cells located at both medullary cords and sinuses, and disclosed interactions with macrophages and lymphoid cells.

Animals↗

PubMed2000

Cardiac autonomic denervation in congestive heart failure: comparison of Chagas' heart disease with other dilated cardiomyopathy.

Congestive heart failure (CHF) is associated with activation of the cardiac sympathetic nerves. However, impairment of the sympathetic nerve terminals in patients with CHF has been indicated by studies showing reduction of cardiac norepinephrine uptake and stores. This investigation studies the histochemical evaluation of the sympathetic nerve terminals in CHF. The cardiac parasympathetic innervation was also studied to address the question of specificity of the presumed sympathetic denervation. Nineteen patients with CHF underwent cardiac transplantation or partial ventriculectomy, which provided the heart tissue. In 11 of them, the dilated cardiomyopathy was associated with Chagas' disease. Inflammatory process and fibrosis were studied histologically. The sympathetic and parasympathetic nerve fibers were visualized through histochemical techniques for, respectively, catecholamines and acetylcholinesterase activity. By using a computer-assisted morphometric program, the inflammation, fibrosis, and parasympathetic innervation were quantified. Moderate to severe fibrosing myocarditis characterized the hearts of the chagasic patients. In cardiomyopathies not associated with Chagas' disease, the inflammation was discrete, if present, but the amount of fibrosis was similar to that found in Chagas' cardiomyopathy. Reduction of both kinds of nerve terminals occurred in the heart of all patients. The parasympathetic denervation was proven to be more severe in chagasic cardiomyopathy. Our data on the heart innervation indicate a progressive autonomic denervation in heart failure. In Chagas' heart disease, the denervation seems to be more severe or rapid, probably because of the sustained inflammatory process.

Acetylcholinesterase↗

PubMed2000

Infection with different Trypanosoma cruzi populations in rats: myocarditis, cardiac sympathetic denervation, and involvement of digestive organs.

We tested four isolates of Trypanosoma cruzi to assess parasite virulence and ability to cause myocarditis, cardiac sympathetic denervation, and histopathologic alterations in organs of the digestive system. The susceptibility of rats depends on the population of T. cruzi, with the ABC strain and the CL-Brener clone killing all animals, regardless of the parasitemic pattern. All tested T. cruzi populations caused acute myocarditis, but failed to induce histologic alterations in the intestine. The Cl-Brener and ABC isolates caused esophageal myositis. The myocarditis caused by the ABC, CL-Brener, and Y isolates was severe, but resolution started at the end of the acute phase. In contrast, the Col 1.7 G2 clone induced mild and sustained myocarditis. Our results also showed that T. cruzi populations able to induce severe acute myocarditis caused marked sympathetic denervation, but recovery of normal cardiac histology and innervation occurred. The sustained myocarditis induced by Col 1.7 G2 clone failed to cause sustained denervation.

Animals↗

PubMed1999

Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice.

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Coll.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.

Acute Disease↗

PubMed1998

Depletion of radiosensitive leukocytes exacerbates the heart sympathetic denervation and parasitism in experimental Chagas' disease in rats.

The acute phase of the Trypanosoma cruzi infection induces damage of the heart sympathetic nerve terminals in rats. The participation of the radiosensitive leukocytes in this process was assessed in young rats submitted to gamma irradiation 1 day before infection with T. cruzi, Y strain. Gamma irradiation increased the heart noradrenergic denervation evaluated at day 12 of infection by histochemical and electron-microscopic methods. The presence of numerous macrophages with ultrastructural features of activation and a significant rise in the myocardial parasitism suggest the involvement of radioresistant macrophages or parasite factors, or both, on the damage of the nerve terminals.

Animals↗

PubMed1998

Cloning of a cDNA from Arabidopsis thaliana homologous to the human XPB gene.

The human gene XPB, defective in xeroderma pigmentosum patients complementation group B, encodes a DNA helicase involved in several DNA metabolic pathways, including DNA repair and transcription. The high conservation of this gene has allowed the cloning of homologs in various species, such as mouse, yeast and Drosophila. Not much information on the molecular basis of nucleotide excision repair in plants is available, but these organisms may have similar mechanisms to other eukaryotes. A homolog of XPB was isolated in Arabidopsis thaliana by using polymerase chain reaction (PCR) with degenerate oligonucleotides based on protein domains which are conserved among several species. Screening of an Arabidopsis cDNA library led to the identification and isolation of a cDNA clone with 2670 bp encoding a predicted protein of 767 amino acids, denoted araXPB. Genomic analysis indicated that this is a nuclear single copy gene in plant cells. Northern blot with the cDNA probe revealed a major transcript which migrated at approx. 2,800 b, in agreement with the size of the cDNA isolated. The araXPB protein shares approximately 50% identical and 70% conserved amino acids with the yeast and human homologs. The plant protein maintains all the functional domains found in the other proteins, including nuclear localization signal, DNA-binding domain and helicase motifs, suggesting that it might also act as part of the RNA transcription apparatus, as well as nucleotide excision repair in plant cells.

