RESUMEN
High-loaded membrane bioreactors (HL-MBRs), i.e., bioreactors equipped with a membrane for biomass retention and operated at extremely short sludge and hydraulic retention times, can concentrate sewage organic matter to facilitate subsequent energy and chemical recovery from these organics. Bioflocculation, accomplished by microorganisms that produce extracellular polymers, is a very important mechanism in these reactors. Bacterial diversity of the sludge and supernatant fraction of HL-MBRs operated at very short sludge retention times (0.125, 0.5, and 1 day) were determined using a PCR-denaturing gradient gel electrophoresis (DGGE) and clone library approach and compared to the diversity in sewage. Already at a sludge retention time (SRT) of 0.125 day, a distinct bacterial community developed compared to the community in sewage. Bioflocculation, however, was low and the majority of the bacteria, especially Arcobacter, were present in the supernatant fraction. Upon increasing SRT from 0.125 to 1 day, a much stronger bioflocculation was accompanied by an increased abundance of Bacteroidetes in the (solid) sludge fraction: 27.5 % at an SRT of 0.5 day and 46.4 % at an SRT of 1 day. Furthermore, cluster analysis of DGGE profiles revealed that the bacterial community structure in the sludge was different from that in the supernatant. To localize specific bacterial classes in the sludge flocs, fluorescence in situ hybridization (FISH) was carried out with three different bacterial probes. This showed that Betaproteobacteria formed clusters in the sludge flocs whereas Alphaproteobacteria and Gammaproteobacteria were mainly present as single cells.
Asunto(s)
Bacterias/aislamiento & purificación , Reactores Biológicos/microbiología , Aguas Residuales/química , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Biodiversidad , Floculación , Aguas del Alcantarillado/química , Aguas del Alcantarillado/microbiología , Aguas Residuales/microbiologíaRESUMEN
High-loaded membrane bioreactors (HL-MBRs), i.e. MBRs which are operated at extremely short sludge and hydraulic retention times, can be applied to flocculate and concentrate sewage organic matter. The concentrated organics can be used for energy recovery, or for the production of more valuable organic chemicals. Little is known about the effect of the dissolved oxygen concentration (DO) on this bioflocculation process. To examine this effect, two HL-MBRs were operated, respectively at a low (1 mg L(-1)) and a higher (4 mg L(-1)) DO. The higher DO resulted in a better flocculation efficiency, i.e. 92% of the colloidal COD in the sewage flocculated compared to 69% at the lower DO. The difference was attributed to a higher microbial production of extracellular polymeric substances at a DO of 4 mg L(-1) and to more multivalent cations (calcium, iron and aluminium) being distributed to the floc matrix. In addition, the HL-MBR that was operated at a DO of 4 mg L(-1) gave a bigger mean floc size, a lower supernatant turbidity, better settleability and better membrane filterability than the HL-MBR that was operated at a DO of 1 mg L(-1).
Asunto(s)
Reactores Biológicos/microbiología , Membranas Artificiales , Oxígeno/metabolismoRESUMEN
High loaded MBRs (HL-MBR) can concentrate sewage organic matter by aerobic bioflocculation for subsequent anaerobic conversion to methane or volatile fatty acids. In the range of very short solid retention times (SRT), the effect of SRT on bioflocculation and EPS production in HL-MBR was investigated. This short SRT range was selected to find an optimum SRT maximising recovery of organics by aerobic bioflocculation and minimizing losses of organics by aerobic mineralization. Bioflocculation was studied in five HL-MBRs operated at SRTs of 0.125, 0.25, 0.5, 1 and 5 d. The extent of flocculation, defined as the fraction of suspended COD in the concentrate, increased from 59% at an SRT of 0.125 d to 98% at an SRT of 5 d. The loss of sewage organic matter by biological oxidation was 1, 2, 4, 11 and 32% at SRT of 0.125-5 d. An SRT of 0.5-1 d gave best combination of bioflocculation and organic matter recovery. Bound extracellular polymeric substances (EPS) concentrations, in particular EPS-protein concentrations, increased when the SRT was prolonged from 0.125 to 1 d. This suggests that these EPS-proteins govern the bioflocculation process. A redistribution took place from free (supernatant) EPS to bound (floc associated) EPS when the SRT was prolonged from 0.125 to 1 d, further supporting the fact that the EPS play a dominant role in the flocculation process. Membrane fouling was most severe at the shortest SRTs of 0.125 d. No positive correlation was detected between the concentration of free EPS and membrane fouling, but the concentration of submicron (45-450 nm) particles proved to be a good indicator for this fouling.
