Dynamics of the seasonal airborne propagation of Staphylococcus aureus in academic dental clinics

Abstract Objective Staphylococcus aureus strains can be disseminated during dental treatments and occasionally lead to the contamination and infection of patients and dentists, which is an important public health problem. The dynamics of the airborne propagation and the genetic diversity of S. aureus isolated in an academic dental clinic environment were investigated using isoenzyme typing. Material and Methods The isoenzymes of 44 previously reported isolates were obtained from fresh cultures and extracted using glass beads. Nine isoenzymes were investigated using multilocus enzyme electrophoresis (MLEE). The genetic diversity and relationship among the strains (electrophoretic type - ET) were determined using statistics previously described by Nei25 (1972) and the SAHN grouping method (UPGMA algorithm). Results Clonal pattern analyses indicated a high level of genetic polymorphism occurring among the 33 ETs, which were grouped into five taxa. Each taxon presented one or more clusters that were moderately related and that contained two or more identical/highly related isolates, revealing seasonal airborne propagation in these dental clinic environments. Conclusions These data suggest the occurrence of active microevolutionary processes in S. aureus as well as the possibility of environmental propagation during a 14-month time span. Such findings are important to show that multiuser academic dental clinics can retain certain strains that are spreadable to different niches.

Valor pronóstico de las isoenzimas de LDH en pacientes con linforma no hodgkin / Prognostic value of LDH isoenzymes in patients with non-Hodgkin linforma

La enzima lactato deshidrogenasa (LDH) es un factor pronóstico en Linfoma No Hodgkin (LNH). El objetivo del trabajo consistió en evaluar prospectivamente el valor pronóstico de las isoenzimas de LDH en pacientes con LNH. Se estudiaron 67 pacientes de primera consulta con diagnóstico de LNH, sin tratamiento previo, VIH negativo y sin otras enfermedades, tiempo promedio de seguimiento 30 meses (rango 3-48 meses). Las muestras de suero se recolectaron previas al tratamiento. La LDH total (LDHT) e isoenzimas de LDH se determinaron respectivamente por método cinético y electroforesis de proteínas en gel de agarosa. Se procesaron muestras de 122 controles sanos para establecer los valores de referencia de las isoenzimas de LDH. 49(73%) LNH agresivos y 18(27%) LNH indolentes y según el Índice Pronóstico Internacional (IPI), 60 (90%) bajo riesgo y 7(10%) alto riesgo. Las isoenzimas LDH1, LDH2, LDH3, LDH4 y LDH5 presentaron niveles absolutos significativamente elevados en 25 (37%), 29 (43%), 32 (48%), 20 (39%) y 11 (16%) de los casos respectivamente (p<0,0001). La actividad porcentual de LDH4 en los pacientes con LNH agresivos fue significativamente superior respecto al grupo de LNH indolentes (p=0,01). En el análisis univariado, valores absolutos elevados de LDH1 se asociaron significativamente con una sobrevida global disminuida (p=0,0064) en el grupo total de pacientes. LDH1 conservó su valor pronóstico aún en el grupo de pacientes con valores normales de LDHT (p=0,04). En pacientes con LNH agresivos, valores elevados de LDHT e IPI alto riesgo se asociaron significativamente con una menor sobrevida global (p<0,05). En el análisis multivariado la LDHT e IPI resultaron factores pronósticos independientes de la sobrevida. Alteraciones específicas del patrón de isoenzimas de LDH sugieren la relación de LDH4 con la biología del tumor y su actividad proliferativa en LNH agresivos y el valor pronóstico de LDH1 como factor adverso de la sobrevida en el análisis univariado.

Lactate dehydrogenase (LDH) is a prognostic factor in non-Hodgkin lymphoma (NHL). Our objective was to evaluate prospectively the prognostic value of LDH isoenzymes in patients with NHL. We studied 67 newly diagnosed NHL patients, previously untreated, HIV-negative and free from other disease, median follow-up of 30 month (range 3-48 month). Before starting treatment serum samples were collected for the determination of total LDH (LDHT) and LDH isoenzymes that were respectively assayed by kinetic method and protein electrophoresis in agarose gel. In order to set reference values of LDH isoenzymes samples from122 healthy controls were processed. Results: 49(73%) of the patients were aggressive NHL and 18(27%) indolent NHL and according to the International Prognostic Index (IPI), 60(90 %) low risk and 7(10%) high risk. High absolute values of LDH1, LDH2, LDH3, LDH4 and LDH5 isoenzymes were significantly elevated in 25 (37%), 29 (43%), 32 (48%), 20 (39%) and 11 (16%) of cases respectively (p<0,0001). The percentage value of LDH4 activity in aggressive NHL patients was significantly higher compared to indolent NHL group (p=0,01). In univariate analysis increased LDH1 absolute values were significantly associated with decreased overall survival in the total group of patients (p = 0.0064). LDH1 remained a prognostic factor for survival even when considering the group of patients with normal serum LDHT values (p = 0.04). In patients with aggressive NHL increased values of LDHT and high risk IPI were significantly associated with decreased overall survival (p<0.05). In a multivariate analysis LDHT and IPI score were independent prognostic factor for survival.

