Caracterização tecnológica de aparas de jacaré-do-Pantanal (Caiman yacare Daudin 1802) criados em cativeiro / Technological characterization of Pantanal alligator shavings (Caiman yacare Daudin 1802) raised in captivity

Objetivou-se caracterizar o corte “aparas” da carcaça do jacaré-do-Pantanal quanto à composição centesimal, teor de colágeno, pH, oxidação lipídica, cor e pigmentos hemetotais (PHT). As aparas apresentaram pH médio de 5,72 e ponto isoelétrico entre 5,2 e 5,5, alto teor de água (76,08%), médio teor proteico (19,89%) e baixo teor de lipídeos (0,54%). O teor de PHT foi de 4,36 mg/g e o de colágeno total de 1,82%, sendo a fração solúvel (0,22%) e a insolúvel (1,61%). Baixo índice de TBARS (0,48 mg MAD/kg) e os índices de cor instrumental foram de 59,55 para luminosidade (L*), 62,87° tonalidade (h) e 12,19 de saturação (C*). Conclui-se que as características das aparas de jacaré são favoráveis para a elaboração de produtos com baixo teor de gordura e coloração mais clara, características da carne destes animais.

Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I / 华中科技大学学报（医学）（英德文版）

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

Origin of Proteinuria as Observed from Qualitative and Quantitative Analysis of Serum and Urinary Proteins

It is well known that proteins present in the primary urine are reabsorbed in the renal proximal tubules, and that this reabsorption is mediated via the megalincubilin complex and the neonatal Fcgamma receptor. However, the reabsorption is also thought to be influenced by an electrostatic interaction between protein molecules and the microvilli of the renal proximal tubules. By analyzing the charge diversity of urinary IgG, we showed that this reabsorption process occurs in a cationic charge-preferential manner. The charge-selective molecular sieving function of the glomerular capillary walls has long been a target of research since Brenner et al. demonstrated the existence of this function by a differential clearance study by using the anionic dextran sulfate polymer. However, conclusive evidence was not obtained when the study was performed using differential clearance of serum proteins. We noted that immunoglobulin (Ig) A and IgG have similar molecular sizes but distinct molecular isoelectric points. Therefore, we studied the differential clearance of these serum proteins (clearance IgA/ clearance IgG) in podocyte diseases and glomerulonephritis. In addition, we studied this differential clearance in patients with Dent disease rather than in normal subjects because the glomerular sieving function is considered to be normal in subjects with Dent disease. Our results clearly showed that the charge-selective barrier is operational in Dent disease, impaired in podocyte disease, and lacking in glomerulonephritis.

Búsqueda y selección de una proteasa fúngica con potencial aplicación en la restauración de documentos históricos en el Archivo de Bogotá / Screening for a fungal protease with potential in the biorestoration of historical valuable documents in Bogota Archive

Se ha buscado y seleccionado sistemáticamente una proteasa que pudiese ser usada en la eliminación "limpia" de encolantes sobre soportes documentales con valor de patrimonio histórico de forma eficiente y económica, a partir de la colección de hongos filamentosos del Archivo de Bogotá. De 74 morfotipos viables evaluados sobre placas selectivas, 32 morfotipos presentaron formación de halos de hidrólisis evidentes sobre placas diferenciales. De ellos, se evaluó el perfil isoenzimático de 8 morfotipos provenientes de muestreos documentales directos y de 2 morfotipos proteolíticos promisorios provenientes de un trabajo previo. Los 10 morfotipos seleccionados fueron representativos de los géneros Penicillium, Stachybotrys, Chaetomium, y Eladia. Luego de inducir la producción de proteasas extracelulares en medios líquidos diferenciales bajo tres fases de fermentación, se realizaron isoelectroénfoques analíticos tendientes a la observación de isoformas en el gradiente de pH establecido (3.0-10.0). Solo los morfotipos 8D (Chaetomium sp.) y 21D (Eladia saccula) presentaron una isoforma alcalina extrema, de puntos isoeléctricos 8.5 y 8.8, respectivamente, susceptible de selección con miras a su purificación y caracterización parcial de forma económica y eficiente. Los demás morfotipos, representativos de los géneros Penicillium sp., y Stachybotrys sp., presentaron unicamente isoformas proteolíticas en el rango acido de pH con puntos isoeléctricos que oscilan entre 4.0 y 5.0.