Asunto(s)
Biopolímeros , Reactores Biológicos , Membranas Artificiales , Aguas del Alcantarillado/química , Bacterias/metabolismo , Floculación , Eliminación de Residuos Líquidos/métodosRESUMEN
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47(PHOX) as compared with the same cells expressing wild type p47(PHOX). In addition, these mutant p47(PHOX) proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.
Asunto(s)
Citosol/enzimología , Leucocitos/enzimología , NADPH Oxidasas/metabolismo , Fosfoproteínas/metabolismo , Ácido Araquidónico/farmacología , Transporte Biológico , Activación Enzimática , Células Madre Hematopoyéticas/enzimología , Linfocitos/enzimología , Mutación , NADPH Oxidasas/efectos de los fármacos , NADPH Oxidasas/genética , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/genética , Fosforilación , Proteínas Recombinantes/metabolismo , Serina/metabolismoRESUMEN
Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.
Asunto(s)
ADN Helicasas/genética , ADN Recombinante , Proteínas de Unión al ADN/genética , Dependovirus/genética , Vectores Genéticos/genética , Transactivadores/genética , Proteínas Virales/genética , Animales , Línea Celular/virología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.
Asunto(s)
Leucocitos/metabolismo , NADPH Oxidasas/sangre , Fosfoproteínas/sangre , Serina , Acetato de Tetradecanoilforbol/farmacología , Linfocitos B/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Activación Enzimática , Humanos , Mediciones Luminiscentes , Mutagénesis Sitio-Dirigida , Fosfoproteínas/química , Fosforilación , Fosfoserina/metabolismo , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Superóxidos/metabolismo , TransfecciónRESUMEN
A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9-fold), in vitro motility (2.7-fold), and the Vmax of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Movimiento/fisiología , Miosinas/metabolismo , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Activación Enzimática , Datos de Secuencia Molecular , Músculo Esquelético , Músculo Liso , Miocardio , Miosinas/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la EspecieRESUMEN
Regulation of a variety of cellular contractile events requires that vertebrate smooth and non-muscle myosin II can achieve an "off" state. To examine the role of the myosin rod in this process, we determined the minimal size at which a myosin molecule is capable of regulation via light chain phosphorylation. Expressed smooth muscle myosin subfragments with as many as 100 amino acids of the coiled-coil rod sequence did not dimerize and were active independently of phosphorylation. To test whether dimerization per se restores regulation of ATPase activity, mutants were expressed with varying lengths of rod sequence, followed by C-terminal leucine zippers to stabilize the coiled-coil. Dimerization restored partial regulation, but the presence of a length of rod approximately equal to the myosin head was necessary to achieve a completely off state. Partially regulated short dimers could be converted into fully regulated molecules by addition of native rod sequence after the zipper. These results suggest that the myosin rod mediates specific interactions with the head that are required to obtain the completely inactive state of vertebrate smooth and non-muscle myosins. If these interactions are prohibited under cellular conditions, unphosphorylated crossbridges can slowly cycle.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Subfragmentos de Miosina/metabolismo , Actinas/farmacología , Adenosina Trifosfatasas/efectos de los fármacos , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Dimerización , Activación Enzimática/efectos de los fármacos , Leucina Zippers , Modelos Moleculares , Datos de Secuencia Molecular , Músculo Liso , Subfragmentos de Miosina/efectos de los fármacos , Subfragmentos de Miosina/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citologíaRESUMEN
The respiratory burst oxidase of phagocytes and B lymphocytes in a multicomponent enzyme that catalyzes the reduction of oxygen by NADPH. It is responsible for O-(2) production in response to stimulation with phorbol 12-myristate 13-acetate (PMA). The study of patients with chronic granulomatous disease (CGD), an inherited disorder characterized by deficient of absent respiratory burst activity, has contributed greatly to our understanding of the NADPH-oxidase. The absence of any one of four components results in the clinical expression of CGD: the two membrane-bound components of the cytochrome b-558, gp91-phox and p22-phox, or the cytosolic factors, p47-phox and p67-phox. We used a system to investigate the activity of mutant p67-phox proteins expressed in a reconstitution assay. This system is characterized by the partial reconstitution of O-(2) production in an Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell line from a patient with p67-phox-deficient CGD by transfection with an expression plasmid containing the 67-phox cDNA in the sense orientation. No O-(2) production was detectable in p67-phox-deficient lymphoblastoid B cell lines transfected with an antisense plasmid or in untransfected p67-phox lymphoblastoid cells stimulated by PMA. We tested two mutants, pEBOp67delta1-22 and pEBOp67delta512-526, and found that both recombinant proteins are active in our system. Thus, we conclude that the first 22 amino acid residues and the last 14 amino acid residues are not critical for initiation of O-(2) production
