Biomedical subjects

J Calvete

Publications and source records attributed to J Calvete.

At least 19 recordsLinked to original sources

PubMed2004

Antagonists Mo and Cu in a heterometallic cluster present on a novel protein (orange protein) isolated from Desulfovibrio gigas.

An orange-coloured protein (ORP) isolated from Desulfovibrio gigas, a sulphate reducer, has been previously shown by extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)] [J. Am. Chem. Soc. 122 (2000) 8321]. We report here the purification and the biochemical/spectroscopic characterisation of this novel protein. ORP is a soluble monomeric protein (11.8 kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS(4)(2-) moiety in the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange colour, with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological function of ORP is discussed.

Amino Acid Sequence↗

PubMed2004

Pancreatic function after severe acute biliary pancreatitis: the role of necrosectomy.

OBJECTIVES: To investigate the recovery of pancreatic function after severe acute biliary pancreatitis (ABP), especially the influence of necrosectomy on endocrine and exocrine functions. METHODS: Prospective cohort study including 39 patients with severe ABP. According to need or no need for surgical necrosectomy, patients were further subdivided into 2 groups. Functional pancreatic evaluation was carried out 12 months after the ABP episode. Endocrine function was evaluated by an oral glucose tolerance test and exocrine function by fecal fat excretion, fecal chymotrypsin (FQ), and secretin-cerulein tests (SCT). RESULTS: Most of the patients with necrosectomy had an abnormal exocrine pancreatic function, with steatorrhea in 25%. In the group without surgery, exocrine function was pathologic in only 13.3% and there were no cases of steatorrhea. Endocrine function was pathologic in 75% of patients undergoing necrosectomy versus 26.7% in the nonoperated group. In this latter group, the patients with abnormal endocrine function did not require insulin therapy, while in 33.3% of patient in the necrosectomy group insulin was necessary. CONCLUSIONS: In our homogeneous series of severe ABP, necrosectomy impaired significantly pancreatic endocrine and exocrine function. On the other hand, most patients with the same origin and severity index, but without surgical debridement, maintained normal pancreatic function.

Aged↗

PubMed2003

Use of photoplethysmography to determine gastrointestinal perfusion pressure: an experimental canine model.

OBJECTIVES: To develop an experimental model to assess the parietal perfusion pressure (PPP) of the digestive tract using photoplethysmography. MATERIALS AND METHODS: Twenty-two mongrel dogs were used. Progressive external compression was applied to the intestinal wall and the PPP was assessed with photoplethysmography. The study group was divided into two groups. In group 1 PPP was measured at the levels of the stomach, duodenum, jejunum and transverse colon. In group 2 PPP was measured after temporary occlusion of the truncal and marginal circulation of the jejunum to provide further variables. RESULTS: The PPP decreased significantly for each successive distal section. Correlation coefficients and indices for PPP and mean arterial pressures were statistically significant (p < 0.005). Truncal occlusion provoked a drop in PPP whereas marginal occlusion scarcely modified the basal results. CONCLUSIONS: Photoplethysmography, through measurements of the residual arterial wave amplitude, is a valid method of determining quantitatively the PPP of the digestive tract and could be useful in a clinical environment.

Animals↗

PubMed2000

Bile duct injury during laparoscopic cholecystectomy: myth or reality of the learning curve?

BACKGROUND: Bile duct injury (BDI) is a severe complication of laparoscopic cholecystectomy (LC) that is probably related to the effects of the learning curve. The aim of this prospective, institutional, and longitudinal study is to compare the incidence of BDI during LC in relation to the progressive experience of surgeons. METHODS: A total of 784 LC were examined during a 6-year period. They were divided into the following three consecutive groups: group A (1993-94), group B (1995-96), and group C (1997-98). Incidence and type of BDI, experience of the surgeon, intra- or postoperative diagnosis, treatment performed to repair the injury, and early and late morbidity and mortality were evaluated. RESULTS: The overall incidence of BDI was 1.4%. There were three cases of transection of the common bile duct, four partial lesions of the bile duct, and four cystic leakages. The number of BDI was maintained over the three different time periods; there were no statistical differences in the proportion of injuries among groups. Most BDI were incurred by experienced surgeons. In all, 36% of BDI were recognized intraoperatively. Hepaticojejunostomy, direct suture over a T-tube, and closure of the cystic stump were done to repair BDI. There was no additional morbidity or mortality in the patients with BDI. CONCLUSIONS: No relation was found between the experience of the surgeon and the number of BDI over the different periods of time. Therefore, BDI during LC cannot be attributed solely to the learning curve.

