Biomedical subjects

M A Couto

Publications and source records attributed to M A Couto.

6 recordsLinked to original sources

PubMed1994

Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin.

Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.

Animals↗

PubMed1993

Selective inhibition of microbial serine proteases by eNAP-2, an antimicrobial peptide from equine neutrophils.

Equine neutrophil antimicrobial peptide 2 (eNAP-2), a recently described antimicrobial peptide isolated from equine neutrophils, was found to selectively inactivate microbial serine proteases (subtilisin A and proteinase K) without inhibiting mammalian serine proteases (human neutrophil elastase, human cathepsin G, and bovine pancreatic trypsin). Although the primary structure of eNAP-2 resembled that of several known antiproteases that belong to the 4-disulfide core peptide family, this pattern of selectivity is unique. eNAP-2 formed a noncovalent complex with native subtilisin A or proteinase K but did not associate with these enzymes if they had been treated with phenylmethylsulfonyl fluoride, a serine protease inhibitor. The eNAP-2-microbial protease complex was disrupted by boiling or by exposure to low pH. We suggest that eNAP-2 exerted selective antiproteinase activity by binding tightly but noncovalently to the active site of subtilisin A or proteinase K. Since microbial exoproteases may act as virulence factors, the combined antimicrobial and antiprotease activities of eNAP-2 could allow it to play an important role in neutrophil-mediated antimicrobial defenses.

Animals↗

PubMed1992

eNAP-2, a novel cysteine-rich bactericidal peptide from equine leukocytes.

We purified a novel cysteine-rich antibiotic peptide, eNAP-2 (M(r), approximately 6,500), from acid extracts of equine neutrophils by sequential gel filtration and reversed-phase high-performance liquid chromatography and determined its partial N-terminal amino acid sequence. Although its cysteine motif distinguished eNAP-2 from all other currently known endogenous antibiotic peptides, including defensins and granulins, it showed substantial sequence similarity to WDNM1, a putative member of the four-disulfide-core protein family that also includes animal and human antiproteases, snake venom neurotoxins, and rat and mouse whey proteins. The antibacterial properties of eNAP-2 were tested against several equine uterine pathogens, namely, Streptococcus zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Killing of S. zooepidemicus was very efficient, as evidenced by a 94% decrease in numbers of CFU per milliliter after exposure to 100 micrograms of eNAP-2 per ml (approximately 15 microM) for 2 h. Exposure of E. coli and P. aeruginosa to 200 micrograms of eNAP-2 per ml for 2 h resulted in 90.2 and 77.6% reduction, respectively, in the numbers of CFU per milliliter. Bacteriostasis, without bactericidal activity, occurred after K. pneumoniae was incubated with 200 micrograms of eNAP-2 per ml. Additional studies will be required in other species and cell types to determine whether eNAP-2 is restricted to equine neutrophils or is the index member of a larger family of endogenous antibiotics.

Amino Acid Sequence↗

PubMed1992

Identification of eNAP-1, an antimicrobial peptide from equine neutrophils.

Endogenous, cysteine-rich antimicrobial peptides known as defensins are prominent components of human, rabbit, and rat neutrophils, yet little is known about their occurrence in other mammalian species. Although we did not detect mature (i.e., processed) defensins in equine neutrophil granules, we found that these granules contained small amounts of other cysteine-rich peptides with antimicrobial activity. One of these, eNAP-1, was purified by a combination of gel permeation and reversed-phase high-performance liquid chromatography from acid extracts prepared from the cytoplasmic granules of equine neutrophils. The molecular mass of eNAP-1 was approximately 7.2 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Amino acid analysis revealed that eNAP-1 had an unusually high cysteine content and that it was relatively enriched in alanine, glycine, lysine, and proline residues. The partial (N-terminal) amino acid sequence of eNAP-1 was DVQCGEGHFCHDXQTCCRASQGGXACCPYSQGVCCADQRHCCPVGF. Thirty-six of these residues (78.3%) were identical to those of a recently cloned human neutrophil peptide of unknown function and belonging to the granulin family. Homologous peptides have also been noted in rat bone marrow cells and rat kidney epithelins. We tested the ability of eNAP-1 to kill several equine uterine pathogens. Streptococcus zooepidemicus was killed most effectively, sustaining a greater than 99.8% decrease in CFU per milliliter after a 2-h exposure to 100 micrograms of eNAP-1 per ml (approximately 15 microM). Escherichia coli and Pseudomonas aeruginosa were somewhat less susceptible, manifesting 87.0 and 87.1% mean decreases in CFU per milliliter, respectively, after incubation for 2 h with 200 micrograms of eNAP-1 per ml. Klebsiella pneumoniae numbers were not significantly reduced after exposure to eNAP-1. These antimicrobial properties suggest that eNAP-1 may contribute to phagocyte-mediated host defense against equine infections.

Amino Acid Sequence↗

PubMedDate not supplied

[Malignant peritoneal pseudomyxoma. A case report].

A case of malignant pseudomyxoma peritonei is described. This rare entity is characterized by widespread dissemination throughout the abdominal cavity and low grade of malignancy. General well-being is usually unimpaired. Diagnosis can be suggested by computed tomographic appearance; the course of action, however, is ultimately determined by the operative findings. The best treatment has been tumour excision, although the extent of this procedure is not well defined.

Cystadenocarcinoma↗

PubMedDate not supplied

[Carcinoid of Meckel's diverticulum. Report of a case and review of the literature].

The presence of carcinoid tumor in a Meckel's diverticulum is a rare entity. This report describes a 56-year-old man who was admitted to hospital with symptomatic gallbladder stones. At laparotomy the gallstones were confirmed and routine exploration of the peritoneal cavity identified a small bowel diverticulum about 60 cm of the ileocecal valve. Cholecystectomy and resection of a small bowel segment containing the diverticulum were performed. Histology revealed carcinoid tumour in Meckel's diverticulum.

Carcinoid Tumor↗