Vesicular Trafficking in Osteoclasts

Osteoclasts are multinucleated bone-degrading cells that undergo large changes in their polarisation and vesicular trafficking during the bone resorption cycle. Rab proteins are small GTPases that offer both temporal and spatial regulation to the transport between membranous organelles. Previously the presence and function of only few of the currently known 60 Rab proteins in osteoclasts have been reported. In this study, the expression of 26 Rab genes in bone-resorbing osteoclasts was demonstrated with gene-specific primer pairs. The further analysis of three Rab genes during human osteoclast differentiation revealed that Rab13 gene is highly induced during osteoclastogenesis. The presence of Rab13 protein in the secretory vesicles directed towards the ruffled border and in the endocytotic or transcytotic pathways in resorbing osteoclasts was excluded. The localisation of Rab13 suggests that that it is associated with a previously unknown vesicle population travelling between the trans-Golgi network and the basolateral membrane in bone resorbing osteoclasts. Rab proteins convey their functions by binding to specific effector proteins. We found a novel Rab13 interaction with endospanins-1 and -2 that are yet poorly characterised small transmembrane proteins. The Rab13 subfamily member Rab8 also bound to endospanins, while Rab10 and unrelated Rabs did not. Rab13 and endospanin-2 co-localised in perinuclear vesicles in transfected cells, demonstrating the interaction also in vivo. The inhibition of Rab13 did not interfere with the localisation of endospanin-2 nor did it affect the cell surface expression of growth hormone receptor, as has been previously described for endospanins. The physiological role of this novel protein-protein interaction thus remains to be clarified. The analysis of the transcytotic route in bone resorbing osteoclasts revealed that multiple vesicle populations arise from the ruffled border and transport the bone degradation products for exocytosis. These vesicles are directed to the functional secretory domain that is encircled by an actin-based molecular barrier. Furthermore, the transcytotic vesicles contain abundant Helix pomatia lectin binding sites and represent lipid raft concentrates. Finally, autophagosomal compartments may also be involved in the transcytosis.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Two variants of HIV-1 B serotype are transmitted heterosexually in São Paulo, Brazil

HIV-1 variability may have an important impact on transmission and pathogenicity. Better characterization of the HIV epidemic in Brazil is necessary for the development of vaccine trials in this country. We analyzed sera from 108 HIV-1-infected volunteers from São Paulo City to determine serotype and reactivity for V3 motifs of HIV in this population, and the relationship to transmission mode. We concluded that the HIV-1 B serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major V3 motifs, GPGR and GWGR, had similar prevalence among women (48% and 52%, respectively) and men (56% and 44%, respectively). We also observed an equal distribution of these strains regardless of their CD4+ T cell counts, clinical status, and mode of transmission. Even though V3 serology for HIV-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct DNA sequencing, should be included to determine HIV-1 genetic diversity.

Decreased spermatogenic and androgenic testicular functions in adult rats submitted to immobilization-induced stress from prepuberty

We investigated whether chronic stress applied from prepuberty to full sexual maturity interferes with spermatogenic and androgenic testicular functions. Male Wistar rats (40 days old) were immobilized 6 h a day for 60 days. Following immobilization, plasma concentrations of corticosterone and prolactin increased 135% and 48%, respectively, while plasma luteinizing hormone and testosterone presented a significant decrease of 29% and 37%, respectively. Plasma concentration of follicle-stimulating hormone was not altered in stressed rats. Chronic stress reduced the amount of mature spermatids in the testis by 16% and the spermatozoon concentration in the cauda epididymidis by 32%. A 17% reduction in weight and a 42% decrease in DNA content were observed in the seminal vesicle of immobilized rats but not in its fructose content. The growth and secretory activity of the ventral prostate were not altered by chronic stress.

Analysis of p53 expression and proliferative assessment using PCNA in localized prostate carcinoma

The surgical specimens from 51 men submitted to radical prostatectomy for localized prostate cancer were examined by immunohistochemistry using proliferation cell nuclear antigen (PCNA) monoclonal antibody to evaluate the proliferative index (PI). The relationship between PI, biological variables and p53 protein expression was evaluated by immunohistochemistry. PI was low in invasive localized prostate carcinoma (mean, 12.4%) and the incidence of PCNA-positive cells was significantly higher in tumors with p53 expression (P = 0.0226). There was no statistical difference in PCNA values when biological parameters such as Gleason score, tumor volume, extraprostatic involvement, seminal vesicle infiltration or lymph node metastasis were considered. We conclude that proliferative activity is usually low in prostate carcinoma but is correlated with p53 immune staining, indicating that p53 is important in cell cycle control in this neoplasm.