Adult↗

PubMed2000

Pentoxifylline ameliorates cerulein-induced pancreatitis in rats: role of glutathione and nitric oxide.

Reactive oxygen radicals, nitric oxide, and cytokines have been implicated in the initiation of pancreatic tissue damage and impairment of the pancreatic microcirculation in acute pancreatitis. Pentoxifylline is a methylxanthine derivative with rheologic and marked anti-inflammatory properties and inhibits the production of proinflammatory cytokines. We have examined whether pentoxifylline ameliorates interstitial edema, inflammatory infiltrate, and glutathione depletion associated with cerulein-induced pancreatitis. Cotreatment of animals with pentoxifylline significantly reduced cerulein-induced pancreatic inflammation and edema and attenuated the depletion of pancreatic glutathione and the increase in serum lipase activity, nitrate, and tumor necrosis factor-alpha levels. Pentoxifylline also prevented both mitochondrial swelling and damage to mitochondrial cristae caused by cerulein. Our findings provide an experimental basis for using pentoxifylline to attenuate inflammatory responses within the pancreas in acute pancreatitis and as an adjuvant in the treatment of acute pancreatitis.

Animals↗

PubMed1999

The N-terminus of collagenase MMP-8 determines superactivity and inhibition: a relation of structure and function analyzed by biomolecular interaction analysis.

Tissue inhibitors of metalloproteinases (TIMPs) are the physiological, specific inhibitors of matrix metalloproteinases (MMPs) forming tight, noncovalent complexes. Therefore they control the proteolytic activity of MMPs toward the extracellular matrix. To analyze the inhibition of the "activated" and "superactivated" variants of human neutrophil collagenase (MMP-8) by TIMP-2, we determined complex dissociation constants using biomolecular interaction analysis (BIA). As it is known that the association rate constants can exceed the limits of the BIA instruments, the biomolecular interaction analysis was used to examine the equlibrium situation. The dissociation constants were determined by fitting the parameters of the mathematical term for the binding of collagenase onto the TIMP-coupled sensor chip surface to the saturation curve derived from individual sensorgrams. The resulting values are in the nanomolar range and correlate with the results of fluorescence kinetics. These data reveal that TIMP-2 (the recombinant inhibitory domain of human TIMP-2 and bovine TIMP-2 isolated from seminal plama) is a better inhibitor of the activated neutrophil collagenase than of the superactivated variant (the recombinant catalytic domain of human MMP-8). It has been demonstrated by X-ray analysis that the N-terminal heptapeptide only of superactivated MMP-8 is attached by a salt bridge and hydrophobic interaction to the C-terminal helix. Because these interactions have to be disrupted in the complex formation with TIMP we assume that the activated variant enables higher flexibility and a tighter induced fit in the complex formation. Therefore superactivation of MMP-8 correlates with weaker inhibition by TIMP-2.

Animals↗

PubMed1999

Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods.

BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

Amino Acid Sequence↗

PubMed1997

Identification of bovine zona pellucida glycoproteins.

