PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan / Diversidade genética baseada em PCR RFLP de genótipos de Plasmodium vivax no distrito de Mardan, Paquistão

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp 3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and [...].

O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3β, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3β, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp 3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3βgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3β de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados [...].

Increased expression levels of metalloprotease, tissue inhibitor of metalloprotease, metallothionein, and p63 in ectopic endometrium: an animal experimental study / Aumento nos níveis de expressão de metaloprotease, inibidor tecidual das metaloproteases, metalotioneina e p63 em endométrio ectópico: um estudo experimental animal

Abstract Objective To characterize the patterns of cell differentiation, proliferation, and tissue invasion in eutopic and ectopic endometrium of rabbits with induced endometriotic lesions via a well- known experimental model, 4 and 8 weeks after the endometrial implantation procedure. Methods Twenty-nine female New Zealand rabbits underwent laparotomy for endometriosis induction through the resection of one uterine horn, isolation of the endometrium, and fixation of tissue segment to the pelvic peritoneum. Two groups of animals (one with 14 animals, and the other with15) were sacrificed 4 and 8 weeks after endometriosis induction. The lesion was excised along with the opposite uterine horn for endometrial gland and stroma determination. Immunohistochemical reactions were performed in eutopic and ectopic endometrial tissues for analysis of the following markers: metalloprotease (MMP-9) and tissue inhibitor of metalloprotease (TIMP-2), which are involved in the invasive capacity of the endometrial tissue; and metallothionein (MT) and p63, which are involved in cell differentiation and proliferation. Results The intensity of the immunostaining for MMP9, TIMP-2, MT, and p63 was higher in ectopic endometria than in eutopic endometria. However, when the ectopic lesions were compared at 4 and 8 weeks, no significant difference was observed, with the exception of the marker p63, which was more evident after 8 weeks of evolution of the ectopic endometrial tissue. Conclusion Ectopic endometrial lesions seem to express greater power for cell differentiation and tissue invasion, compared with eutopic endometria, demonstrating a potentially invasive, progressive, and heterogeneous presentation of endometriosis.

Resumo Objetivo Caracterizar o padrão de diferenciação celular, proliferação e invasão tecidual em endométrio eutópico e ectópico de coelhas com lesões de endometriose induzidas por um modelo experimental 4 e 8 semanas após o procedimento de implantação endometrial. Métodos Vinte e nove coelhas fêmeas Nova Zelândia foram submetidas a laparotomia para indução de endometriose através da ressecção de um dos cornos uterinos, isolamento do endométrio e fixação do tecido no peritônio pélvico. Dois grupos de animais (14 animais em um grupo e 15 animais no outro) foram sacrificados 4 e 8 semanas após a indução da endometriose. A lesão foi excisada junto com o corno uterino contralateral para determinação da presença de glândulas e de estroma endometrial. Reações de imunohistoquímica foram realizadas no tecido endometrial eutópico e ectópico para análise dos seguintes marcadores: metaloprotease (MMP9) e inibidor tecidual da metaloprotease 2 (TIMP-2), os quais estão envolvidos na capacidade de invasão do tecido endometrial; e metalotioneina (MT) e p63, os quais estão envolvidos na diferenciação e proliferação celular. Resultados A intensidade da imunomarcação para MMP9, TIMP-2, MT e p63 foi mais alta nos endométrios ectópicos do que nos endométrios eutópicos. Contudo, quando as lesões foram comparadas entre 4 e 8 semanas, nenhuma diferença foi observada, com exceção do marcador p63, o qual foi mais evidente depois de 8 semanas de evolução do tecido endometrial ectópico. Conclusão Lesões endometriais ectópicas parecem expressar maior poder de diferenciação celular e de invasão tecidual comparadas com endométrios eutópicos, demonstrando o potencial de invasão, de progressão e de apresentação heterogênea da endometriose.

Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon)

BACKGROUND: The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. OBJECTIVE: The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. METHODS: OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. RESULTS: OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. CONCLUSIONS: Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

Hypoxia Increases Epithelial Permeability in Human Nasal Epithelia

PURPOSE: The nasal mucosa is the first site to encounter pathogens, and it forms continuous barriers to various stimuli. This barrier function is very important in the innate defense mechanism. Additionally, inflammation of the nasal sinus is known to be a hypoxic condition. Here, we studied the effect of hypoxia on barrier function in normal human nasal epithelial (NHNE) cells. MATERIALS AND METHODS: The expression levels of various junction complex proteins were assessed in hypoxia-stimulated NHNE cells and human nasal mucosal tissues. We performed real-time polymerase chain reaction analysis, western blotting, and immunofluorescence assays to examine differences in the mRNA and protein expression of ZO-1, a tight junction protein, and E-cadherin in NHNE cells. Moreover, we evaluated the trans-epithelial resistance (TER) of NHNE cells under hypoxic conditions to check for changes in permeability. The expression of ZO-1 and E-cadherin was measured in human nasal mucosa samples by western blotting. RESULTS: Hypoxia time-dependently decreased the expression of ZO-1 and E-cadherin at the gene and protein levels. In addition, hypoxia decreased the TER of NHNE cells, which indicates increased permeability. Human nasal mucosa samples, which are supposed to be hypoxic, showed significantly decreased levels of ZO-1 and E-cadherin expression compared with control. CONCLUSION: Our results demonstrate that hypoxia altered the expression of junction complex molecules and increased epithelial permeability in human nasal epithelia. This suggests that hypoxia causes barrier dysfunction. Furthermore, it may be associated with innate immune dysfunction after encountering pathogens.

The role of intratumoral lymphovascular density in distinguishing primary from secondary mucinous ovarian tumors

OBJECTIVE: Ovarian mucinous metastases commonly present as the first sign of the disease and are capable of simulating primary tumors. Our aim was to investigate the role of intratumoral lymphatic vascular density together with other surgical-pathological features in distinguishing primary from secondary mucinous ovarian tumors. METHODS: A total of 124 cases of mucinous tumors in the ovary (63 primary and 61 metastatic) were compared according to their clinicopathological features and immunohistochemical profiles. The intratumoral lymphatic vascular density was quantified by counting the number of vessels stained by the D2-40 antibody. RESULTS: Metastases occurred in older patients and were associated with a higher proportion of tumors smaller than 10.0 cm; bilaterality; extensive necrosis; extraovarian extension; increased expression of cytokeratin 20, CDX2, CA19.9 and MUC2; and decreased expression of cytokeratin 7, CA125 and MUC5AC. The lymphatic vascular density was increased among primary tumors. However, after multivariate analysis, the best predictors of a secondary tumor were a size of 10.0 cm or less, bilaterality and cytokeratin 7 negativity. Lack of MUC2 expression was an important factor excluding metastasis. CONCLUSIONS: The higher intratumoral lymphatic vascular density in primary tumors when compared with secondary lesions suggests differences in the microenvironment. However, considering the differential diagnosis, the best discriminator of a secondary tumor is the combination of tumor size, laterality and the pattern of expression of cytokeratin 7 and MUC2. .

Expressão gênica de moléculas da matriz extracelular e da membrana celular durante a diferenciação de células-tronco adultas da polpa dentária humana / Gene expression of extracellular matrix and cell membrane molecules during cellular differentiation from human dental pulp stem cells