Studies on a protease as an efficient, environmental friendly and relatively economical remover of residual proteins for historical valuable documents were performed and was selected for this work, from the filamentous fungi collection of the Bogota Archive. 32 morphotypes of 74 evaluated show hydrolytic activities over differential solid media. From them, 8 morphotypes obtained directly from documental samples and representative of the genera Penicillium and Stachybotrys were selected and their isoenzyme profile were tested. Also 2 previous morphotypes with promisorius proteolytic activities and representative of the genera Chaetomium and Eladia were analysed. Extracelullar proteases production was induced in differential liquid media on three fermentation steps and analitycal isoelectrofocusing were performed over pH 3.0-10.0 ranges. Only the morphotypes 8D (Chaetomium sp.), and 21D (Eladia sp.), showed an alkaline isoform with pIs 8.5 and 8.8, respectly, suceptible of selection for its purification and characterization through efficient and economical way. The others morphotypes only showed acid isoforms with pIs in the range of 4.0 and 5.0.

Application of membrane protein-based two-dimensional electrophoresis in chondrocyte- related investigations / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the feasibility of membrane protein-based two-dimensional electrophoresis (2-DE) in the investigations of chondrocyte-related diseases and its complementarity with total protein-based 2-DE.</p><p><b>METHODS</b>Knee cartilage samples were obtained to isolate the chondrocytes with type II collagenase/hyaluronidase digestion. The membrane proteins and total proteins were extracted and loaded separately onto PH3.0-10.0 non-linear gel strip for 2-DE analysis. The qualities of membrane protein-based 2-DE gels were evaluated, and the difference between the distribution profiles of the membrane protein spots and that of the total protein were observed and their complementarities were evaluated.</p><p><b>RESULTS</b>Membrane protein-based 2-DE generated high-quality gel images, and on each gel 412.3±13.5 protein spots were identified. These spots were distributed in the region of isoelectric point pH 5.0-9.0. In the gel images generated by the total proteins, 564.3±5.9 protein spots were identified in each image, and the spots were distributed in the region of isoelectric point pH 3.0-7.0.</p><p><b>CONCLUSION</b>Membrane protein-based 2-DE of the chondrocytes can generate high-quality gel images, and the isoelectric distribution of the protein spots is complementary to that of total protein, which provides valuable information for chondrocyte-related diseases.</p>

SHV-28, an extended-spectrum beta-lactamase produced by a clinical isolate of Klebsiella pneumoniae in south India.

SHV-28, an extended spectrum beta-lactamase from a clinical isolate of Klebsiella pneumoniae , had an isoelectric point of 7.6 and a substrate profile showing preferential hydrolysis for cefotaxime over ceftazidime. It differed from SHV-1 by one amino acid substitution. The conserved S-T-F-K and K-T-G motifs were identified by SHV-28 protein sequencing.

Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum.

A RING zinc finger ankyrin protein gene,designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE.Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4- type RING finger domain was found at the C-terminal region of the AdZFP1 protein,and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869.Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root,stem and leaf of the plant.Semi-quantitative reverse- transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity,cold and heat to some extent.Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

[Determination of Helicobacter pylori immunogenes using two- dimensional gel electrophoresis and immunoblotting]