Fertility of male adult rats submitted to forced swimming stress

We investigated whether stress interferes with fertility during adulthood. Male Wistar rats (weighing 220 g in the beginning of the experiment) were forced to swim for 3 min in water at 32ºC daily for 15 days. Stress was assessed by the hot-plate test after the last stressing session. To assess fertility, control and stressed males (N = 15 per group) were mated with sexually mature normal females. Males were sacrificed after copulation. Stress caused by forced swimming was demonstrated by a significant increase in the latency of the pain response in the hot-plate test (14.6 ± 1.25 s for control males vs 26.0 ± 1.53 s for stressed males, P = 0.0004). No changes were observed in body weight, testicular weight, seminal vesicle weight, ventral prostate weight or gross histological features of the testes of stressed males. Similarly, no changes were observed in fertility rate, measured by counting live fetuses in the uterus of normal females mated with control and stressed males; no dead or incompletely developed fetuses were observed in the uterus of either group. In contrast, there was a statistically significant decrease in spermatid production demonstrated by histometric evaluation (154.96 ± 5.41 vs 127.02 ± 3.95 spermatids per tubular section for control and stressed rats, respectively, P = 0.001). These data demonstrate that 15 days of forced swimming stress applied to adult male rats did not impair fertility, but significantly decreased spermatid production. This suggests that the effect of stress on fertility should not be assessed before at least the time required for one cycle of spermatogenesis.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Luteinizing hormone reduction by the male potency herb, Butea superba Roxb.

To determine if Butea superba Roxb., a traditional Thai male potency herb, has androgenic activity in 60-day-old male Wistar rats, we measured its effects on the pituitary-testicular axis and sex organs. Intact and orchidectomized adult male rats were subdivided into five groups (10 rats/group): distilled water, Butea superba (BS)-10, BS-50, BS-250, and testosterone propionate (TP). They received 0, 10, 50, and 250 mg·kg body weight-1·day-1 BS in distilled water by gavage and 6 mg·kg body weight-1·day-1 TP sc, respectively, during the 30-day treatment period. Blood was collected every 15 days and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone were measured. Changes of weight and histological appearance of sex organs were determined at the end of the 30-day treatment and 15-day post-treatment periods. TP treatment reduced serum FSH and LH levels and significantly increased the weight of the seminal vesicles and epididymis, in accordance with histopathological changes, in both intact and orchidectomized rats. No changes in serum testosterone, LH, and FSH levels were observed in any of the intact rats treated with BS, but a significant increase in seminal vesicle weight was observed only in the BS-250 group. Although a significant reduction in serum LH was detected in the BS-50 and BS-250 groups of orchidectomized rats, no significant change in weight or histology of sex organs was observed. Thus, we conclude that B. superba needs endogenous testosterone to work synergistically to stimulate the accessory sex organ of intact animals and can potentially exhibit an LH reduction effect in orchidectomized animals.

Anemia na doença renal crônica em hospital da região noroeste do estado do Rio Grande do Sul

Introdução: A doença renal crônica (DRC) tem sido identificada em um número crescente de pacientes e, dentre suas consequências, encontra-se a anemia. Objetivo: Verificar a ocorrência de anemia em pacientes com DRC que realizavam hemodiálise em um hospital da Região Sul, Brasil, bem como seu perfil laboratorial renal e de ferro. Métodos: Realizou-se estudo retrospectivo descritivo analítico. Foram analisados 45 prontuários com resultados de exames desde o início do tratamento hemodialítico até nove meses após. Resultados: Mais de 50,0% dos pacientes apresentava hipertensão arterial e diabetes e 68,8% era do gênero masculino. A anemia esteve presente em 97,8% dos pacientes e foi tratada com eritropoetina e/ou ferro. No período avaliado, ocorreu aumento nas medianas dos níveis de hemoglobina (p < 0,001), hematócrito (p < 0,001), ferritina, creatinina (p < 0,001) e ureia pré (p = 0,039). A saturação de transferrina estava baixa em 35,6% dos pacientes após aproximadamente um ano de tratamento hemodialítico. Houve correlação entre creatinina e ureia, ambas elevando-se. Conclusão: Após a Introdução de tratamento para anemia, ocorreu aumento dos níveis plasmáticos de hemoglobina e melhora do quadro, ainda que sem normalização completa desses níveis.

