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Biomedical subjects

A Padilla

Publications and source records attributed to A Padilla.

At least 37 records · Page 2Linked to original sources
PubMed1997

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

Crystallization↗
PubMed1997

Binge drinking among college students: a comparison of California with other states.

California college students (1,864 students from 15 colleges) were compared with students who participated in the Harvard School of Public Health College Alcohol Study, which surveyed 17,592 students in 140 colleges nationwide. California college students, in comparison with the remainder of the nation, were less frequent drinkers; less frequent binge drinkers; exhibited fewer personal problems and risks associated with heavy episodic drinking, including drinking and driving; and reported fewer "secondhand" effects of binge drinking, such as being physically assaulted or experiencing an unwanted sexual advance. Many of these differences appear to be related to the California college students' being older, more likely to be married, and less likely to live on campus than those in the Harvard study. The findings suggest that, in developing programs tailored to local needs, there is significant value in augmenting national surveillance of college student health risk behaviors with the development of regional, state, and local surveillance systems.

Adolescent↗
PubMed1997

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D H NMR and molecular dynamics.

Peptides derived from the inhibitor of cAMP-dependent protein kinase. PKI, have been studied by 2D 1H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala20-Ser21]PKI(5-24), and a phosphorylated form of the latter [Ala20-Ser21P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly14-Gly17 turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp9 and Arg15. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2 degrees C) shows the occurrence of pH-dependent contacts with Phe10. We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala20-Ser21]PKI(5-24) and the product [Ala20-Ser21P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains.

Amino Acid Sequence↗
PubMed1996

Assignment of 13C resonances and analysis of relaxation properties and internal dynamics of pike parvalbumin by 13C-NMR at natural abundance.

Pike parvalbumin is an 11.5-kDa globular protein which binds Ca2+ through EF-hand structural motifs. Nearly complete assignment of the protonated 13C resonances has been achieved by means of heteronuclear two-dimensional experiments. The study shows that 13Ca chemical shifts can be very sensitive to localised conformational aspects. To characterise internal dynamics of pike parvalbumin, longitudinal-relaxation and transverse-relaxation rates and 1H-13C NOEs were measured for alpha-carbons at natural abundance by means of two-dimensional NMR spectroscopy. Relaxation data were obtained at a spectrometer frequency of 600 MHz for 69 residues with an even spread along the parvalbumin polypeptide chain. A double approach that included Lipari-Szabo analysis and direct mapping of spectral densities was used to interpret relaxation data in terms of internal dynamics. The former analysis provides valuable information about the overall rotational correlation time and S2 order parameters, while the mapping approach characterises the relative contributions of different motional frequencies. The results suggest that Ca(2+)-loaded pike parvalbumin has a rigid structure, even in the functional regions, i.e., the Ca(2+)-binding loops. The patterns of density-function values are more sensitive to the secondary structure than those of S2. Moreover, depending on the sampling frequency, these patterns reveal different aspects of structure-specific motions.

Amino Acid Sequence↗
PubMed1996

The biological properties, assay, and standardization of interferon-alpha: a need for a WHO collaborative study.

Interferon-alpha (IFN-alpha) exists as a range of closely related, biologically active proteins and has been the subject of extensive research and clinical investigation. Standardization of IFN-alpha and the uniform reporting of IFN-alpha activity in International Units (IU) is critical to preclinical research and the clinical development of IFN-alpha products as therapeutic agents. Currently, several different IFN-alpha-containing reference preparations, established as World Health Organization (WHO) International Standards (IS) for particular IFN-alpha proteins (mixtures or single molecular species) are available for assay calibration. Nevertheless, the heterogeneous nature of IFN-alpha has raised standardization issues relating to the activity of individual IFN-alpha proteins, hence-forth termed subtypes, in the various biologic assays used for determining IFN-alpha levels. These issues include the question of parallelism of dose-response curves among particular IFN-alpha subtypes and different, naturally produced IFN-alpha subtype mixtures, for example, leukocyte IFN-alpha, and the applicability of IU of IFN-alpha activity defined by antiviral assays to alternative biologic assays, for example, antiproliferative assays. To address such issues, a WHO Consultative Group on Cytokine Standardization requested that the National Institute for Biological Standards and Control (NIBSC) and the Centre for Biologics Evaluation and Research (CBER) organize an international collaborative study to compare the activities and relative potencies of the several available IFN-alpha preparations, including those derived from human cells containing mixtures of IFN-alpha subtypes and those derived by rDNA methods containing single IFN-alpha subtypes, in different assays. To date, 111 participants in 32 countries have been recruited to the study and have agreed to assay a total of 17 different natural and recombinant IFN-alpha preparations or a defined subset thereof in specific in-house assays. The assay results generated will be statistically analyzed and evaluated to address the stated issues and to assess whether any individual IFN-alpha preparation is suitable to serve as an IS for all IFN-alpha preparations or whether more than one IS will be needed for this purpose.