As células-tronco mesenquimais (MSCs) são células multipotentes que tem o potencial de se diferenciarem em várias linhagens celulares in vitro e in vivo. Estas são encontradas em nichos específicos em muitos órgãos e tecidos adultos, tais como medula óssea, tecido adiposo, músculo, dente, cordão umbilical, pele, cartilagem articular, sendo facilmente isoladas, expandidas e com alta capacidade proliferativa in vitro. Assim, estas características têm despertado grande interesse na sua utilização como uma potencial fonte de células para o reparo e regeneração tecidual de diversos órgãos e tecidos. Pouco se conhece sobre as moléculas que são secretadas pelas MSCs para a matriz extracelular (MEC) e que estão na interface célula-matriz e estão presentes em vias de transdução de sinais intracelulares. Desta forma, o objetivo deste trabalho foi avaliar o perfil de expressão gênica de enzimas que remodelam a MEC (metaloproteinases de matriz MMPs: 15 membros) e seus inibidores (inibidores teciduais das metaloproteinases de matriz TIMPs: 4 membros e RECK) e proteína da membrana plasmática (Caveolina-1) durante a diferenciação osteogenica in vitro a partir de células-tronco mesenquimais da polpa dentária humana (DPSCs). Para tanto, utilizamos polpas dentárias humanas provenientes de terceiros molares de indivíduos adultos (18-32 anos n=3) e as DPSCs isoladas foram imunofenotipadas por citometria de fluxo, avaliada a taxa de proliferação, induzidas as diferenciações osteogênica (1, 7, 14, 21 e 28 dias) e adipogênica (28 dias) e os transcritos avaliados por PCR em tempo real. Estas células foram positivas para o marcadores CD29, CD105, STRO-1, CD44, CD90 negativas os marcadores para CD31, CD45, CD34 e CD14 e são capazes de se diferenciarem em osteoblastos e adipócitos.

Verificamos que as MMP-2, MMP-3, MMP-13, MMP-14, MMP-25, TIMP-3, TIMP-4 e Caveolina-1 foram diferencialmente expressas durante a diferenciação osteogênica, sendo reguladas positivamente apenas no período de 28 dias pós indução e a TIMP-1 regulada positivamente desde o primeiro dia de indução. A MMP-11 e MMP-16 não foram detectadas nas DPSCs e nem durante a diferenciação osteogênica. Desta forma, concluímos que MMPs encontradas bem como a Caveolina-1 e as TIMP-3 e TIMP-4 podem estar participando dos dos eventos de diferenciação óssea em DPSCs, a TIMP-1 pode estar participando de eventos biológicos relacionados as propriedades do estado indiferenciado das DPSCs e da diferenciação óssea e que as MMP-11 e MMP-16 não são expressas pelas DPSCs e também não estão envolvidas na diferenciação osteogênica.

Mesenchymal stem cells (MSCs) are multipotent cells that have the potential to differentiate into various cell lineages in vitro and in vivo. These are found in specific niches in many adult organs and tissues, such as bone marrow, adipose tissue, muscle, tooth, umbilical cord, skin, cartilage, being easily isolated, expanded and high proliferative capacity in vitro. Thereby, these features have attracted great interest in its use as a potential source of cells for tissue repair and regeneration of various organs and tissues. Little is known about the molecules secreted by MSCs into the extracellular matrix (ECM), present at cell-matrix interface and present on intracellular signal transduction. Thus, the aim of this study was to evaluate gene expression profile of ECM remodeling enzymes (matrix metalloproteinases MMPs: 15 members) and their inhibitors (tissue inhibitors of matrix metalloproteinases TIMPs: 4 members and RECK) and plasma membrane proteins (Caveolin-1) that participate in signaling pathways during osteogenic differentiation in vitro from human dental pulp stem cells (DPSCs). Normal human impacted third molars were collected from adults (18-32 years-old n=3) and DPSCs isolated were immunophenotyping by flow cytometry, evaluated the proliferation ratio, induced to osteogenic (1, 7, 14, 21 and 28-days) and adipogenic differentiation (28-days) and the transcript levels evaluated by Real Time PCR.

These cells are positive for CD29, CD105, STRO -1, CD44, and CD90 markers and negative for CD31, CD45, CD34, and CD14 markers and are capable of differentiating into osteoblasts and adipocytes. We found that MMP- 2, MMP -3, MMP -13, MMP -14, MMP -25, TIMP-3, TIMP-4 and Caveolin-1 were differentially expressed during osteogenic differentiation, being upregulated only at 28 days post-induction and TIMP-1 upregulated from the first day of induction. MMP-11 and MMP-16 were not detected in DPSCs neither during differentiation. Thus, we conclude that MMPs, Caveolin-1 found as well as TIMP-3 and TIMP-4 may be participating in the event of bone differentiation in DPSCs, TIMP-1 may participate in biological events related to the properties of the undifferentiated state DPSCs and osteogenic differentiation, MMP-11 and MMP-16 are not also expressed by DPSCs and are not involved in osteogenic differentiation.