Molecular characteristics of the inhibition of human neutrophil elastase by nonsteroidal antiinflammatory drugs

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.

Elevación asintomática de fosfatasas alcalinas plasmáticas secundaria a hiperfosfatasemia familiar benigna en un paciente / Asymptomatic elevation of alkaline phosphatases secondary to benign familial hyperphosphatasemia in a patient

We report a 33 years old male consulting for abdominal pain. Initial diagnostic work up showed high levels of alkaline phosphatases, that were confirmed in a new blood sample. Using electrophoresis, total alkaline phosphatases were 198 U/l (normal values=30-117) and the bone fraction was 101 U/l (normal values=0-35). Bone scintiscan and endocrinological assessment were normal. One year later, the same values persisted. Studying the family, 3 of 4 brothers had the same alterations in alkaline phosphatases. It was concluded that these subjects had the rare condition known as benign familial hyperphosphatasemia

Porphyrinogen carboxylyase. Studies on existence of isoenzymes

Porphyrinogen carboxylyase from normal rat liver was subjected to purification methods. Two different purification protocols were used. In both cases, the inital steps consisted in obtaining a liver homogenate, followed by centrifugation, salt precipitation and phosphate gel absorption. Scheme I consisted in then submiting the preparation to DEAE-cellulose, followed by Sephacryl S-200 and Phenyl-sepharose sequential column chromatographies. Scheme II involved an affinity column followed by a Sephadex G-75 gel filtration column. In both cases, the enzyme was stored at-20 degrees Celsius until its assay. The addition of 2mM dithiotreytol to the incubation media or to the enzyme extract before storage, did not help improve the activity nor the stability of the enzyme. Those fractions containing the maximal enzyme activity, detected using Uroporphyrinogen III or Pentacarboxy-porphyrinogen III as substrate, were not alawys present in the same tubes for the different columns employed. In addition, the degree of purification obtained in some steps was different according to the substrate employed. The results suggest the existence of at least two isoenzymes for rat liver porphyrinogen carboxy-lyase.

Distribution of phospholipase C isozymes in normal human lung tissue and their immunohistochemical localization

Phospholipase C(PLC) plays a central role in signal transduction and it is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC identified and cloned. However, there are no report of PLC distribution in human lung tissue or their significances in pulmonary diseases. Presence of various PLC isozymes in normal human lung tissue was studied from surgical specimens. PLC isozymes in tissue extracts of the lung were partially purified by successive chromatographic steps on heparin-sepharose CL-6B conventional and TSKgel heparin-5PW HPLC columns and their activities were assayed. PLC activity peaks identified in the chromatography were immunoblotted with specific antibodies against ten known mammalian PLC isozymes(PLC-beta 1-4, -gamma 1-2, and -delta 1-4). In addition, immunohistochemical staining of the lung tissue was performed to determine subcellular and histological localization of PLC isozymes. The results indicate that normal human lungs contain beta 1, beta 3, gamma 1, and delta 1, isozymes of PLC. The order of amount present in the lung tissue was PLC-delta 1 > gamma 1 >beta 1 >> beta 3, in descending order. On immunohistochemistry, PLC-gamma 1 was most widely distributed and was present in bronchiolar epithelium, in type I and type II pneumocytes as well as in fibroblasts of the interstitial tissue. PLC-delta 1 was present in the cytoplasm of the bronchiolar epithelium whereas PLC-beta 1 was localized to the apical membranous portion of the same epithelium. PLC-beta 3 was seen in the nucleus of the respiratory and alveolar lining epithelium as well as in the nucleus of lung fibroblasts.

Caracterización de formas macroenzimáticas de la fosfatasa alcalina: descripción de un nuevo método / A new method for the characterization of alkaline phosphatase macroenzymes

Se describe un nuevo método para la caracterización de macroenzimas de la fosfatasa alcalina. El método combina una separación previa de las formas isoenzimáticas por gel filtración en capa fina de Sephadex G-200, y el posterior revelado de la correspondiente actividad enzimática in situ. Entre otras ventajas, este sistema no sólo separa adecuadamente las formas macroenzimáticas, sino que mantiene todas las isoenzimas en estado nativo, a diferencia de las técnicas con desnaturalizantes, por lo que es posible su detección y eventual aislamiento. Permite además la resolución simultánea de un gran número de muestras, así como la inclusión de controles de distinto peso molecular (PM) dentro de una misma corrida, lo que facilita en forma significativa la posterior interpretación del perfil obtenido, aportando una gran ventaja con respecto a la técnica de gel filtración en columna. El método propuesto se presenta como una técnica relativamente simple y rápida para el screening de macroenzimas en el laboratorio clínico

Lysine- and threonine-sensitive aspartokinase isoenzymes from spinach leaves share common antigenic determinants.

