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Biomedical subjects

F J Medrano

Publications and source records attributed to F J Medrano.

At least 37 records · Page 2Linked to original sources
PubMed1999

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold.

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.

Adhesins, Bacterial↗
PubMed1999

Treatment of visceral leishmaniasis in HIV-infected patients: a randomized trial comparing meglumine antimoniate with amphotericin B. Spanish HIV-Leishmania Study Group.

BACKGROUND: Visceral leishmaniasis is common in patients with HIV infection living in endemic areas, but the most effective and safe treatment remains unknown. OBJECTIVE: To compare the efficacy and safety of meglumine antimoniate versus amphotericin B in HIV-infected patients with first episodes of visceral leishmaniasis (VL). DESIGN: An open, multicentre, prospective and randomized trial. SETTING: Twelve tertiary hospitals. PATIENTS: Eighty-nine consecutive HIV-infected patients diagnosed with VL. Patients were randomly assigned to treatment with either meglumine antimoniate (20 mg pentavalent antimony per kilogram of body weight per day) or amphotericin B (0.7 mg/kg per day) both for 28 days. Treatment was considered successful if a bone marrow aspirate performed 1 month after the end of therapy did not detect parasites. Relapse was defined as the reappearance of parasites after an initial cure. RESULTS: An initial cure was attained in 29 of 44 patients (65.9%) randomly assigned to treatment with meglumine antimoniate and 28 of 45 (62.2%) randomly assigned to treatment with amphotericin B. The incidence of moderate to severe adverse events was similar in both groups. The patients treated with meglumine antimoniate had higher incidences of cardiotoxicity (14 versus 0%, P = 0.02) and chemical pancreatitis (30 versus 0%, P < 0.01). However, in the amphotericin B group, nephrotoxicity was more frequent (36 versus 5%, P < 0.01). There was no difference in survival or relapse-free interval according to the allocated group of therapy. CONCLUSION: Treatment of VL with meglumine antimoniate or amphotericin B was shown to have similar efficacy and toxicity rates in Spanish HIV-infected patients. The differences in the toxicity patterns could be useful in choosing one of these agents as first-line treatment.

AIDS-Related Opportunistic Infections↗
PubMed1998

Tumor necrosis factor beta and soluble APO-1/Fas independently predict progression to AIDS in HIV-seropositive patients.

The relationship between serum concentration of different components of the nerve growth factor/tumor necrosis factor (TNF) receptor family, including soluble APO-1/Fas (sAPO-1/Fas) and progression of HIV infection, was analyzed in a case-control study of individuals selected from a cohort of HIV-seropositive patients who were progressing or not progressing to AIDS while being treated with nucleoside analogs. HIV-seronegative healthy controls were also analyzed. The results showed that, despite close matching for immunologic (CD4 cell count, beta2-microglobulin concentration) and virologic (p24 antigen, detection of HIV syncytium-inducing phenotype, plasma HIV viremia) parameters, the baseline serum concentrations of TNF-beta and sAPO-1/Fas were statistically different between progressing and nonprogressing patients. In addition, serum concentrations of TNF-beta and sAPO-1/Fas showed the strongest independent predictive power for progression to AIDS in a multivariate conditional logistic regression model. Because TNF-beta and Fas were suggested to be mediators of antigen-induced cell death (AICD) in HIV infection and sAPO-1/Fas was hypothesized to protect lymphocyte against AICD, these data suggest an important pathogenetic role for AICD in the progression of HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V.

Residue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic acid [thiaproline, Pro(S)], a non-natural amino acid analog of proline, into human recombinant annexin V was achieved with a proline-auxotrophic Escherichia coli strain by fermentation procedures in minimal medium. Quantitative replacement of proline with thiaproline was confirmed by mass-spectrometric, amino acid, and x-ray crystallographic analyses. The wild-type protein and its per-Pro(S) mutant were found to crystallize isomorphously and to show identical three-dimensional structures in crystals. In solution the dichroic properties of the wild-type and per-Pro(S) protein confirmed nearly identical overall folds. From thermal denaturation experiments, however, a reduced Tm (-4.5 K) value was determined whereas the van't Hoff enthalpy and entropy were not significantly affected. Therefore, protein mutants containing bioactive amino acid analogs like thiaproline at multiple sites would be expected to fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.