Benefits of a Continuous Ambulatory Peritoneal Dialysis (CAPD) Technique with One Icodextrin-Containing and Two Biocompatible Glucose-Containing Dialysates for Preservation of Residual Renal Function and Biocompatibility in Incident CAPD Patients

In a prospective randomized controlled study, the efficacy and safety of a continuous ambulatory peritoneal dialysis (CAPD) technique has been evaluated using one icodextrin-containing and two glucose-containing dialysates a day. Eighty incident CAPD patients were randomized to two groups; GLU group continuously using four glucose-containing dialysates (n=39) and ICO group using one icodextrin-containing and two glucose-containing dialysates (n=41). Variables related to residual renal function (RRF), metabolic and fluid control, dialysis adequacy, and dialysate effluent cancer antigen 125 (CA125) and interleukin 6 (IL-6) levels were measured. The GLU group showed a significant decrease in mean renal urea and creatinine clearance (-Delta1.2+/-2.9 mL/min/1.73 m2, P=0.027) and urine volume (-Delta363.6+/-543.0 mL/day, P=0.001) during 12 months, but the ICO group did not (-Delta0.5+/-2.7 mL/min/1.73 m2, P=0.266; -Delta108.6+/-543.3 mL/day, P=0.246). Peritoneal glucose absorption and dialysate calorie load were significantly lower in the ICO group than the GLU group. The dialysate CA125 and IL-6 levels were significantly higher in the ICO group than the GLU group. Dialysis adequacy, beta2-microglobulin clearance and blood pressure did not differ between the two groups. The CAPD technique using one icodextrin-containing and two glucose-containing dialysates tends to better preserve RRF and is more biocompatible, with similar dialysis adequacy compared to that using four glucose-containing dialysates in incident CAPD patients. [Clincal Trial Registry, ISRCTN23727549]

Benefits of a Continuous Ambulatory Peritoneal Dialysis (CAPD) Technique with One Icodextrin-Containing and Two Biocompatible Glucose-Containing Dialysates for Preservation of Residual Renal Function and Biocompatibility in Incident CAPD Patients

In a prospective randomized controlled study, the efficacy and safety of a continuous ambulatory peritoneal dialysis (CAPD) technique has been evaluated using one icodextrin-containing and two glucose-containing dialysates a day. Eighty incident CAPD patients were randomized to two groups; GLU group continuously using four glucose-containing dialysates (n=39) and ICO group using one icodextrin-containing and two glucose-containing dialysates (n=41). Variables related to residual renal function (RRF), metabolic and fluid control, dialysis adequacy, and dialysate effluent cancer antigen 125 (CA125) and interleukin 6 (IL-6) levels were measured. The GLU group showed a significant decrease in mean renal urea and creatinine clearance (-Delta1.2+/-2.9 mL/min/1.73 m2, P=0.027) and urine volume (-Delta363.6+/-543.0 mL/day, P=0.001) during 12 months, but the ICO group did not (-Delta0.5+/-2.7 mL/min/1.73 m2, P=0.266; -Delta108.6+/-543.3 mL/day, P=0.246). Peritoneal glucose absorption and dialysate calorie load were significantly lower in the ICO group than the GLU group. The dialysate CA125 and IL-6 levels were significantly higher in the ICO group than the GLU group. Dialysis adequacy, beta2-microglobulin clearance and blood pressure did not differ between the two groups. The CAPD technique using one icodextrin-containing and two glucose-containing dialysates tends to better preserve RRF and is more biocompatible, with similar dialysis adequacy compared to that using four glucose-containing dialysates in incident CAPD patients. [Clincal Trial Registry, ISRCTN23727549]

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy.

CONTEXT: Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders. AIM: To study the utility of MLPA in diagnosis and carrier detection for DMD. MATERIALS AND METHODS: Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared. RESULTS AND CONCLUSIONS: We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.

Dysferlinopathy: Spectrum of pathological changes in skeletal muscle tissue.