Repercussões vasculares do uso de CDL em pacientes hemodialíticos: análise ecográfica dos sítios de inserção

Introdução: A doença renal crônica é uma doença cuja prevalência tem aumentado no Brasil. A hemodiálise é sua principal modalidade terapêutica e tem, como via de acesso preferencial, a confecção de fístula arteriovenosa. Apesar disso, muitos pacientes necessitam do uso de cateteres duplo-lúmen, seja como acesso temporário ou permanente. Complicações vasculares relacionadas a este procedimento podem ocorrer, sendo o melhor método de avaliação não invasiva a análise ecográfica. Objetivo: Analisar as complicações pelo uso do cateter duplo-lúmen em pacientes com doença renal crônica, em hemodiálise, no Hospital Santa Casa de Misericórdia de Ponta Grossa, PR, por meio do eco-Doppler. Métodos: Estudo observacional, analítico, tipo caso-controle, com obtenção dos dados por TASY®, questionário, exame físico direcionado e exame de imagem (eco-Doppler). Resultados: Nenhuma das variáveis analisadas se mostrou significativa isoladamente como preditora de repercussão vascular no eco-Doppler, que detectou alterações em 31,25% dos casos. O exame físico se mostrou de péssima acurácia em relação ao eco-Doppler na detecção das complicações (K = -0,123). Conclusão: Concluímos que as repercussões vasculares do uso de CDL são frequentes (31,25%), manifestando-se na forma de oclusões com/sem recanalização e estenoses. Sendo assim, é necessária uma análise prévia do sítio de inserção com o eco-Doppler, a fim de se evitar procedimentos desnecessários e com possíveis complicações.

O custo de soluções alcalinas em sessões de hemodiálise ambulatorial: uma análise sobre o desperdício a partir do controle dos processos

Introdução: São escassos estudos dos custos dos insumos consumidos em hemodiálise e, dentre estes gastos, os compostos que compõem o dialisato estão entre os valores considerados como representativos nessa terapia. Contudo, não foram encontrados estudos que orientem sobre o comportamento de custos dessas soluções. Objetivo: O objetivo do artigo é avaliar se há desperdício no consumo de soluções alcalinas em hemodiálise ambulatorial e, consequentemente, a possibilidade de redução no custo a partir da simulação de padronização no processo de estabelecimento do fluxo do dialisato nos períodos entre turnos em sessões de hemodiálise ambulatorial. Métodos: Partindo de um estudo observacional analítico, foi realizada uma simulação de 20 cenários, sendo 10 estabelecidos pela padronização dos processos de controle no fluxo do dialisato nos intervalos das sessões. A combinação dos dados foi realizada tomando por base os preços de três fornecedores de soluções alcalinas líquidas ou em pó. Resultados: Observou-se, dentre os cenários com processos padronizados, uma variação entre 7,7% e 33,3% de economia no custo da solução alcalina (em pó ou líquida), pela redução do desperdício. Conclusão: É possível refrear o desperdício no uso de soluções alcalinas, tanto em pó quanto líquidas e, consequentemente, seus custos, a partir da padronização na redução do fluxo de dialisato durante os intervalos verificados entre os turnos na hemodiálise ambulatorial. Todavia, estes resultados estão condicionados ao comprometimento de profissionais de saúde, principalmente no que tange ao exercício da supervisão e controle das atividades no desdobramento da função qualidade.

Encaminhamento tardio ao nefrologista e a associação com mortalidade em pacientes em hemodiálise

Introdução: A doença renal crônica (DRC) caracteriza-se pela perda progressiva da função renal. Intervenções em estágios iniciais melhoram significativamente o prognóstico dos pacientes com DRC e vários estudos mostram que o encaminhamento precoce (EP) ao nefrologista reduz a taxa de mortalidade. Objetivo: Analisar o perfil dos pacientes em diálise e o tempo transcorrido entre a primeira consulta na unidade de diálise e o início do programa dialítico. Métodos: Foi realizado um estudo de coorte retrospectivo com dois eixos de análise: perfil socioepidemiológico e clínico dos pacientes em hemodiálise e o tempo transcorrido entre a primeira consulta na unidade de diálise e o início do programa dialítico. Utilizaram-se métodos analíticos para comparar estes dados com o EP e com a mortalidade em 12 meses após o início da diálise. Resultados: Foram analisados 111 pacientes. A taxa de mortalidade dos pacientes encaminhados tardiamente e precocemente foi, respectivamente, de 47,8% e 20,5% (razão de risco [HR] = 2,38; intervalo de confiança [IC] = 1,06-5,36; p = 0,035). Os pacientes que iniciaram a diálise por cateter e fístula arteriovenosa (FAV) tiveram mortalidade, respectivamente, de 51,4% e 10,3% (HR = 4,61; IC = 1,54-13,75; p = 0,006). Conclusão: O tempo de encaminhamento foi predominantemente tardio. Foi demonstrado que o encaminhamento tardio (ET) esteve relacionado a maior mortalidade. Outros fatores associados a maior mortalidade foram a idade maior ou igual a 70 anos, presença de DM e uso de cateter ao início da diálise.