Amino Acid Sequence↗

PubMed1998

Heart autonomic innervation during the acute phase of experimental American trypanosomiasis in the dog.

Heart autonomic innervation was studied in dogs during the acute phase of the experimental infection with the Berenice-78 strain of Trypanosoma cruzi. A glyoxylic acid-induced fluorescence method for catecholamines and a thiocholine method for demonstrating acetylcholinesterase activity showed the sympathetic and the parasympathetic nerve fibers, respectively. At day 34 of infection, moderate-to-intense rarefaction of both cholinergic and noradrenergic nerve fibers occurred in the atria of all animals coincident with moderate to intense myocarditis. In the ventricles, sympathetic denervation was clearly present only when the inflammatory processes were moderate to intense. Preliminary results on the chronic phase indicate that normal autonomic innervation coexists with an incipient chronic fibrosing myocarditis.

Acetylcholinesterase↗

PubMed1997

Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance.

The THI4 gene of Saccharomyces cerevisiae encodes an enzyme of the thiamine biosynthetic pathway. The plant homolog thi1, from Arabidopsis thaliana, is also involved in thiamine biosynthesis; but was originally cloned due to its capacity to complement DNA repair deficient phenotypes in Escherichia coli. Here, the behavior of a thi4 disrupted strain was examined for increased sensitivity to treatment with the DNA damaging agents ultraviolet radiation (UV, 254 nm) and methyl methanesulfonate (MMS). Although the thi4 null mutant showed a similar level of survival as the wild-type strain, a higher frequency of respiratory mutants was induced by the two treatments. A similar phenotype was seen with wild-type strains expressing an antisense THI4 construct. Further analysis of respiratory mutants revealed that these were due to mutations of mitochondrial DNA (mtDNA) rather than nuclear DNA, consisting of rho-petite mutants. Moreover, the frequency of mutations was unaffected by the presence or absence of thiamine in the growth medium, and the defect leading to induction of petites in the thi4 mutant was corrected by expression of the Arabidopsis thi1 gene. Thus, Thi4 and its plant homolog appear to be dual functional proteins with roles in thiamine biosynthesis and mitochondrial DNA damage tolerance.

Arabidopsis↗

PubMed1996

Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair.

An Arabidopsis thaliana cDNA was isolated by complementation of the Escherichia coli mutant strain BW535 (xth, nfo, nth), which is defective in DNA base excision repair pathways. This cDNA partially complements the methyl methane sulfonate (MMS) sensitive phenotype of BW535. It also partially corrects the UV-sensitive phenotype of E. coli AB1886 (uvrA) and restores its ability to reactivate UV-irradiated lambda phage. It has an insert of ca. 1.3 kb with an open reading frame of 1047 bp (predicting a protein with a molecular mass of 36 kDa). This cDNA presents a high homology to a stress related gene from two species of Fusarium (sti35) and to genes whose products participate in the thiamine biosynthesis pathway, THI4, from Saccharomyces cerevisiae and nmt2 from Schizosaccharomyces pombe. The Arabidopsis predicted polypeptide has homology to several protein motifs: amino-terminal chloroplast transit peptide, dinucleotide binding site, DNA binding and bacterial DNA polymerases. The auxotrophy for thiamine in the yeast thi4::URA3 disruption strain is complemented by the Arabidopsis gene. Thus, the cloned gene, named thi1, is likely to function in the biosynthesis of thiamine in plants. The data presented in this work indicate that thi1 may also be involved in DNA damage tolerance in plant cells.

Amino Acid Sequence↗

PubMed1996

Preganglionic fibres of the adrenal medulla and cervical sympathetic ganglia: differential involvement during experimental American trypanosomiasis in rats.

The acute phase of experimental Chagas' disease in rats is associated with early lesions of the post-ganglionic sympathetic nerve terminals of the heart, the varicosities being the target. In the superior cervical and stellate ganglia the preganglionic fibres showed no signs of lesion in the course of experimental Chagas' disease. In the adrenal medulla, however, the acute phase of the Trypanosoma cruzi infection induced a clear rarefaction of the acetylcholinesterase-positive nerve fibres (20 and 32 days after infection). Recovery of the normal pattern occurred in most animals by day 125 after infection. At days 20, 32 and 46 after infection, electron-microscopic studies demonstrated the occurrence of damage in cholinergic nerve terminals contacting the chromaffin cells. The signs of damage included dense bodies, clumps of synaptic vesicles and filaments, rarefaction of all organelles, vacuoles and irregular contour. The ultrastructural peculiarities of the sympathetic ganglia may explain the ganglionar microenvironment protective against the hazardous factors elicited by acute Chagas' disease.

Acetylcholinesterase↗