Background: Dysferlinopathy is an autosomal recessive-limb girdle muscular dystrophy (AR-LGMD) caused due to the defect in gene encoding dysferlin, a sarcolemmal protein. Awareness of the variants and their relative frequency is essential for accurate diagnosis. Aim: To study the spectrum of morphologic changes in immunohistochemically proven cases of dysferlinopathies, to correlate the findings with clinical phenotype and durations of illness and determine the frequency. Materials and Methods: Dysferlinopathies seen over a period of 2 years at a tertiary neurological center were analyzed. Results: Clinically, majority had Miyoshi phenotype (46.6%) with distal involvement and LGMD phenotype (40%) with proximal muscle involvement. In addition, a proximo-distal and tibial muscle phenotype was encountered. Morphologically, rimmed vacuoles were noted in the Miyoshi phenotype. The presence of ragged red fibers, lobulated fibers and inflammation had no preference to a particular phenotype. Significant atrophy and lobulated fibers were noted in patients with longer duration of illness. Conclusions: Dysferlinopathy was the second most common identifiable cause (21%) of LGMD next to sarcoglycanopathies (27%).

Utility of p63 immunohistochemical stain in differentiating urothelial carcinomas from adenocarcinomas of prostate.

Background: Prostatic adenocarcinoma and urothelial carcinoma of the urinary bladder are common cancers in men. High grade forms of these tumors may present ambiguous morphologic features that do not permit a definite diagnosis. This distinction between the two tumors has significant staging and therapeutic implications. Hence, an accurate diagnosis is essential for optimal patient care. p63 is a new marker which can be used in this context. It is expressed in most of the urothelial carcinomas and negative in majority of prostatic adenocarcinomas. Aim: To compare the expression of p63 in urothelial carcinomas and adenocarcinomas of prostate. Materials and Methods: Comparative cross--sectional study was carried out at a tertiary cancer hospital from 15 June 2006 to 15 December 2006. Immunohistochemical stain p63 was performed on 50 cases of urothelial carcinoma and 50 prostatic adenocarcinomas. Patients' name, age, histology numbers, grade of tumor, and expression of p63 were recorded. p63 expression was seen in 44 of 50 urothelial carcinomas (88%). None of the prostatic adenocarcinomas expressed p63. The ages of patients with prostatic adenocarcinoma ranged from 49 to 86 years with a median age of 71 years and 41 to 83 years for urothelial carcinomas with a median age of 60.5 years. Conclusion: p63 can be used as a reliable marker to distinguish prostatic adenocarcinomas from urothelial carcinomas in difficult cases in conjunction with other markers like PSA.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots / PtSRR1, um possível receptor simbiose-regulado de Pisolithus tinctorius é expresso nas primeiras horas de interação ectomicorrízica com raízes de Castanea sativa

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1 foi isolado preliminarmente de P. tinctorius nas primeiras horas da interação com raízes de C. sativa. Análises de QRT-PCR confirmaram sua expressão positiva (12 h) e seu peptídeo putativo indicou forte possibilidade para proteína transmembranar. Estes dados estimulam o estudo do PtSRR1 durante a formação de ectomicorrizas.

Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells.

BACKGROUND & OBJECTIVE: The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. METHODS: Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12). RESULTS: The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions. INTERPRETATION & CONCLUSION: The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.

Claudin-7 is Highly Expressed in Chromophobe Renal Cell Carcinoma and Renal Oncocytoma

Claudin-7 has recently been suggested to be a distal nephron marker. We tested the possibility that expression of claudin-7 could be used as a marker of renal tumors originating from the distal nephron. We examined the immunohistochemical expression of claudin-7 and parvalbumin in 239 renal tumors, including 179 clear cell renal cell carcinoma (RCC)s, 29 papillary RCCs, 20 chromophobe RCCs, and 11 renal oncocytomas. In addition, the methylation specific-PCR (MSP) of claudin-7 was performed. Claudin-7 and parvalbumin immunostains were positive in 3.4%, 7.8% of clear cell RCCs, 34.5%, 31.0% of papillary RCCs, 95.0%, 80.0% of chromophobe RCCs, and 72.7%, 81.8% of renal oncocytomas, respectively. The sensitivity and specificity of claudin-7 in diagnosing chromophobe RCC among subtypes of RCC were 95.0% and 92.3%. Those of parvalbumin were 80.0% and 88.9%. The expression pattern of claudin-7 was mostly diffuse in chromophobe RCC and was either focal or diffuse in oncocytoma. All of the cases examined in the MSP revealed the presence of unmethylated promoter of claudin-7 without regard to claudin-7 immunoreactivity. Hypermethylation of the promoter might not be the underlying mechanism for loss of its expression in RCC. Claudin-7 can be used as a useful diagnostic marker in diagnosing chromophobe RCC and oncocytoma.