Annexin A5↗
PubMed1998

Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.

Amino Acid Sequence↗
PubMed1998

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone.

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.

Adhesins, Bacterial↗
PubMed1998

The susceptibility of pure tubulin to high magnetic fields: a magnetic birefringence and x-ray fiber diffraction study.

The orientational behavior of microtubules assembled in strong magnetic fields has been studied. It is shown that when microtubules are assembled in a magnetic field, they align with their long axis parallel to the magnetic field. The effect of several parameters known to affect the microtubule assembly are investigated with respect to their effect on the final degree of alignment. Aligned samples of hydrated microtubules suitable for low-resolution x-ray fiber diffraction experiments have been produced, and the results obtained from the fiber diffraction experiments have been compared with the magnetic birefringence experiments. Comparisons with earlier fiber diffraction work and small-angle x-ray solution scattering experiments have been made.

Animals↗
PubMed1998

Dynamics of serum cytokines in patients with visceral leishmaniasis and HIV-1 co-infection.

Serum cytokine levels and peripheral T cell subpopulations of HIV-1-infected patients before, during and after active visceral leishmaniasis (VL) were analysed and compared with appropriate controls. At VL diagnosis, co-infected patients showed higher serum levels of interferon-gamma (IFN-gamma) than matched HIV-1 controls without VL, and lower serum concentrations of IL-10 than non-immunocompromised VL controls. High levels of tumour necrosis factor-alpha (TNF-alpha) and IFN-gamma were present in the sera of HIV-1-infected patients with active VL. TNF-alpha remained elevated after VL recovery. A steady decline in the CD4+ cell count, an increase of serum HIV viraemia and a progressive seroconversion for the HIV-1 p24 antigen was observed during the course of VL disease. Thus, an aberrant activation of the TNF system with possible negative immunological and virological consequences is present in HIV-1-infected patients with VL. A more extensive prospective validation of these findings in a bigger cohort of patients will nevertheless be necessary. The results support the hypothesis that different opportunistic infection agents may trigger the production of proinflammatory cytokines during immunodeficiency, and in this way accelerate the course of HIV-1 disease.

AIDS-Related Opportunistic Infections↗
PubMed1998

Evidence for a role of T-helper type 2 cytokines in the acquisition of human immunodeficiency virus syncytium-inducing phenotype.

BACKGROUND: The objective of the present study was to investigate the relationship between the serum cytokine pattern of T-helper (Th) response and the acquisition of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants in HIV-1-seropositive patients treated with antiretroviral drugs. METHODS: Serum cytokines of Th1/Th2 responses were analysed in a case-control study of 20 individuals selected from a cohort of HIV-1-infected patients without SI variants at entry, who developed or did not develop SI virus during a prospective follow-up. A group of 10 patients with SI variants at study entry was also evaluated. Serum concentration of interferon (IFN) gamma, interleukin (IL) 2, IL-4 and IL-10 was evaluated by mean of a commercial enzyme immunoassay. RESULTS: Despite close matching for immunological (CD4+ cell count) and virological (p24 antigen, serum HIV viraemia) parameters, SI-converting patients showed at baseline significantly lower serum levels of IL-2 and higher concentrations of IL-4 than those who remained persistently negative for SI variants. Shortly after the phenotype conversion, SI-converting patients were characterized by significantly high serum concentration of IL-4 and by low levels of IFN-gamma (Th2-like pattern). Patients with SI phenotype at study entry featured lower mean levels of both IL-4 and IL-10, but mean IFN-gamma and IL-2 values were higher, although the clinical and immunological baseline was also poorer and no statistical analysis could be applied. CONCLUSION: Serum cytokine pattern of Th1/Th2 response differs between patients with NSI and SI phenotype. Our data strongly suggest that the Th2 cytokine pattern could be associated with the acquisition of the SI HIV-1 phenotype.

Case-Control Studies↗
PubMed1998

The role of serology in the diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type-1.