Helicobacter pylori is one of the most common infectious agents that colonizes in the mucus layer of stomach. This bacterium has been identified to be the etiologic agent of chronic active gastritis, peptic ulceration and gastric cancer. The present study was aimed to identify H. pylori immunogenes for clinical diagnisis of the infection in the above 3 groups of patients. H. pylori bacteria isolated from biopsy specimens of patients suffering from gastritis, peptic ulcer and gastric cancer were extracted in an extraction solution containing lysozyme, urea and CHAPS. Two-dimensional gel electrophoresis were performed. The resolved proteins were transferred to PVDF membrane using tank blotting and their reaction with purified IgG fraction of the patients. Sera were determined by immunoblotting. The bacterial extract showed several hundreds of silver-stained spots with molecular weights [MW] ranging from 10 to 100 KDa and isoelectric points [pI] ranging from 3.5 to 9.5. This pattern contained 6-7 major proteins, some of which as protein groups consisted of several spots. The results of immunoblots revealed that several protein spots with different MW and pI, were stained with all three groups of patients. sera but some proteins were stained only with one or two groups of sera. The protein spot with MW of 30 KDa reacted with sera of only two groups of patients; gastritis and gastric cancer; the protein with MW of 18 KDa reacted only with sera of gastritis patients. These proteins can be potential candidates for recognition of the type of gastric disorder. In addition, the results indicated that protein profiles of H. pylori, isolated from gastric cancer and peptic ulcer, are more similar to each other, comparing to that of gastritis patients

Assessment of the phospholipase A2 patterns of some Egyptian snakes venoms

Despite of the fact that functionally diverse phospholipase A2 variants of snake venoms are well characterized at the level of protein and gene sequences; the patterns of individual snake venom PLA2s are poorly known. We investigated the activity, molecular weights and isoelectric points of the phospholipase A2s of some medically important snake venoms in Egypt. Portrayal of the phospholipase A2 activity of the vipers, "Pseudocerastes persicus fieldi, Cerastes cerastes and Echis carinatus" and the elapids "Naja haje, Walterinnesia aegyptia and Naja nigricollis" venoms revealed that: 1- The elapid venoms, with the exception of Naja haje, displayed higher PLA2 activity than viper venoms; 2- The molecular weights of the phospholipase A2 variants were close to 14 kDa; 3- The major phospholipase A2s of Naja nigricollis were basic proteins while those of Walterinnesia aegyptia venom were acidic proteins; 4- The Naja nigricollis and Pseudocerastes persicus fieldi venoms possessed the highest phospholipase A2 activity while the Walterinnesia aegyptia and Pseudocerastes persicu fieldi had the highest hemolytic activity of the tested elapids and vipers, respectively; 5- The in vitro hemolytic activities of the venoms were inhibited by the heterologous antivenoms, suggesting that the venom hemolytic factor [s] have shared epitopes. The data provided biochemical information of snake venoms phospholipase A2 which allowed designing procedure for isolation of the phospholipase A2s to study their pharmacological effects

Structural Bioinformatics Analysis of Disease-related Mutations

In order to understand the protein functions that are related to disease, it is important to detect the correlation between amino acid mutations and isease. Many mutation studies about disease-related proteins have been carried out through molecular biology techniques, such as vector design, protein engineering, and protein crystallization. However, experimental protein mutation studies are time-consuming, be it in vivo or in vitro. We therefore performed a bioinformatic analysis of known disease-related mutations and their protein structure changes in order to analyze the correlation between mutation and disease. For this study, we selected 111 diseases that were related to 175 proteins from the PDB database and 710 mutations that were found in the protein structures. The mutations were acquired from the Human Gene Mutation Database (HGMD). We selected point mutations, excluding only insertions or deletions, for detecting structural changes. To detect a structural change by mutation, we analyzed not only the structural properties (distance of pocket and mutation, pocket size, surface size, and stability), but also the physico-chemical properties (weight, instability, isoelectric point (IEP), and GRAVY score) for the 710 mutations. We detected that the distance between the pocket and disease-related mutation lay within 20 A (98.5%, 700 proteins). We found that there was no significant correlation between structural stability and disease-causing mutations or between hydrophobicity changes and critical mutations. For large-scale mutational analysis of disease-causing mutations, our bioinformatics approach, using 710 structural mutations, called "Structural Mutatomics," can help researchers to detect disease-specific mutations and to understand the biological functions of disease-related proteins.

