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Biomedical subjects

M C Sousa

Publications and source records attributed to M C Sousa.

15 recordsLinked to original sources
PubMed2006

Steroid hormone secretion during the ovulatory cycle and pregnancy in farmed Alaskan reindeer.

Seasonal endocrine changes in 5 non-bred and 10 pregnant Alaskan reindeer have been documented. Blood samples were collected from early September until early May, spanning the breeding season, gestation, or the anovulatory period. Plasma was analyzed by RIA for progesterone (P4), estradiol-17beta, estrone, and estrone sulfate. Elevated P4 in 80% of the reindeer at the onset of the study indicated that ovarian activity had been initiated. The median date for the onset of the first recorded full-length ovulatory cycle was September 23. In nonbred reindeer, the mean ovulatory cycle length from September to May was 24 +/- 1 d (range 18 to 29 d). Nonbred females continued to cycle throughout the winter, displaying 6 to 8 ovulatory cycles after the beginning of blood sampling. Cycle length (mean 22 to 24 d) did not vary between individuals (P = 0.170) or over the course of the winter (P = 0.244). In early April, ovulatory cycles ceased with normal demise of the corpus luteum in 2 females, whereas the remaining 3 females formed apparently persistent corpora lutea. Natural service breeding occurred between September 10 and October 2, and P4 profiles indicated that all breeding females conceived to the first mating. Concentrations of P4 rose steadily after conception and remained elevated throughout gestation, with mean concentrations not varying significantly (P = 0.104) from 4 to 28 wk of gestation. Estrogens all followed patterns similar to each other, remaining at baseline concentrations until approximately 24 wk of gestation and rising coincidently as P4 declined just before parturition. There was a continual overlap throughout the winter in peak P4 concentrations observed in cycling and pregnant reindeer. Calving occurred between April 8 and May 2, resulting in a mean gestation length of 211 +/- 2.2 d (range 198 to 221 d). Information from this study can be used by Alaskan reindeer producers to improve management and profitability of reindeer production.

Agriculture↗
PubMed2003

The male effect, mounting behavior, and the onset of estrus in farmed muskoxen.

Muskox farming is an emerging industry in Alaska. For such an endeavor to succeed, it is necessary to develop fundamental husbandry tools specifically for this species. This study examined the role of the bull in the onset of seasonal ovarian activity and the synchrony of estrus and tested the efficacy of a radiotelemetric estrus detection system. Twenty parous female muskoxen, ranging in age from 4 to 15 yr, were divided into three groups (balanced by age and body weight). Groups received early bull exposure (EBE; August 1, n = 7), late bull exposure (LBE; August 22, n = 6), or remained physically isolated from a bull (NBE; n = 7). Just before bull introduction, radiotelemetry transmitters were attached to the rump of muskox cows in the EBE and LBE groups. Plasma samples were collected from all the cows twice weekly and analyzed for progesterone (P4). The mean date of onset of seasonal ovarian activity was earlier in the EBE and LBE groups than in the NBE group (P < 0.001) and was earlier in EBE (P < 0.021) than in LBE. All EBE and LBE cows conceived to the first fertile cycle, giving 100% fertility. The time interval for the onset of ovarian activity between the first and last cow within each group was 7 d in the EBE group, 12 d in the LBE group, and 24 d in the NBE group. Radiotelemetry correctly identified estrus 95% of the time (18 of 19 instances). Mean length of estrus for each cow, calculated from the time of first mount until last mount, was 771 +/- 98 min (range = 4 to 1,508 min). Mean number of mounts during estrus was 8.89 +/- 1.29 (range = 3 to 25 mounts). Length of estrus at the first short cycle did not differ from the length of the first fertile estrus between, or within, individuals. Estrous behavior was initiated more frequently during the night (2300 to 0500; P < 0.05). Bull introduction can be used as a simple, low-cost management tool for synchronizing estrus in farmed muskoxen. Radiotelemetry was an effective tool for identifying breeding behavior in this species.

Animal Husbandry↗
PubMed2001

Structure of Haemophilus influenzae HslV protein at 1.9 A resolution, revealing a cation-binding site near the catalytic site.

The structure of the Haemophilus influenzae HslV protease of the HslUV 'prokaryotic proteasome' has been solved by molecular replacement and refined with data to 1.9 A resolution. The protease is a 'double donut' of hexameric rings; two alternative sets of intermolecular interactions between protomers in the rings result in 'quasi-equivalent' packing within the assembly. Anomalous scattering data from crystals with potassium present in the mother liquor reveal a K(+) ion bound with octahedral coordination near the active-site Thr1 residue. The site also binds Na(+) ions and is likely to bind Mg(2+), suggesting that monovalent and divalent metal ions may influence the catalytic activity of the protease.

