Candida dubliniensis does not show phospholipase activity: true or false?

INTRODUCTION: The phospholipase activity in Candida albicans and Candida dubliniensis isolated from oral candidiasis cases were studied. METHODS: The phospholipase activity was evaluated in egg yolk agar. RESULTS: All the C. albicans isolates (n = 48) showed phospholipase activity (mean Pz = 0.66). However, none of the C. dubliniensis isolates (n = 24) showed this activity. CONCLUSIONS: The authors discuss whether these findings are a true characteristic of C. dubliniensis or a consequence of the methodology employed, which includes the possibility that NaCl may have inhibited the enzymatic activity of C. dubliniensis.

Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs) and extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

Phospholipase gene expression during Paracoccidioides brasiliensis morphological transition and infection

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

Influência da linfadenectomia radical (D2) na morbidade e mortalidade da ressecção curativa do carcinoma gástrico

A despeito da controvérsia existente na literatura com relação aos benefícios da linfadenectomia na sobrevida dos doentes submetidos a ressecções curativas para tratamento do adenocarcinoma gástrico, é inegável que a linfadenectomia ampliada (nível II na classificação japonesa) contribui para o melhor estadiamento e prognóstico destes pacientes. Este procedimento permite-nos melhor identificar aqueles pacientes que têm pior prognóstico e oferecer-lhes novas formas de terapia adjuvante. Como o principal argumento para a não realização de cirurgias mais alargadas é que estas são acompanhadas de maior morbidade e mortalidade, os autores estudaram prospectivamente parâmetros relacionados a esses índices nas gastrectomias com linfadenectomia nível 11 (D2) que tiveram intenção curativa. Para tanto, estudaram-se a taxa de mortalidade, o tempo operatório, as unidades de glóbulos transfundidas, as complicações e o tempo de internação pós-operatória. Entre dezembro de 1992 e fevereiro de 1997 foram internados 86 pacientes com diagnóstico de adenocarcinoma gástrico, dos quais, em 27, atendidos por uma mesma equipe interessada no tratamento destes tumores, houve ressecção cirúrgica com intenção curativa e o tratamento consistiu de gastrectomia acompanhada de linfadenectomia D2. A gastrectomia subtotal foi realizada em 17 doentes, a total em três e a total ampliada em sete. Nove doentes tinham tumores superficiais. Não houve mortalidade entre os pacientes submetidos a ressecções D2; o tempo médio operatório foi de 208,7 minutos; receberam em média 0,2 unidades de glóbulos e a incidência de complicações foi de 33,3%. A permanência hospitalar pós-operatória média foi de 8,6 dias. Foram estudados 854 linfonodos, dos quais 22,1% eram positivos para tumor metastático. Os autores concluem que a dissecção D2 pode ser feita de forma segura e não deve ser evitada por causa do risco de complicações. Permite estadiamento anatomopatológico mais preciso e melhor avaliação do prognóstico destes pacientes.

Linfadenectomia ampliada (D2) no tratamento do carcinoma gástrico: análise das complicações pós-operatórias

OBJETIVO: Descrever e analisar as principais complicações pós-operatórias e mortalidade dos pacientes submetidos à ressecção gástrica por câncer gástrico com linfadenectomia D2. MÉTODO: Foi realizada uma coorte histórica onde as principais variáveis em estudo foram: idade, localização do tumor, estadiamento, complicações do procedimento cirúrgico, padrão de recidiva tumoral, análise da sobrevida livre de doença e sobrevida total. RESULTADOS: Foram avaliados 35 pacientes submetidos à dissecção linfonodal D2 no período de Janeiro de 2000 a Dezembro de 2004. A média de idade foi 57 anos. Apenas um (2,9%) paciente apresentava tumor precoce e o local mais comum do tumor foi no terço médio do estômago. O número de linfonodos ressecados por paciente variou de 15 a 80 linfonodos (média 28,8). Vinte e seis (74,3%) pacientes apresentaram linfonodos metastáticos, sendo a média de 13,4 (±11,8) linfonodos comprometidos por paciente. Seis (17,1%) pacientes apresentaram complicações no período pós-operatório, sendo duas pneumonias, uma fístula pancreática, uma fístula do coto duodenal e duas deiscências da anastomose esôfago-jejunal. Apenas um (2,86%) paciente morreu devido a complicações operatórias. O tempo de seguimento médio foi de 26 meses. Vinte e dois pacientes apresentavam-se vivos no fechamento do estudo, com uma sobrevida atuarial de 62,9%. CONCLUSÃO: Os resultados deste estudo sugerem que, em centros especializados, a linfadenectomia D2 é um procedimento com nível de complicações aceitável e pode ser realizada sem aumento da mortalidade operatória.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Abundant immunohistochemical expression of dopamine D2 receptor and p53 protein in meningiomas: follow-up, relation to gender, age, tumor grade, and recurrence