Prevalence of Class A Extended-Spectrum beta-Lactamases in Clinical Isolates of Acinetobacter baumannii and Pseudomonas aeruginosa / 대한진단검사의학회지

BACKGROUND: Prevalence of class A extended-spectrum beta-lactamases (ESBLs) has been investigated repeatedly in members of family Enterobacteriaceae in Korea, but only rarely in Acinetobacter baumannii and Pseudomonas aeruginosa. The aims of this study were to determine the prevalence of class A ESBL-producing A. baumannii and P. aeruginosa and to characterize the genotypes. METHODS: During the period of June to September 2004, clinical isolates of A. baumannii and P. aeruginosa were collected from patients in Kosin University Gospel Hospital, Busan, Korea. Antimicrobial susceptibility was determined by the disk diffusion and the agar dilution methods, and ESBLproduction by the double-disk synergy test. Transferability of ceftazidime-resistance of ESBL-producers were tested by conjugation. The isoelectric points of ESBLs were determined by isoelectric focusing. Searches for blaTEM, blaSHV, blaCTX-M, blaPER-1, blaVEB, and blaGES/IBC genes were performed by PCR amplification, and the genotypes of ESBLs were determined by a direct nucleotide sequence analysis of the amplified products. RESULTS: A total of 58 clinical isolates of A. baumannii and 77 P. aeruginosa were collected. Three (5.2%) isolates of A. baumannii and four (5.2%) P. aeruginosa isolates showed positive results in the double-disk synergy test using ceftazidime and imipenem disks, and one (1.7%) A. baumannii and two (2.6%) P. aeruginosa isolates showed positive results in that test using ceftazidime and cefoxitin disks. The most prevalent class A ESBL genotype in A. baumannii isolates was blaPER-1 (n=6), and blaSHV-12 gene was also found in one P. aeruginosa isolate. CONCLUSIONS: It is concluded that class A PER-1 ESBL-producing A. baumannii isolates are spreading, and SHV-12-producing P. aeruginosa has emerged in Korea. The spread of class A ESBLs could compromise the future usefulness of expanded-spectrum -lactam antibiotics for the treatment of A. baumannii and P. aeruginosa infections.

Caracterización fenotípica y genotípica de la resistencia enzimática a las cefalosporinas de tercera generación en Enterobacter spp. / Phenotypic and genotypic characterization of resistance to third-generation cephalosporins in Enterobacter spp.

Enterobacter spp. es un patógeno intrahospitalario que presenta múltiples mecanismos de resistencia a los antibióticos b-lactámicos. Se caracterizaron fenotípica y genotípicamente las diferentes b-lactamasas presentes en 27 aislamientos consecutivos e ininterrumpidos de Enterobacter spp. (25 Enterobacter cloacae y 2 Enterobacter aerogenes). También se evaluó la habilidad de diferentes métodos fenotípicos para detectar b-lactamasas de espectro extendido (BLEE) en estos microorganismos. En 15/27 aislamientos (63%) se observó resistencia a las cefalosporinas de tercera generación. En 12 de los aislamientos resistentes se detectó un alto nivel de producción de cefalosporinasa cromosómica, siendo 6 de ellos también productores de PER-2. Dicha resistencia en los 3 aislamientos restantes se debió exclusivamente a la presencia de BLEE, PER-2 en 2 de ellos y CTX-M-2 en un caso. Sólo CTX-M-2 se detectó con todas las cefalosporinas probadas en los ensayos de sinergia, utilizando el método de difusión, mientras que cefepima mejoró la detección de PER-2 en 7/8 aislamientos productores de esta BLEE, 4/8 utilizando la prueba de doble disco y 7/8 comparando discos de cefepima con y sin el agregado de ácido clavulánico. El método de dilución empleado solo detectó 1/9 BLEE al comparar las cefalosporinas con y sin el agregado de inhibidor.