Despite the economical importance of in vitro gamete technologies in cattle, only little is known about the molecular mechanisms of binding of spermatozoa to the zona pellucida (ZP) of the oocyte. The aim of the present work was to identify proteins from the bovine zona pellucida (bZP) and to investigate which bZP proteins play a role in sperm-egg binding. High resolution 2-dimensional polyacrylamide gel electrophoresis of bZP proteins under reducing conditions showed that the bovine ZP could be separated into 4 glycoprotein spots, provisionally named bZP1, bZP2, bZP3, and bZP4, with different molecular masses and isoelectrical points. The N-terminal amino acid sequence of bZP1, bZP2, and bZP4 could be determined. The N-terminal amino acid sequences of bZP1 and bZP4 were identical and were homologous to that of pZP4. Comparison of our data to that of Noguchi et al., 1994 (Biochim Biophys Acta 1201:7-14) revealed that bZP2 and bZP4 are fragments of bZP1. Immunoblot analysis showed that, respectively, anti-porcine-ZP3alpha and -ZP3beta antibodies recognized 2 distinct regions of the bZP3 spot. Both antibodies inhibited sperm-egg binding in the bovine. We conclude that the bovine ZP consists of 3 proteins that correspond by size, N-terminal amino acid sequence, and antigenic determinants of pZP1, pZP3alpha, and pZP3beta, respectively, that are encoded by the porcine ZPA, ZPB, and ZPC genes (Harris et al., 1994: J Seq Map 4:6331-393), respectively.

Animals↗

PubMed1996

Characterization of representative enzymes from a sulfate reducing bacterium implicated in the corrosion of steel.

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed.

Corrosion↗

PubMed1995

Immunocytochemical characterization of porcine zona pellucida during follicular development.

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 alpha and ZP3 beta. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 alpha and anti-ZP3 beta. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 alpha and ZP3 beta. In secondary follicles, distinct labelling with anti-ZP3 beta and weak labelling with anti-ZP3 alpha could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 alpha and ZP3 beta. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 alpha and anti-ZP3 beta. Some granulosa cells showed staining for ZP3 alpha and ZP3 beta. The ZP displayed strong immunoreactivity for ZP3 beta and ZP3 alpha. Cells of the corona radiata were strongly immunopositive for ZP3 alpha and ZP3 beta. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 alpha and ZP3 beta strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.

Animals↗

PubMed1995

Evaluation of residual vascularisation in oesophageal substitution gastroplasty by surface oximetry-capnography and photoplethysmography. An experimental study.

OBJECTIVE: To evaluate residual vascularisation in gastroplasty and its possible relation to the development of anastomotic fistulas or dehiscences after oesophageal resection. DESIGN: Experimental open study. MATERIAL: Eleven mongrel dogs. INTERVENTIONS: Gastric parietal blood flow was evaluated by photoplethysmography and measurement of surface oxygen and carbon dioxide tensions under basal conditions and after Akiyama's tubular gastroplasty. Temperature, heart rate, and electrocardiogram; arterial pressure, pulmonary arterial pressure, pulmonary capillary pressure, central venous pressure, cardiac output, venous oxygen saturation, and arterial blood gas tensions were monitored under stable haemodynamic conditions. RESULTS: After gastroplasty, the mean serosal oxygen tension (pO2) of 8.6 mmHg and carbon dioxide tension (pCO2) of 99.5 mmHg and residual photoplethysmographic wave amplitude (8%) indicated considerable severe ischaemia at the fundus. There was relative ischaemia of the mid-stomach with residual mean values of 52.7 mmHg, 77.8 mmHg, and 57% for pO2; pCO2 and PPG wave amplitude, respectively. CONCLUSIONS: Important devascularisation of the fundus, theoretically incompatible with healing, occurs after gastroplasty. Operative photoplethysmography and surface measurements of pO2 and pCO2 are good ways of evaluating the level and degree of ischaemia in gastric tubes for oesophageal replacement.

Anastomosis, Surgical↗

PubMed1994

Primary sequence, oxidation-reduction potentials and tertiary-structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin.

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.

Amino Acid Sequence↗

PubMed1993

The use of photoplethysmography and Doppler ultrasound to predict anastomotic viability after segmental intestinal ischaemia in dogs.