Animals↗
PubMed1996

Inhibition of thrombin generation by heparin and LMW heparins: a comparison of chromogenic and clotting methods.

Inhibition of thrombin generation by heparin and low-molecular-weight (LMW) heparins is an important parameter which relates to their anticoagulant actions in vivo. Previous studies in our laboratory used a clotting assay for assessment of thrombin generation but other published studies have used a chromogenic method. We have therefore measured the inhibition of thrombin generation by unfractionated heparin (UFH) and LMW heparins by a modified chromogenic method using microtitre plates and compared the results with the clotting method. The degree of inhibition of thrombin generation when calculated from both peak thrombin concentrations and areas under the curve in the chromogenic assay was the same. When the activities of each heparin were expressed as EC80, i.e. concentrations required for 80% inhibition of thrombin generation, the EC80 were higher in the chromogenic assay than in the clotting system. However when the potencies of the LMW heparins were expressed as percentages of that of the UFH standard by parallel line analysis, the differences between clotting and chromogenic assay results were small and not statistically significant (P > 0.05). This study demonstrates the feasibility of measuring inhibition of thrombin generation by a modified chromogenic method using microtitre plates, and shows that the results with this method are similar to those obtained with the clotting method.

Anticoagulants↗
PubMed1996

Serologic reactivity of a synthetic peptide from human immunodeficiency virus type 1 gp41 with sera from a Mexican population.

The reactivities of 1,172 serum samples obtained from asymptomatic human immunodeficiency virus type 1 (HIV-1)-positive and HIV-1-negative individuals residing in Mexico to a synthetic disulfide-looped peptide from the HIV-1 gp41 (amino acids 602 to 616 [IWGCSGKLICTTAVP] were examined by an enzyme-linked immunoadsorbent assay (ELISA) procedure. Antibodies to the synthetic peptide were detected in 261 of 268 serum samples from HIV-positive individuals (sensitivity, 97.4%). The peptide also reacted with 12 of 904 serum samples from control HIV-negative individuals (specificity, 98.7%). Western blots (immunoblots) of four of the seven serum samples that produced false-negative results in the ELISA showed that three of them reacted weakly with gp41 and strongly with gp120, p55, and/or p24. Potential diagnostic difficulties raised by the reported C1q binding capacity of this peptide were also evaluated: few and weak false-positive results were found among sera from patients with rheumatoid arthritis (1 of 31) and neurocysticercosis (2 of 111). In fact, strong reactivity with the peptide spotted an undetected HIV infection underlying clinical neurocysticercosis.

Amino Acid Sequence↗
PubMed1996

WHO activities towards the three Rs in the development and control of biological products.

WHO supports the concept of replacement, reduction and refinement of the use of in vivo methods for biologicals production and control, and regularly conducts reviews of its recommended procedures to allow reduction in the use of animals. The coordination of collaborative studies, publication of standardized methods, and holding of workshops on the use of these methods contributes to their use. The neurovirulence test for oral poliovaccine is probably the single most visible animal test for which alternative methods are sought. Collaborative studies on alternative methods for screening products are currently being sponsored by WHO. The use of Vero cells rather than primary monkey kidney cells for poliovaccine production can avoid the use of many monkeys. Cell banks of Vero and HEp-2 cells have been developed by WHO, tested for virus sensitivity and freedom from adventitious agents, and are available for vaccine production and control, replacing primary animal cells. For the future, final product testing will increasingly be directed towards establishment of consistency of production rather than potency. By supporting the validation and use of this approach, WHO can effectively influence more rational animal use in biological production and control.

Animal Testing Alternatives↗
PubMed1995

Immunization of pigs against Taenia solium cysticercosis: factors related to effective protection.

Fifty-six (56) pigs were immunized against Taenia solium cysticercosis with antigens from Taenia crassiceps metacestodes, in a variety of protocols, and then challenged orally with Taenia solium proglottids or eggs. Results of immunization (expressed as individual parasite loads) ranged from significant reduction of parasite loads (host protection) to clear increase (parasite facilitation) in apparent relation to the immunogen dose, adjuvant employed and genetic background of the pigs. In all trials, however, immunized pigs harboured more damaged cysticerci than controls, indicating that immunization does induce some restrictions to parasite these are eventually overwhelmed by other parasite-promoting factors. Western blots in immunized-protected pigs indicated antigens of 242, 234, 118, 77, 55 and 45 kDa as possibly being involved in immunological protection.

Animals↗
PubMed1993

Symmetrical rearrangement of the cation-binding sites of parvalbumin upon Ca2+/Mg2+ exchange. A study by 1H 2D NMR.