Enterobacter spp. are becoming increasingly frequent nosocomial pathogens with multiple resistance mechanism to b-lactam antibiotics. We carried out the phenotypic and genotypic characterization of beta-lactamases in 27 Enterobacter spp. (25 Enterobacter cloacae y 2 Enterobacter aerogenes), as well as the ability of different extended spectrum b-lactamase (ESBL) screening methods. Resistance to third generation cephalosporins was observed in 15/27 (63%) isolates. Twelve resistant isolates produced high level chromosomal encoded AmpC b-lactamase; 6 of them were also producers of PER-2. Resistance to third generation cephalosporins in the remaining 3 isolates was due to the presence of ESBLs, PER-2 in 2 cases, and CTX-M-2 in the other. Only CTX-M-2 production was detected with all tested cephalosporins using difusion synergy tests, while cefepime improved ESBLs detection in 7/8 PER-2 producers, 4/8 in the inhibitor aproximation test and 7/8 with double disk test using cefepime containing disk with and without clavulanic acid. Dilution method, including cephalosporins with and without the inhibitor detected 1/9 ESBLs producers.

Molecular Epidemiology of Infection Caused by OXA-23 or IMP-1 beta-Lactamase-Producing Acinetobacter baumannii / 대한임상미생물학회지

BACKGROUND: The purposes of this study were to investigate the prevalence of imipenem-resistant clinical Acinetobacter baumannii isolates and to determine the mechanism of the resistance. METHODS: During the period of June to September 2004, susceptibility to imipenem of A. baumannii isolates from a hospital in Busan, Korea were investigated. The isolates were screened for the production of carbapenemase and metallo-beta-lactamase by Modified-Hodge and EDTA-disk synergy tests, respectively; minimum inhibitory concentrations (MICs) were determined by agar dilution method. Genes coding for GES, IMP, VIM, SMP-1, GIM-1 and OXA type beta-lactamases were searched by PCR amplification, and the PCR products were subjected to direct sequencing. Isoelectric points of beta-lactamases were estimated by isoelectric focusing and the epidemiological relationships of isolates were investigated by enterobacterial repetitive intergenic consensus (ERIC) PCR. RESULTS: Fifty eight strains of A. baumannii were isolated from clinical specimens during the surveillance period, and 14 isolates (24.1%) were resistant to imipenem. Of the 14 isolates, 9 were tested positive in Modified-Hodge test and 2 were also positive in EDTA-disk synergy test. Genes encoding OXA-23 and IMP-1 were detected in 7 and 2 isolates, respectively. In IEF studies, OXA-23 and IMP-1 enzymes had corresponding pIs at 6.7 and 9.0, respectively. Seven OXA-23-producing and 2 IMP-1-producing isolates showed the same ERIC PCR patterns. CONCLUSION: It is concluded that 7 and 2 A. baumannii isolates from the patients in a hospital in Busan acquired resistance to imipenem by producing OXA-23 and IMP-1 beta-lactamases, respectively. The isolates producing these beta-lactamases might be originated from a common source.

Oxidative Inactivation of Peroxiredoxin Isoforms by H2O2 in Pulmonary Epithelial, Macrophage, and other Cell Lines with their Subsequent Regeneration / 결핵및호흡기질환