OBJECTIVE: To compare photoplethysmography and Doppler ultrasonography in the prediction of the viability of experimental jejunoileal anastomoses in the presence of arterial (n = 11), venous (n = 11), and mixed arterial and venous (n = 9) segmental ischaemia. DESIGN: Experimental open study. MATERIAL: 31 Mongrel dogs. INTERVENTIONS: Laparotomy, selective devascularisation, and anastomosis. In 20 of the experiments a bolus injection of papaverine was given into the superior mesenteric artery. Second laparotomy on day 15, or earlier if indicated. OUTCOME MEASURES: Correlation between photoplethysmography, Doppler ultrasonography, and macroscopic and histological findings. RESULTS: All non-viable anastomoses had photoplethysmographic wave amplitudes of less than 10% of the control value, with sensitivity and specificity of 100%. Venous volume and maximum venous outflow had a sensitivity of 72% and a specificity of 77%. Doppler ultrasonography had a sensitivity of 100%, but a specificity of only 67%. Papaverine had no effect on photoplethysmographic wave amplitude in these experiments. CONCLUSION: Photoplethysmography is the preferred method of assessing intestinal viability in the presence of ischaemia.

Anastomosis, Surgical↗

PubMed1992

Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L.

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase-type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single-chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro-uPA. As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys158-Ile159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro-uPA of 1:2,000). Nevertheless, even at pH 7.0, pro-uPA was activated by cathepsin L, although a 10-fold higher concentration of cathepsin L was required. As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro-uPA; (ii) activation by serine proteases (plasmin, kallikrein, Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).

Amino Acid Sequence↗

PubMed1992

[An inflammatory fibroid polyp (eosinophilic granuloma) and Crohn's disease located in the ileum].

Lower gastrointestinal tract bleeding as a manner of presentation of the Crohn's disease is uncommon. In most cases there is an other concomitant pathology responsible for that bleeding. We report a case of lower gastrointestinal tract bleeding to an inflammatory fibroid bowel polyp in a patient with ileal Crohn's disease. A commentary is made about the etiopathogenic factors of the both diseases.

Adolescent↗

PubMed1991

Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1.

Cyclic Arg-Gly-Asp-Phe-Val peptides with either D-Phe or D-Val residues were 20- to more than 100-fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly-Arg-Gly-Asp-Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding of platelet receptor alpha IIb beta 3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all-trans conformation with a beta II' and gamma turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity of inhibition.

Amino Acid Sequence↗

PubMed1991

Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA).

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.

Blotting, Western↗

PubMed1991

Biological and clinical relevance of the urokinase-type plasminogen activator (uPA) in breast cancer.

Tumor cell invasion and metastasis is a multifactorial process, which at each step may require the action of proteolytic enzymes such as collagenases, cathepsins, plasmin, or plasminogen activators. An enzymatically inactive proenzyme form of the urokinase-type plasminogen activator (pro-uPA) is secreted by tumor cells which may be converted to an enzymatically active two-chain uPA-molecule (HMW-uPA) by plasmin-like enzymes. Action of proteases on pro-uPA may generate the enzymatically active or inactive high-molecular-weight form of uPA (HMW-uPA). Some proteases (plasmin, cathepsin B and L, kallikrein, trypsin or thermolysin) activate pro-uPA by cleaving the peptide bond Lys158 and IIe159. Other proteases (elastase, thrombin) cleave pro-uPA at different positions to yield enzymatically inactive HMW-uPA. HMW-uPA may be split into the enzymatically active LMW-uPA and the enzymatically inactive ATF (amino terminal fragment). ATF may be cleaved between peptide sequence 20 and 40 within the receptor binding domain of uPA (GFD). Such impaired ATF does not bind to uPA-receptors. Action of the bacterial endoproteinase Asp-N from Pseudomonas fragi mutant on pro-uPA or HMW-uPA, however, generates intact ATF which efficiently competes for binding of HMW-uPA or pro-uPA to receptors on tumor cells. High uPA-antigen content (pro-uPA, HMW-uPA, or LMW-uPA) in breast cancer tissue (not in plasma) indicates an elevated risk for the patient of recurrences and shorter overall survival. Thus pro-uPA/uPA-antigen content in breast cancer tissue serves as an independent prognostic parameter for the outcome of the disease. Cathepsin D is also an independent prognostic factor for recurrences and overall survival. High content of cathepsin D in breast cancer tumors is, however, not correlated with elevated levels of pro-uPA/uPA indicating that synthesis and release of cathepsin D and pro-uPA/uPA are independent events.

Binding Sites↗