GIS-based approach for optimization of onshore wind park infrastructure alignment in Finland

Wind power is a rapidly developing, low-emission form of energy production. In Fin-land, the official objective is to increase wind power capacity from the current 1 005 MW up to 3 500–4 000 MW by 2025. By the end of April 2015, the total capacity of all wind power project being planned in Finland had surpassed 11 000 MW. As the amount of projects in Finland is record high, an increasing amount of infrastructure is also being planned and constructed. Traditionally, these planning operations are conducted using manual and labor-intensive work methods that are prone to subjectivity. This study introduces a GIS-based methodology for determining optimal paths to sup-port the planning of onshore wind park infrastructure alignment in Nordanå-Lövböle wind park located on the island of Kemiönsaari in Southwest Finland. The presented methodology utilizes a least-cost path (LCP) algorithm for searching of optimal paths within a high resolution real-world terrain dataset derived from airborne lidar scannings. In addition, planning data is used to provide a realistic planning framework for the anal-ysis. In order to produce realistic results, the physiographic and planning datasets are standardized and weighted according to qualitative suitability assessments by utilizing methods and practices offered by multi-criteria evaluation (MCE). The results are pre-sented as scenarios to correspond various different planning objectives. Finally, the methodology is documented by using tools of Business Process Management (BPM). The results show that the presented methodology can be effectively used to search and identify extensive, 20 to 35 kilometers long networks of paths that correspond to certain optimization objectives in the study area. The utilization of high-resolution terrain data produces a more objective and more detailed path alignment plan. This study demon-strates that the presented methodology can be practically applied to support a wind power infrastructure alignment planning process. The six-phase structure of the method-ology allows straightforward incorporation of different optimization objectives. The methodology responds well to combining quantitative and qualitative data. Additional-ly, the careful documentation presents an example of how the methodology can be eval-uated and developed as a business process. This thesis also shows that more emphasis on the research of algorithm-based, more objective methods for the planning of infrastruc-ture alignment is desirable, as technological development has only recently started to realize the potential of these computational methods.

The effect of lysobisphosphatidic acid (LBPA) on α-tocopherol transfer protein (α-TTP) binding to lipid membranes

The a-tocopherol transfer protein (a-TTP) is responsible for the retention of the atocopherol form of vitamin E in living organisms. The detailed ligand transfer mechanism by a-TTP is still yet to be fully elucidated. To date, studies show that a-TTP transfers a-tocopherol from late endosomes in liver cells to the plasma membrane where it is repackaged into very low density lipoprotein (VLDL) and released into the circulation. Late endosomes have been shown to contain a lipid known as lysobisphosphatidic acid (LBP A) that is unique to this cellular compartment. LBPA plays a role in intracellular trafficking and controlling membrane curvature. Taking these observations into account plus the fact that certain proteins are recruited to membranes based on membrane curvature, the specific aim of this project was to examine the effect of LBP A on a-TTP binding to lipid membranes. To achieve this objective, dual polarization interferometry (DPI) and a vesicle binding assay were employed. Whilst DPI allows protein binding affinity to be measured on a flat lipid surface, the vesicle binding assay determines protein binding affinity to lipid vesicles mimicking curved membranes. DPI analysis revealed that the amount of a-TTP bound to lipid membranes is higher when LBPA is present. Using the vesicle binding assay, a similar result was seen where a greater amount of protein is bound to large unilamellar vesicles (LUV s) containing LBP A. However, the effect of LBP A was attenuated when small unilamellar vesicles (SUVs) were replaced with LUVs. The outcome of this project suggests that aTTP binding to membranes is influenced by membrane curvature, which in turn is induced by the presence of LBP A.