BACKGROUND: Peroxiredoxins (Prxs) are a relatively newly recognized, novel family of peroxidases that reduce H2O2 and alkylhydroperoxide into water and alcohol, respectively. There are 6 known isoforms of Prxs present in human cells. Normally, Prxs exist in a head-to-tail homodimeric state in a reduced form. However, in the presence of excess H2O2, it can be oxidized on its catalytically active cysteine site into inactive oxidized forms. This study surveyed the types of the Prx isoforms present in the pulmonary epithelial, macrophage, endothelial, and other cell lines and observed their response to oxidative stress. METHODS: This study examined the effect of exogenous, excess H2O2 on the Prxs of established cell lines originating from the pulmonary epithelium, macrophages, and other cell lines, which are known to be exposed to high oxygen partial pressures or are believed to be subject to frequent oxidative stress, using non-reducing SDS polyacrylamide electrophoresis (PAGE) and 2 dimensional electrophoresis. RESULT: The addition of excess H2O2 to the culture media of the various cell-lines caused the immediate inactivation of Prxs, as evidenced by their inability to form dimers by a disulfide cross linkage. This was detected as a subsequent shift to its monomeric forms on the non-reducing SDS PAGE. These findings were further confirmed by 2 dimensional electrophoresis and immunoblot analysis by a shift toward a more acidic isoelectric point (pI). However, the subsequent reappearance of the dimeric Prxs with a comparable, corresponding decrease in the monomeric bands was noted on the non-reducing SDS PAGE as early as 30 minutes after the H2O2 treatment suggesting regeneration after oxidation. The regenerated dimers can again be converted to the inactivated form by a repeated H2O2 treatment, indicating that the protein is still catalytically active. The recovery of Prxs to the original dimeric state was not inhibited by a pre-treatment with cycloheximide, nor by a pretreatment with inhibitors of protein synthesis, which suggests that the reappearance of dimers occurs via a regeneration process rather than via the de novo synthesis of the active protein. CONCLUSION: The cells, in general, appeared to be equipped with an established system for regenerating inactivated Prxs, and this system may function as a molecular "on-off switch" in various oxidative signal transduction processes. The same mechanisms might applicable other proteins associated with signal transduction where the active catalytic site cysteines exist.

Developmental Analysis of Calretinin Using Quantitative Analysis in Rat Cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Calretinin is a neuron specific, high affinity cytosolic calcium-binding protein of the EF-hand family. In the mammalian inner ear, it is expressed in the organ of Corti and most of the spiral ganglion neurons. Authors observed the change of expression and amount of calretinin according to the maturation in the rat cochleae. MATERIALS AND METHOD: Sprague-Dawley rat cochleae collected from each stage (postnatal day (P) P5, P17, P35) were analyzed using 2D gel electrophoresis, proteomic analysis, Western blot analysis, and fluorescence immunocytochemistry. RESULTS: In P17 and P35, calretinin was identified at an isoelectric point (pI) of 4.9 and a molecular weight of 29 kDa in the analysis of the rat cochlea proteins using proteomic analysis. P17 and P35 revealed remarkable existence of calretinin in 2D gel electrophoresis and the Western blot, whereas P5 demonstrated no existence in 2D gel electrophoresis and weak expression in the Western blot. In fluorescence immunocytochemistry, P17 and P35 showed intense calretinin immunoreactivity in the inner hair cells and most ganglion neurons, but P5 displayed no immunoreactivity in inner hair cells and very weak expression in spiral ganglion cells. CONCLUSION: Compared with the early neonatal stage, an amount of calretinin remarkably increases during the critical period of the cochlear maturation and is maintained until the young adult stage. These results suggest that calretinin may have a specific role as a calcium-binding protein since the cochlear maturation in the rat inner ear.

Inmunoglobulinas en pacientes con actinomicetoma por Nocardia brasiliensis / Immunoglobulins in patients with Nocardia brasiliensis actinomycetoma

Considerando que algunos autores han reportado un aumento en la cantidad de algunas inmunoglobulinas en los pacientes con actinomicetoma, en este trabajo nos propusimos determinar diferencias en la producción de IgG1, IgG2, IgG3, IgG4 e IgM en 25 pacientes con actinomicetoma por Nocardia brasiliensis y 25 personas sanas provenientes de una zona endémica de micetoma. La determinación de inmunoglobulinas se realizó por medio de la técnica de ELISA. Para sensibilizar las placas se emplearon 6 antígenos de N. brasiliensis: un antígeno crudo denominado NB y cinco derivados del mismo (NB2, NB4, NB6, NB8 y NB10) separados por punto isoeléctrico. Los niveles de las cuatro subclases de IgG fueron mayores en los sueros de los pacientes que en el suero de los controles, con una diferencia máxima en IgG3 e IgG4; para esta última subclase, los seis antígenos fueron altamente reactivos. La concentración de IgM fue igual en ambos grupos. Es probable que como ocurre en otras infecciones, en la fisiopatogenia del actinomicetoma influya no sólo el aumento o deficiencia de una clase de inmunoglobulina, sino la relación que existe entre las diferentes subclases.