To define the possible role of serology in the diagnosis and prognosis of visceral leishmaniasis (VL) in patients with human immunodeficiency virus type-1 (HIV-1) infection, the dynamics of humoral immune responses was investigated in 20 coinfected patients. Sequential sera obtained before, during, and after VL diagnosis were analyzed by an indirect immunofluorescent antibody test (IFAT), a recombinant ELISA (using the rK39 protein), and immunoblotting. During the active course of the disease, positive results were found by IFAT or ELISA in 22% of the cases and by immunoblotting in 78% of the cases. A great variability in the response was observed during the follow-up with a trend to more positive results near the time of VL diagnosis. Forty-six percent of the patients were positive by IFAT or ELISA on at least one time point before VL and 37.5% were positive during the period following treatment. These results confirm the limited usefulness of the IFAT and ELISA in the diagnosis of VL in coinfected patients and demonstrate their low ability to predict the development or the outcome of disease. In these patients, immunoblotting could be a useful tool for studying the natural course of leishmaniasis, although it has limited value for diagnosis or treatment control.

Acute Disease↗
PubMed1997

Crystallization and preliminary X-ray diffraction analysis of proline iminopeptidase from Xanthomonas campestris pv. citri.

Proline iminopeptidase from Xanthomonas campestris pv. citri, displaying no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapour diffusion method. Two different orthorhombic crystal forms (space group C222 and I222) were obtained from a solution containing NaCl or polyethylene glycol monomethyl ether (MW 5000) as precipitating agent for the native and lanthanum-derivatized protein, respectively. Complete diffraction data sets have been collected up to 2.6 A (native) and 3.0 A (lanthanum derivative) resolution. Cell dimensions are a= 147.2 A, b = 167.8 A, and c = 85.6 A (C222) and a = 146.7 A, b = 167.7 A, and c = 171.4 A (I222), respectively. Considerations of the possible values of V(m) and analysis of the self-rotation function of the native crystals account for the presence of one dimer per asymmetric unit, whereas a tetramer probably would occupy the smallest crystallographically independent crystal portion in the lanthanum-derivatized protein crystals.

Aminopeptidases↗
PubMed1997

Significance of indeterminate reactivity to human T-cell lymphotropic virus in western blot analysis of individuals at risk.

Current tests to confirm human T-cell lymphotropic virus (HTLV) infection in individuals at risk of retroviral infection commonly yield indeterminate results. To assess the significance of HTLV-seroindeterminate reactivities in a high-risk population, 16 at-risk individuals who had this serologic pattern by Western blot were studied using a polymerase chain reaction (PCR) assay. Human T-cell lymphotropic virus type II infection was confirmed by the presence of virus-specific nucleic acid in four patients. However, PCR analysis was negative in the remaining 12 individuals. These results indicate strongly that all specimens from at-risk individuals with nondiagnostic HTLV reactivity by current Western blot assay should continue to be considered inconclusive, requiring further testing by more sensitive tests.

Antibodies, Viral↗
PubMed1997

Presence of glomerular basement membrane (GBM) antibodies in HIV- patients with Pneumocystis carinii pneumonia.

Following the unexpected finding of antibodies to GBM in a patient with Pneumocystis carinii pneumonia in the absence of kidney abnormalities, the presence of anti-GBM antibodies was analysed in 14 patients with pulmonary P. carinii infection who did not have clinical evidence of autoimmune glomerulonephritis. Patients were divided into three groups: HIV- with P. carinii pneumonia (n = 4), HIV+ with P. carinii pneumonia (n = 5) and HIV- carriers of P. carinii without pneumonia (n = 5). As control groups, HIV- patients with community-acquired non-P. carinii pneumonia (n = 6) and healthy individuals (n = 16) were included. Anti-GBM antibodies, studied with a quantitative enzyme immunoassay (EIA) for anti-alpha3 chain of collagen IV antibodies, were detected in three out of the four HIV-patients with P. carinii pneumonia, but not in any individuals of the other categories. These results suggest that P. carinii alveolar injury or the host response to the organism could affect the basal membrane Goodpasture antigen or a similar antigen, and induces anti-GBM antibody production in HIV- patients, and support the hypothesis that, at least in some cases, Goodpasture's syndrome could be triggered by an alveolar lesion induced by a P. carinii pneumonia.

Adult↗
PubMed1996

Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase. Conformational transitions induced by temperature.

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

Choline↗
PubMed1996

Structural organization of the major autolysin from Streptococcus pneumoniae.

LytA amidase is the best known bacterial autolysin. It breaks down the N-acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH2-terminal module, responsible for the catalytic activity, and the COOH-terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C-LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH2-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer <--> dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function.

Calorimetry, Differential Scanning↗