Asunto(s)
NADPH Deshidrogenasa/química , Fosfoproteínas/metabolismo , Estallido Respiratorio , Linfocitos B/enzimología , Secuencia de Bases , Cartilla de ADN/química , Genes , Humanos , Datos de Secuencia Molecular , Fosfoproteínas/genética , Eliminación de Secuencia , Relación Estructura-Actividad , Superóxidos/metabolismo , TransfecciónRESUMEN
Myosin II crossbridges interact with F-actin producing powerstrokes of around 100 A (refs 1, 2), during which the products of ATP hydrolysis are released. This has been postulated to involve an articulation of the myosin head (S1) on actin, or substantial conformational changes in S1 itself. Small movements of the regulatory light chain have been detected (see, for example, refs 9, 10), but most data suggest that the bulk of S1 does not move on actin during crossbridge cycling. Here we present three-dimensional maps of S1-decorated F-actin in the presence and absence of MgADP. The myosin motor domain is similar in both states but there are major orientational differences in the light-chain-binding domain. This domain acts as a rigid level arm pivoting about the end of the motor domain and swinging approximately 23 degrees, resulting in a approximately 35-A step. Small, nucleotide-mediated conformational changes in the motor domain may thus be converted by the light-chain domain into large movement steps.
Asunto(s)
Adenosina Difosfato/metabolismo , Músculo Liso/fisiología , Miosinas/fisiología , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Movimiento , Músculo Liso/metabolismo , Conformación ProteicaRESUMEN
The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.
Asunto(s)
Linfocitos B/enzimología , NADH NADPH Oxidorreductasas/metabolismo , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Fagocitos/enzimología , Fosfoproteínas/metabolismo , Serina , Secuencia de Aminoácidos , Secuencia de Bases , Membrana Celular/enzimología , Citosol/enzimología , Activación Enzimática , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/biosíntesis , NADPH Deshidrogenasa/biosíntesis , Oligonucleótidos Antisentido , Fosfoproteínas/biosíntesis , Fosforilación , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
BACKGROUND: The purpose of this study was to assess the structure and function of the small intestine before and after enteral feeding given via a percutaneous feeding gastrostomy (PEG). It is not known whether this method of feeding provides a good luminal drive to the small intestine. METHODS: Studies were performed of patients at the time of PEG placement, in a cross-sectional group after a period of feeding and in a smaller longitudinal subgroup. Enteral feeds were adjusted in volume and caloric content for each patient. Duodenal biopsies were taken during endoscopy for quantitative morphometry, and lactulose-rhamnose permeability tests were performed during the next day. Duodenal fluid was cultured quantitatively in the first study, and disaccharidases determined in the second study. RESULTS: The first study of 15 patients, who had enteral feeding for a median (range) period of 13 (8 to 104) weeks, showed partial villous atrophy with normal crypt length, no increase in duodenal bacteriology, and abnormal lactulose-rhamnose sugar permeability due to rhamnose malabsorption. These changes were also present in 38 similar patients before enteral feeding. A second study before enteral feeding showed lowered maltase activity (24 patients), and similar intestinal permeability findings (22 patients). Twelve of these patients were followed longitudinally for 3 months of enteral feeding that maintained but did not improve nutrition, as assessed by body mass index and mid-arm muscle circumference, and there was no change in duodenal morphometry (11 patients), rhamnose malabsorption (4 patients), or disaccharidases (11 patients). CONCLUSIONS: These studies suggest villous atrophy was not due to an inflammatory enteropathy but resulted from a poor luminal "drive" associated with the enteral feeding. Enteral feeding maintained but did not improve nutrition status.