Two forms of parvalbumin, i.e., the fully Ca-loaded form PaCa2 and the fully Mg-loaded form PaMg2, are investigated by 2D 1H NMR in solution. A detailed analysis of the resonances, which belong to residues involved in direct coordination of Ca2+ and Mg2+, establishes that the sixth ligand, a highly conserved Glu residue at the relative position 12 in both cation-binding sites CD and EF, undergoes a conformational rearrangement through a 120 degrees rotation of its side chain about the C alpha-C beta bond with PaMg2 adopting the less energetically favored g- conformation, as inferred from scalar coupling constants and dipole-dipole contacts measured on the COSY and NOESY spectra, respectively. Similarly, chemical shift effects, which selectively involve NH and C alpha H resonances (as well as side-chain resonances) in both CD and EF sites, point to a symmetrical behavior of both cation-binding sites upon Ca2+/Mg2+ exchange.

Animals↗
PubMed1993

Solution conformation of a synthetic bis-headed inhibitor of trypsin and carboxypeptidase A: new structural alignment between the squash inhibitors and the potato carboxypeptidase inhibitor.

The trypsin carboxypeptidase peptide inhibitor (TCPI) which inhibits both trypsin and carboxypeptidase A has been chemically engineered by modification of the Ecballium elaterium trypsin inhibitor II (EETI-II). The solution conformation of TCPI, studied by two-dimensional nuclear magnetic resonance, was shown to be very close to those of squash inhibitors. Only limited deviations of the trypsin binding loop compared to its location in the EETI-II/trypsin complex were detected. It was also shown that the position of the C-terminal tail did not significantly change from the position observed in the complex between carboxypeptidase A and the potato carboxypeptidase inhibitor (PCI). The conformation of TCPI was carefully compared with the PCI one and a new structural alignment between the two microproteins is proposed. This alignment points out the very good conservation in the two inhibitors of a subdomain comprising segments 7-15, 19-22 and 25-28. Most importantly, the 2-19 disulfide bridge of TCPI was not structurally conserved in PCI and appeared to be rather unimportant for the early folding process of these molecules. This result agrees with the recent observation that the 2-19 bridge is the last to be formed in the folding of the squash inhibitor EETI-II and suggests that this is also the case during the folding of the potato carboxypeptidase inhibitor.

Amino Acid Sequence↗
PubMed1993

Antithrombotic activity and its relationship to in vitro anticoagulant effects.

Antithrombotic activities of low molecular weight heparins (LMWHs) in a venous stasis thrombosis model, when compared to unfractionated heparin (UFH), correlated better with anti-IIa activities and inhibition of thrombin generation than with anti-Xa activities. The relative antithrombotic potencies of LMWHs were closer to inhibitory activity in thrombin generation with platelet-poor plasma than platelet-rich plasma. Comparing activities in intrinsic and extrinsic systems, higher concentrations of heparins were required in vitro to inhibit intrinsic thrombin generation than extrinsic thrombin production; however, higher doses were required to prevent in vivo thrombotic events initiated via the extrinsic pathway.

Animals↗
PubMed1992

Immunodiagnosis of neurocysticercosis. Disappointing performance of serology (enzyme-linked immunosorbent assay) in an unbiased sample of neurological patients.

To ascertain the reliability of serological diagnosis of neurocysticercosis in the everyday a priori situation of neurological consultation, the enzyme-linked immunosorbent assay test was used to predict the eventual diagnosis of neurocysticercosis in an unselected sample of 1064 consecutive neurological cases. Results showed 69% sensitivity and 71% specificity of the enzyme-linked immunosorbent assay test for the diagnosis of neurocysticercosis. In sharp contrast with publications that have proclaimed the excellent diagnostic performance of immunodiagnostic tests, our results suggest that identification of serum antibodies with standard enzyme-linked immunosorbent assay techniques is of little value when applied to a large and heterogeneous group of neurological patients in an endemic area of cysticercosis, and our results urge a reevaluation of currently used immunodiagnostic tests that are practiced in the serum of suspected cases.

Central Nervous System Diseases↗
PubMed1992

Anti-dsDNA and Sm autoantibodies in systemic lupus erythematosus.

During the present study the coincidence of anti-dsDNA and Sm antibodies was detected in 16 percent of 51 consecutive SLE patients. These antibodies were detected by the standard indirect immunofluorescence and Ouchterlony tests. All patients with anti-dsDNA and Sm antibodies showed disease activity, including renal, CNS and pulmonary disease. We excluded a cross reactivity of these antibodies by ELISA, using competitive experiments with dsDNA and Sm antigens. The results support the presence of multiple autoantibody production during SLE activity, and suggest that different mechanisms may underlie the induction and regulation of both autoantibodies.

Adolescent↗
PubMed1992

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4).

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.

Factor Xa Inhibitors↗