Considering that some authors have reported an increasing of some immunoglobulins in actinomycetoma patients, in this study we propose to determine differential production of IgG1, IgG2, IgG3, IgG4 and IgGM in 25 patients with actinomycetoma and 25 healthy individuals from a mycetoma endemic area. Immunoglobulins were determined by ELISA technique. To sensibilize the plates, six Nocardia brasiliensis antigens were used: a crude antigen denominated NB and five derivatives (NB2, NB4, NB6, NB8 and NB10) obtained by their isoelectric point. Results showed that all IgG subclasses were higher in the patients’ sera than in control sera, with a maximal difference to IgG3 and IgG4. To the latter subclass, six antigens were highly reactives. IgM levels were similar in both groups. As it occurs in other infections, in the actinomycetoma pathogenesis probably participate the increase or deficiency of a determined immunoglobulin class, as well as the relationship between different subclasses.

Isolation, purification, and characterization of L-glutamate oxidase from Streptomyces sp. 18G

An extracellular L-glutamate oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G. The enzyme had a molecular weight of approximately 120,000 and consisted of two identical subunits, each with a molecular weight of 61,000. The isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.0-7.4. GLOD showed the maximum activity at 37ºC. The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among 21 amino acids tested for substrate specificity, L-glutamate was almost exclusively oxidized. D-glutamate and L-aspartate were oxidized but only to extents of 0.79 percent and 0.53 percent, respectively.

Prevalence of OXA-23 Extended-Spectrum beta-Lactamase-Producing Clinical Isolates of Acinetobacter baumannii in a University Hospital, Busan, Korea / 대한임상미생물학회지

BACKGROUND: Acinetobacter baumannii is a glucose-nonfermenting gram-negative rod and is a well-recognized nosocomial pathogen. In recent years, A. baumannii strains showing resistance to carbapenems by producing metallo-beta-lactamases or OXA-type beta-lactamases have increased, and it is considered to be a serious clinical problem. But genotypes of carbapenemases produced by A. baumannii isolates in Korea have been rarely reported. The purpose of this study was to investigate the prevalence of imipenem-resistant A. baumannii and to determine the mechanism of resistance. METHODS: During the period of January through September, 2003, susceptibilities to imipenem of A. baumannii isolates from patients admitted in Kosin University Gospel Hospital in Busan, Korea were investigated. The modified Hodge and EDTA-disk synergy tests were performed for screening of carbapenemase and metallo-beta-lactamase-production. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. For detection of IMP, VIM and OXA-type beta-lactamases genes, polymerase chain reactions (PCR) were performed, and the DNA sequences of OXA-type beta-lactamases genes were determined by using the dideoxy-chain termination method. The isoelectric points of beta-lactamases were determined by isoelectric focusing. Pulsed-field gel electrophresis (PFGE) of the SmaI-digested genomic DNA was performed. RESULTS: A total of 193 strains of A. baumannii were collected from patients during the surveillance period. Twenty-seven percents (52/193) of A. baumannii isolates were resistant to imipenem. Among the 52 imipenem-resistant isolates, 41 isolates (78.8%) showed positive results in the modified Hodge test, but none of the isolates showed positive results in the EDTA-disk synergy test. Thirty-eight modified Hodge test-positive isolates harbored blaOXA-23 gene, but none of the isolates harbored IMP- or VIM-type metallo-beta-lactamases genes. Analytical isoelectric focusing revealed that all the 38 isolates had a nitrocefin-positive band at pI of 6.65. Thirty-five OXA-23-producing isolatesshowed a similar PFGE pattern when digested by SmaI endonuclease. CONCLUSION: Thirty-eight clinical isolates of A. baumannii acquired resistance to imipenem by producing OXA-23 beta-lactamase. Among them were 35 isolates thought to be originated from the same source, because they contained a similar chromosomal type. To the best of our knowledge, this is the first time that OXA-23 beta-lactamase has been detected in Korea.