The lysine- and threonine-sensitive isoenzymes of aspartate kinase were purified to homogeneity from spinach leaves and polyclonal antibodies were raised in rabbits. The antibodies were characterized by various immunological tests like Ouchterlonys-double-diffusion, titrations of the inhibition of enzyme activity and ELISA. The antibodies against the lysine-sensitive isoenzyme could recognise as little as 50 ng of the pure antigen protein and that against the threonine-sensitive form could recognise 200 ng of the protein in the ELISA tests. The immunological tests have also shown that the lysine and threonine sensitive isoenzymes of aspartate kinase share some common antigenic determinants and differ in others.

Rapid purification of secretory acid proteinase from Candida albicans and its characterization.

Secretory acid proteinase from C. albicans was purified from culture supernatant to apparent homogeneity by ion-exchange chromatography. Two isozymes of the proteinase were resolved using a novel chromatofocusing method. The enzyme, which appears to be a glycoprotein, consists of a single polypeptide chain with glutamine at the N-terminus. Its molecular weight is about 45,000 and isoelectric point is pH 4.6. At pH 5, the proteinase is stable at 45 degrees C for at least 15 min. It has a broad substrate specificity. With BSA as a substrate, Km was determined to be 1.6 x 10(-4) M. The enzyme is inhibited by pepstatin and thus is a carboxyl proteinase. It undergoes autocatalytic digestion at or below pH 5.0. The kinetics of induction of proteinase by various proteins are also reported.

Aislamiento de isoenzimas de fosfatasa alcalina en plasma humano, por cromatografía de intercambio iónico / Alkaline phosphatase isolation isoenzymes in human plasma ionic chromatography

A partir de la preparación de fosfatasa alcalina (EC 3.1.3.1) parcialmente purificada con etanol, de plasma de personas con grupo 0, se separaron cuatro fracciones con actividad enzimática, mediante cromatografía en columna de DEAE-celulosa, con gradiente discontinuo de NaCI. También se extrajo fosfatasa alcalina de hígado, hueso y mucosa intestinal humanos, a fin de caracterizar las distintas formas moleculares. el uso de inhibidores (L-fenil-alcalina y urea) y electroforesis en gel de poliacrilamida, permitió identificar las isoenzimas presentes en los extractos de tejidos y en los picos de elución cromatográfica de la enzima de plasma. La isoenzima hepática se encuentra en todas las fracciones. La primera eluye con NaCI 40 mM y puede separarse en dos (IA e IB). Ambas contienen las isoenzimas intestinal y ósea, además de formas parcialmente desializadas de la hepática. La segunda fracción (NaCI 60 mM) contiene exclusivamente fosfatasa alcalina hepática. La tercera (NaCI 200 mM), de alto peso molecular, está compuesta por las isoenzimas ósea, intestinal y hepática. El perfil es muy reproducible. La comparación del cromatograma correspondiente a plasma normal con el de un paciente portador de un tumor con metástasis óseas, muestra notables diferencias, indicando la utilidad diagnóstica del método, ya que permite identificar el órgano de origen, en casos de incremento de fosfatasa alcalina en plasma.

Purification and properties of a carboxymethylcellulase from phytopathogenic fungus Macrophomina phaseolina.

From the culture filtrate of Macrophomina phaseolina, two forms of carboxymethylcellulase were separated by ion-exchange chromatography and designated as CMCase-I and CMCase-II. CMCase-I was purified following a four-step procedure involving gel filtration on Sephadex G-75, Con-A Sepharose 4B affinity chromatography, fast protein liquid chromatography on mono Q anion-exchanger and on Superose 12 gel filtration. The final preparation was homogeneous by SDS-PAGE, isoelectric focussing in thin layers of polyacrylamide gels and immunoelectrophoresis. The enzyme showed optimum activity at pH 5.5 and 65 degrees C, was stable to heating at 65 degrees C for 10 min, and retained 31% of original activity after heating at 80 degrees C for 10 min. The molecular weight of the enzyme was 3.5 x 10(4) Da. A Km of 0.25 mg/ml was determined using carboxymethyl-cellulose as the substrate.

Multiple forms of glutamine synthetase in Bacillus brevis AG 4.

Glutamine synthetase in Bacillus brevis AG 4, a Gram-positive spore forming bacteria, has been found to exist in multiple molecular forms. It was purified to electrophoretic homogeneity by single-step Blue Sepharose affinity chromatography. The native enzyme has a molecular weight of 600,000 with subunits of 50,000. The enzyme samples purified from different stages of growth differed in Mg2+ sensitivity and other kinetic properties. Four different enzyme samples selected on the basis of Mg2+ sensitivity showed distinct mobilities at pH 6.3 on PAGE using discontinuous buffer system. A correlation amongst Mg2+ sensitivity, electrophoretic mobility, and kinetic properties was highly suggestive of multiple forms of glutamine synthetase in Bacillus brevis arising due to modification.