Asunto(s)
Nutrición Enteral/efectos adversos , Intestino Delgado/patología , Síndromes de Malabsorción/etiología , Atrofia , Estudios Transversales , Duodeno/microbiología , Duodeno/patología , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Lactulosa/metabolismo , Estudios Longitudinales , Fenómenos Fisiológicos de la Nutrición , Ramnosa/metabolismo , Factores de Tiempo , alfa-Glucosidasas/metabolismoRESUMEN
The respiratory burst oxidase catalyzes the production of O2.- from oxygen and NADPH. It is dormant in resting cells but becomes active when the cells are stimulated. Activation is accompanied by the phosphorylation of multiple serines in the cytosolic oxidase component p47phox, which moves from cytosol to the membrane during oxidase activation. Using immunopurified p47phox isolated from 32Pi-loaded neutrophils activated with phorbol myristate acetate, we showed that all the 32P was in the C-terminal CNBr fragment of the protein, and that in that fragment, Ser-303, Ser-304, Ser-320, Ser-328, Ser-345, and Ser-348 and at least one of the three serines, Ser-359, Ser-370, and Ser-379, were phosphorylated, while Ser-282, Ser-287, Ser-381, and Ser-388 were not. Of the phosphorylated serines, Ser-303, Ser-304, Ser-320, and Ser-328 are located in protein kinase C substrate sequences. Ser-345 and Ser-348, however, are located in sequences recognized by mitogen-activated protein (MAP) kinase (-PXSP-). This finding suggests that MAP kinase or a related proline-directed kinase may participate in the regulation of O2.- production by activated neutrophils. The tryptic peptide map of p47phox phosphopeptides from neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine closely resembled that of p47phox phosphopeptides from phorbol-activated cells, suggesting that the same serines were phosphorylated in response to each agent.
Asunto(s)
NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Neutrófilos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , Mapeo Peptídico , Fosforilación , Fosfoserina/metabolismo , Proteínas Quinasas Dirigidas por Prolina , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
A point mutation in the heavy chain of cardiac myosin, resulting in replacement of an arginine (Arg) with glutamine (Gln), has been linked to hypertrophic cardiomyopathy in humans (Geisterfer-Lowrance, A. A. T., Kass, S., Tanigawa, G., Vosberg, H.-P., McKenna, W., Seidman, J. G., and Seidman, C. E. (1990) Cell 62, 999-1006). To determine the functional impact of this mutation, baculovirus-driven coexpression of myosin heavy and light chains has been developed. The Arg-403-->Gln mutation resulted in cardiac myosin with normal ATPase activity in the absence of actin. However, in the presence of actin, ATPase activity was greatly reduced (Vmax decreased > 3.5-fold and K(app) increased > 3-fold). In vitro motility was reduced nearly 5-fold by this single amino acid mutation. Thus, Arg-403 likely contributes to an important interaction at the actin interface of myosin. Replacement of Arg-403 with Gln leads to decreased rate(s) of transition within the actin-myosin crossbridge cycle. In humans, this mutation will result in decreased power output per unit area of cardiac muscle, likely providing a stimulus for hypertrophy.