Proteomic analysis of the shistosome tegument and its surface membranes

The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS), and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

Distrofia muscular de Emery-Dreifuss: relato de caso / Emery-Dreifuss muscular dystrophy: case report

A distrofia muscular de Emery-Dreifuss é uma forma de distrofia muscular freqüentemente associada a contraturas articulares e defeitos de condução cardíaca, que pode ser causada pela deficiência da proteína emerina na membrana nuclear interna das fibras musculares. Descrevemos o caso de um homem de 19 anos com diminuição de força muscular, hipotrofia nas cinturas escapular e pélvica, disfagia, contraturas articulares em cotovelos e tornozelos, apresentando história familiar compatível com herança ligada ao cromossomo X. A investigação mostrou creatinaquinase sérica elevada, eletrocardiograma com bloqueio atrioventricular de primeiro grau e bloqueio de ramo direito, eletroneuromiografia normal, biópsia muscular com alterações miopáticas e a análise por imuno-histoquímica mostrou deficiência de emerina. São discutidas as manifestações clínicas e genéticas, alterações laboratoriais e eletroneuromiográficas, bem como, a importância do estudo do padrão de herança no aconselhamento genético destas famílias.

Giant Vascular Eccrine Spiradenoma: Report of a Case with Immunohistochemical Study

We report a rare case of giant vascular eccrine spiradenoma (GVES) which developed in 56-yr-old Korean woman. It is a rare variant of eccrine spiradenoma (ES), which might be mistaken for angiomatous lesions in view of its florid vascularity and hemorrhagic features. Histogenesis of GVES is not clearly elucidated although it is known that ES presumably originates in the eccrine glands. To clarify the histogenesis of GVES, immunohistochemical stainings using various monoclonal antibodies were also performed. The tumor was composed of three types of cells, namely pale epithelial cells, small basal cells, and myoepithelial cells. Therefore, we conclude that GVES originated from eccrine gland and mainly differentiates toward secretory portion of secretory coil.

Cell type-specific upregulation of myristoylated alanine-rich C kinase substrate and protein kinase C-alpha, -beta I, -beta II, and -delta in microglia following kainic acid-induced seizures

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed protein kinase C (PKC) substrate and has been implicated in actin cytoskeletal rearrangement in response to extracellular stimuli. Although MARCKS was extensively examined in various cell culture systems, the physiological function of MARCKS in the central nervous system has not been clearly understood. We investigated alterations of cellular distribution and phosphorylation of MARCKS in the hippocampus following kainic acid (KA)-induced seizures. KA (25 mg/kg, i.p.) was administered to eight to nine week-old C57BL/6 mice. Behavioral seizure activity was observed for 2 h after the onset of seizures and was terminated with diazepam (8 mg/kg, i.p.). The animals were sacrificed and analyzed at various points in time after the initiation of seizure activity. Using double-labeling immunofluorescence analysis, we demonstrated that the expression and phosphorylation of MARCKS was dramatically upregulated specifically in microglial cells after KA-induced seizures, but not in other types of glial cells. PKC alpha, beta I, beta II and delta, from various PKC isoforms examined, also were markedly upregulated, specifically in microglial cells. Moreover, immunoreactivities of phosphorylated MARCKS were co-localized in the activated microglia with those of the above isoforms of PKC. Taken together, our in vivo data suggest that MARCKS is closely linked to microglial activation processes, which are important in pathological conditions, such as neuroinflammation and neurodegeneration.