Meningiomas are common, usually benign tumors, with a high postoperative recurrence rate. However, the genesis and development of these tumors remain controversial. We aimed to investigate the presence and implications of a mutated p53 protein and dopamine D2 receptor in a representative series of meningiomas and to correlate these findings with age, gender, tumor grade, and recurrence. Tumor tissue samples of 157 patients diagnosed with meningioma (37 males and 120 females, mean age 53.6±14.3 years) who underwent surgical resection between 2003 and 2012 at our institution were immunohistochemically evaluated for the presence of p53 protein and dopamine D2 receptor and were followed-up to analyze tumor recurrence or regrowth. Tumors were classified as grades I (n=141, 89.8%), II (n=13, 8.3%), or grade III (n=3, 1.9%). Dopamine D2 receptor and p53 protein expression were positive in 93.6% and 49.7% of the cases, respectively. Neither of the markers showed significant expression differences among different tumor grades or recurrence or regrowth statuses. Our findings highlight the potential role of p53 protein in meningioma development and/or progression. The high positivity of dopamine D2 receptor observed in this study warrants further investigation of the therapeutic potential of dopamine agonists in the evolution of meningiomas.

Características morfoagronômicas do milho submetido a diferentes níveis de desfolha manual

O presente trabalho foi realizado com o objetivo de avaliar os danos causados pela desfolha na cultura do milho. O experimento foi desenvolvido no ano agrícola 2005/2006, na Fazenda Roma, localizada no município de Ituverava, Estado de São Paulo. Para a sua concretização utilizou-se o híbrido triplo CODETEC 304®. O experimento foi composto de parcelas subdivididas, que foram distribuídas em três blocos de forma casualizada, apresentando como tratamento principal a desfolha manual (30, 40, 60 e 80% de desfolha) e como secundário as fases fenológicas da cultura D1 (sete folhas completamente desenvolvidas), D2 (quando as plantas apresentam-se na fase de pendoamento) e D3 (quando as plantas apresentam-se na fase de enchimento de grãos-grãos leitosos) com três repetições para cada tratamento e uma parcela controle (0% de desfolha) para cada bloco. Foram avaliados a produção de grãos das parcelas (g/m²), o tamanho médio das espigas despalhadas (cm) e o peso médio de 1.000 grãos (g). Para o presente estudo, concluiu-se que as relações fonte-dreno foram afetadas negativamente em que a desfolha de 80% foi a que mais afetou todas variáveis, portanto é um nível de desfolha responsável pelas maiores perdas, não havendo meios de compensação para a cultura do milho.

Coral snake venoms: mode of action and pathophysiology of experimental envenomation

Coral snakes, the New World Elapidae, are included in the genera Micniroides and Micrurus. The genus Mlcrurus comprises nearly all coral snake species and those which are responsible for human snake-bite accidents. The following generalizations concerning the effects induced by their venoms, and their venom-properties can be made. Coral snake venoms are neurotoxic, producing loss of muscle strenght and death by respiratory paralysis. Local edema and necrosis are not induced nor blood coagulation or hemorrhages. Proteolysis activity is absent or of very low grade. They display phospholipase A2 activity. Nephrotoxic effects are not evoked. The main toxins from elapid venoms are postsynaptic and presynaptic neurotoxins and cardiotoxins. Phospholipases A2 endowed with myonecrotic or cardiotoxin-like properties are important toxic components from some elapid venoms. The mode of action of Micrurus frontalis, M. lemniscatus, M. corallinus and M. fulvius venoms has been investigated in isolated muscle preparations and is here discussed. It is shown that while M. frontalis and M. lemniscatus venoms must contain only neurotoxins that act at the cholinergic end-plate receptor (postsynaptic neurotoxins), M. corallinus venom also inhibits evoked acetylcholine release by the motor nerve endings (presynaptic neurotoxin-like effect) and M. fulvius induces muscle fiber membrane depolarization (cardiotoxin-like effect). The effects produced by M. corallinus and M. fulvius venoms in vivo in dogs and M. frontalis venom in dogs and monkeys are also reported.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Candiduria in a public hospital of São Paulo (1999-2004): characteristics of the yeast isolates

The study involved 100 yeast isolates, obtained from urine samples provided by a Public Pediatric Hospital of São Paulo, Brazil, from 1999 to 2004. The most frequent species was Candida albicans, followed by C. tropicalis, C. glabrata and C. parapsilosis. In regard to virulence, 97% of the isolates showed index 3 for proteinase and 63% index 2 for phospholipase. The most frequent killer biotypes were 511 and 888.

Secretion of five extracellular enzymes by strains of chromoblastomycosis agents

The gelatinase, urease, lipase, phospholipase and DNase activities of 11 chromoblastomycosis agents constituted by strains of Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana and Exophiala jeanselmei were analyzed and compared. All strains presented urease, gelatinase and lipase activity. Phospholipase activity was detected only on five of six strains of F. pedrosoi. DNase activity was not detected on the strains studied. Our results indicate that only phospholipase production, induced by egg yolk substrate, was useful for the differentiation of the taxonomically related species studied, based on their enzymatic profile.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1% and 55.9% of Candida albicans and 69.8% and 37.7% of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.