ATP-Dependent Proteases↗
PubMed2001

Structure of the universal stress protein of Haemophilus influenzae.

BACKGROUND: The universal stress protein UspA is a small cytoplasmic bacterial protein whose expression is enhanced several-fold when cellular viability is challenged with heat shock, nutrient starvation, stress agents which arrest cell growth, or DNA-damaging agents. UspA enhances the rate of cell survival during prolonged exposure to such conditions, suggesting that it asserts a general "stress endurance" activity. However, neither the structure of UspA nor the biochemical mechanism by which it protects cells from the broad spectrum of stress agents is known. RESULTS: The crystal structure of Haemophilus influenzae UspA reveals an asymmetric dimer with a tertiary alpha/beta fold similar to that of the Methanococcus jannaschi MJ0577 protein, a protein whose crystal structure revealed a novel ATP binding motif. UspA differs significantly from the MJ0577 structure in several details, including the triphosphate binding loop of the ATP binding motif; UspA shows no ATP binding activity. CONCLUSIONS: Within the universal stress protein family that is delineated by sequence similarity, UspA is the only member which has been correlated with a cellular activity, and MJ0577 is the only member which has been assigned a biochemical activity, i.e., ATP binding. UspA has a similar fold to the MJ0577 protein but does not bind ATP. This suggests that members of this protein family will segregate into two groups, based on whether or not they bind ATP. By implication, one subset of the universal stress proteins presumably has an ATP-dependent function, while another subset functions in ATP-independent activities.

Adenosine Triphosphate↗
PubMed2001

Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells.

Attachment of Giardia lamblia trophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for parasite-induced enterocyte dysfunction and clinical disease. In this work, coincubation of Giardia with Int-407 cells, was used as an in vitro model to study the role of cytoskeleton and surface lectins involved in the attachment of the parasite. This interaction was also studied by scanning and transmission electron microscopy. Adherence was dependent on temperature and was maximal at 37 degrees C. It was reduced by 2.5 mM colchicine (57%), mebendazole (10 microg/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 microg/ml) (21%). No significant modification was observed when Giardia was pretreated with cytochalasins B and D and with EDTA. Giardia attachment was also diminished by preincubating Int-407 cells with cytochalasin B and D (5 microg/ml) (16%) and by glutaraldehyde fixation of intestinal cells and of G. lamblia trophozoites (72 and 100%, respectively). Ultrastructural studies showed that Giardia attaches to the Int-407 monolayer predominantly by its ventral surface. Int-407 cells contact trophozoites with elongated microvilli, and both trophozoite imprints and interactions of Giardia flagella with intestinal cells were also observed. Transmission electron microscopy showed that Giardia lateral crest and ventrolateral flange were important structures in the adherence process. Our results suggest a combination of mechanical and hydrodynamic forces in trophozoite attachment; surface lectins also seem to mediate binding and may be involved in specific recognition of host cells.

Animals↗
PubMed2000

Crystal and solution structures of an HslUV protease-chaperone complex.

HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.

ATP-Dependent Proteases↗
PubMed2000

Tc-99m MDP uptake in uterine leiomyoma.

A 47-year-old woman with adenocarcinoma of the right breast had bone scintigraphy with Tc-99m MDP. Bone imaging did not show any metastases. However, a large area of increased tracer uptake was seen extending from the abdomen to the pelvis. Abdominal ultrasound revealed a large solid and heterogeneous mass, measuring 18 x 11 x 14.3 cm, that originated in an empty uterus. A biopsy of the surgical specimen showed a leiomyoma of the uterus.

Adenocarcinoma↗
PubMed1999

A new method for assessing metronidazole susceptibility of Giardia lamblia trophozoites.