Asunto(s)
Actinas/metabolismo , Miocardio/metabolismo , Miosinas/genética , Miosinas/metabolismo , Mutación Puntual , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Arginina , Sitios de Unión , ATPasas Transportadoras de Calcio/metabolismo , Cardiomiopatía Hipertrófica/genética , Proteínas de Transporte de Catión , Línea Celular , Expresión Génica , Glutamina , Humanos , Miosinas/biosíntesis , Unión Proteica , Ratas , TransfecciónRESUMEN
BACKGROUND: To aid in development of patient testing policy, in-service education, and resource planning, it is necessary to have a useful and meaningful tool for determining the population-specific HIV seroprevalence rate for our hospital patients. We were offered by the Centers for Disease Control a newly developed survey tool: "Rapid Assessment of HIV Seroprevalence in Hospital Patients." We subsequently served as one pilot site for this tool. METHODS: A population-based sample of 1000 patients (500 inpatients, 500 outpatients) was stratified into age and sex groups on the basis of admission statistics from the previous year in a general community hospital system in southeastern Pennsylvania that consists of two clinical campuses: an urban site with 343 beds and a suburban site with 506 beds. The study was conducted as an anonymous, unlinked screening for HIV antibody in 1000 serum samples. RESULTS: We found our overall seroprevalence rate to be 2.60% (Poisson 95% confidence interval, 1.77% to 3.81%), or 1 in 38 patient specimens. The highest rates for both sexes were found in the age range 25 to 44 years. CONCLUSIONS: This protocol is a useful survey tool for community hospitals to determine the HIV seroprevalence rate in patient populations, a practical necessity for planning and education. Survey results would aid in implementation of current Centers for Disease Control guidelines for HIV testing of inpatients and outpatients in the acute care hospital setting.
Asunto(s)
Seroprevalencia de VIH , Hospitales Comunitarios/estadística & datos numéricos , Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Adulto , Recolección de Datos , Femenino , Humanos , Masculino , Tamizaje Masivo , Persona de Mediana Edad , Pennsylvania/epidemiología , Proyectos PilotoRESUMEN
Lehigh Valley Hospital initiated a program of total quality management using the model taught by Philip Crosby Associates. When the training began in the Research Department, we used the application of this model to identify, define and chart the various processes that a research project or study moves through from conception to publication. The resulting charts and process outlines enabled the Research Department personnel to recognize problem areas in the processes, and to chart the progress of a study at any given time. This provided us with both a better understanding of input and output in the research processes, and an opportunity to improve efficiency by correcting problem areas.
Asunto(s)
Biometría , Hospitales Comunitarios/normas , Garantía de la Calidad de Atención de Salud/organización & administración , Hospitales con más de 500 Camas , Participación en las Decisiones , Cómputos Matemáticos , Pennsylvania , Proyectos de InvestigaciónRESUMEN
The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells.
Asunto(s)
Linfocitos B/metabolismo , NADH NADPH Oxidorreductasas/deficiencia , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasas , Superóxidos/metabolismo , Transfección , Secuencia de Aminoácidos , Anticuerpos , Antígenos Virales/genética , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Proteínas de Unión al ADN/genética , Antígenos Nucleares del Virus de Epstein-Barr , Vectores Genéticos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Immunoblotting , Cinética , Mediciones Luminiscentes , Sustancias Macromoleculares , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/metabolismo , Oligodesoxirribonucleótidos , Péptidos/síntesis química , Péptidos/inmunología , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The history and etiology of inflammatory bowel disease which is characterized by two major disease processes: ulcerative colitis and Crohn's disease, remain unknown. Research is focussing on seven major areas of genetic, environmental and physiologic factors that apparently relate to this disease. Based on this background, a population based Inflammatory Bowel Disease Registry was established in 1987 in the Lehigh Valley area of southeastern Pennsylvania. Consent forms, patient data forms and protocols for operation and implementation were developed, and databases were designed to accommodate demographic, basic history, follow-up and relative history data. The databases were correlated with an IBD registry ID number which both enabled relational analyses and ensured confidentiality of data information. The registry continues to grow, providing feedback for both continued medical research and supportive information for IBD patients and their physicians.
Asunto(s)
Procesamiento Automatizado de Datos , Enfermedades Inflamatorias del Intestino/etiología , Sistema de Registros , Sistemas de Administración de Bases de Datos , Femenino , Encuestas Epidemiológicas , Humanos , Masculino , Linaje , PennsylvaniaRESUMEN
Recombinant ethanolamine ammonia-lyase from S. typhimurium has been overexpressed and purified in large quantities by a simple procedure. The molecular weight of the native enzyme is about 480 kDa, and it contains two active sites/molecule as shown by kinetic studies and by titration with CNCbl. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirms earlier cloning studies indicating that it is composed of two kinds of subunits, one of MW 31 kDa and the other of MW 50 kDa. These subunits, inactive by themselves, combine to produce an active enzyme whose composition is most likely alpha 6 beta 6. The Km for AdoCbl is 0.5 microM, and the turnover number is 55 s-1 per active site at 22 degrees C.