Prevalence of PER-1 Extended-Spectrum beta-Lactamase-Producing Clinical Isolates of Acinetobacter baumannii in a University Hospital, Busan, Korea / 대한임상미생물학회지

BACKGROUND: In recent years, Acinetobacter baumannii isolates acquired resistance to cefepime have increased significantly. The aim of this study was to survey the prevalence of PER-1 extendedspectrum beta -lactamase (ESBL)-producing A. baumannii isolates in a University Hospital, Busan, Korea. METHODS: Antimicrobial susceptibilities were tested by the disk diffusion method, and double disk synergy test was performed for screening of ESBL-production. MICs were determined by agar dilution method. The isoelectric points of beta -lactamases were determined by isoelectric focusing. Transferability of cefepime-resistance were tested by conjugation. blaPER-1 and blaPER-2 alleles were detected by PCR, and the DNA sequences of amplified products were determined by using the dideoxy-chain termination method. RESULTS: Among 51 clinical isolates of A. baumannii intermediate or resistant to cefepime, 10 isolates (19.6%) showed positive results in double disk synergy test. PCR-based experiments detected blaPER-1 gene in all the 10 isolates. All the isolates contained three beta -lactamase bands: pI 5.3, 7.9, and 9.4. MICs of ampicillin, piperacillin, cephalothin, cefoxitin, cefoperazone, ceftazidime, cefotaxime, cefepime, and aztreonam were >256 mg/L, respectively, and them of imipenem were 8-16 mg/L. CONCLUSION: The prevalence of PER-1-producing A. baumannii strains in Busan was less than that of in Seoul. But an outbreak of infection caused by this strain in an intensive care unit shows that spread of PER-1-producing A. baumannii strains can be anticipated in a near future. Prevention of hospital infection by these resistant microorganisms are needed.

The Proteomic Analysis of Extracellular Proteins from Culture Filtrates of Dermatophytes / 대한의진균학회지

BACKGROUND: Proteases from dermatophytes have an important role in pathogenicity of these fungi as they facilitate penetration and colonization in the keratin structures of the stratum corneum, nails, and hair. Since the 1960s, efforts have been made to isolate and purify these enzymes, shedding light on hydrolytic properties and other characteristics of these extracellular proteins. It is now well known that under certain conditions, various proteases are produced which possess the capacity to digest casein, collagen, elastin, bovine serum albumin, hair, and keratin, while specific nature of these enzymes still remains to be elucidated. OBJECTIVE: We aimed to draw a proteome map of extracellular proteins from common dermatophytes using 2-dimensional electrophoresis, thus verifying the nature and interspecies differences in composition of extracellular proteins. METHODS: Following strains of dermatophytes were isolated from patients who visited the dermatologic outpatient clinic of Severance hospital and subcultured for 2 weeks on Sabouraud's dextrose agar: 2 strains of Trichophyton rubrum, 2 T. mentagrophytes, and 2 Microsporum canis. For growth media, glucose-peptone broth was added to each strain and 10 ml of media was taken, filtered using a 0.4micrometer syringe filter and the protein content obtained was concentrated by an ultrafiltration device before electrophoresis on the culture day 0 and 10. Stained with silver nitrate, the gel was scanned and analysed. RESULTS: 7 spots have increased in intensity including a 16 kDa-spot with isoelectric point at 9.3 from the supernatants of M. canis culture media, 4 spots have increased including a 32 kDa-spot with isoelectric point at 6.7, from the supernatants of T. mentagrophytes, and 5 spots including a 10 kDa-spot with isoelectric point at 6.3 showed significant increase from the supernatants of T. rubrum. M. canis and T. mentagrophytes subspecies shared 2 spots that increased. CONCLUSION: We concluded dermatophyte fungi produce different proteins according to their subspecies, and that proteomics appears to be a useful tool for comparative analysis of dermatophyte extracellular proteins.