A quantitative, simple, and rapid assay has been developed to assess Giardia lamblia trophozoite sensitivity to metronidazole [1-(2-hydroxyetyl)-2-methyl-5-nitroimidazole] (MTZ). This new assay utilizes the ability of live (surviving) trophozoites to take up oxygen after have been exposed to MTZ. The effect of MTZ on oxygen uptake was compared with its effect on viability as evaluated by a culture method and morphological assays. Oxygen uptake rates decreased in trophozoites treated with MTZ, and this effect was drug concentration dependent: O(2) uptake rates went from 3.04 microM O(2) min(-1) per 10(6) cells to 0.72 microM O(2) min(-1) per 10(6) cells with increasing drug concentration (0.15 to 0.6 mM) in the preincubation. Concentrations of the drug which inhibited oxygen uptake by 28 to 76% in trophozoites killed from 39 to 82% of trophozoites, as evaluated by the culture method, and altered the morphology of 21 to 86% of the trophozoites. Thus, the trophozoites killed by MTZ are nonmotile cells and do not take up oxygen. A good correlation was found between the inhibitory effects of MTZ, as evaluated by oxygen uptake, and cellular viability. Similar 50% inhibitory concentrations were obtained: 0.33 mM by oxygen uptake, 0. 26 mM by the culture method, and 0.35 mM by morphological criteria. Oxygen uptake appears to be a good indicator of parasite viability. Therefore, this new method can provide a convenient means to assess MTZ susceptibility in G. lamblia and can be applied for screening potential antigiardial agents.

Animals↗
PubMed1998

The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change.

The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.

Adenosine Triphosphatases↗
PubMed1997

Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone.

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.

Adenosine Triphosphate↗
PubMed1996

The interaction of the UDP-GLC:glycoprotein glucosyltransferase with the acceptor glycoprotein.

The UDP-Glyc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum that in cell-free assays glucosylates denatured but not native glycoproteins. It has been postulated that the enzyme participates in the quality control mechanism of glycoprotein folding in the endoplasmic reticulum by which only properly folded species are transported to the Golgi apparatus. This paper reports studies on the protein determinants that upon recognition by the glucosyltransferase allow the selective glucosylation of denatured glycoproteins. It is shown here that extensive chemical modification of denatured soybean agglutinin does not modify its glucose acceptor capacity, thus suggesting that the protein determinants recognized by the glucosyltransferase are not formed by specific amino acids. Moreover, it is reported that the enzyme binds patches of hydrophobic amino acids exposed in denatured but not in native conformations. As exposure of such residues is the only feature common to all misfolded conformations, it is suggested that binding to hydrophobic amino acids forms part of the recognition mechanism of denatured conformations by the glucosyltransferase.

Amino Acid Sequence↗
PubMed1996

The use of UDP-Glc:glycoprotein glucosyltransferase for radiolabeling protein-linked high mannose-type oligosaccharides.

A simple method for specifically radiolabeling high mannose-type oligosaccharides linked to protein backbones has been developed. The method is based on the fact that incubation of rat liver UDP-Glc:glycoprotein glucosyltransferase, glucose-labelled UDP-Glc and a denatured high mannose-type glycoprotein target leads to the glucosylation of the oligosaccharide. In the case described here it allowed to follow easily the purification, by HPLC and affinity chromatography, of labelled glycopeptides obtained by controlled proteolysis of cruzipain, a cysteine proteinase isolated from the human pathogen Trypanosoma cruzi. It was thus determined that the N-glycosylation site in Asn33 of cruzipain is occupied by high mannose-type oligosaccharides.

Amino Acid Sequence↗
PubMed1992

Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase.

Animals↗
PubMedDate not supplied

The cytotoxic effects of ciprofloxacin in Giardia lamblia trophozoites.

The cytotoxicity of ciprofloxacin was investigated in culture of Giardia lamblia (ATCC 30957), used as an in vitro cellular model, on the basis of cell growth, morphology, viability, adherence and metabolic studies. The effects on cell membrane integrity were evaluated by permeability of the cells to trypan blue, and the morphological alterations were studied by optical and transmission electronic microscopy. The metabolic studies were performed by measured oxygen uptake with a Clark-type oxygen electrode. Our data show that ciprofloxacin induces loss of viability with morphological alterations and loss of membrane integrity. Ultrastructural observations revealed that this drug promoted modifications on the cell shape, pronounced dorsal vesiculation, plasma membrane blebbing, disaggregation of ribosome, coagulation of cytoplasmic matrix, and heavy deposit of electron-dense precipitates on the cytoplasm and nucleus. Ciprofloxacin also inhibits parasite growth, adherence and O(2) uptake in a concentration-dependent manner. Some of these observations suggest that cytotoxicity of ciprofloxacin results in cell death by a necrosis process. We demonstrate that ciprofloxacin has a lethal effect in G. lamblia trophozoites and could be used as an alternative drug in giardiasis treatment, particularly in infections that are resistant to other antibiotics.

Animal Testing Alternatives↗