Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Blood-brain barrier disruption induces in vivo degeneration of nigral dopaminergic neurons

We have evaluated the possibility that changes in the vascular system may constitute a contributing factor for the death of nigral dopaminergic neurons in Parkinson's disease. Thus, we have employed intranigral injections of vascular endothelial growth factor (VEGF), the most potent inducer of blood-brain barrier (BBB) permeability. A single dose of 1 mug of VEGF, chosen from a dose-response study, highly disrupted the BBB in the ventral mesencephalon in a time-dependent manner. A strong regional correlation between BBB disruption and loss of tyrosine hydroxylase-positive neurons was evident. Moreover, Fluoro-Jade B labelling showed the presence of dying neurons in the substantia nigra in response to VEGF injection. High number of TUNEL-positive nuclei was observed in this area along with activation of caspase 3 within nigral dopaminergic neurons. Analysis of the glial population demonstrated a strong inflammatory response and activation of astroglia in response to BBB disruption. We conclude that disruption of the BBB may be a causative factor for degeneration of nigral dopaminergic neurons.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

Proteomic analysis of log to stationary growth phase Lactobacillus plantarum cells and a 2-DE database

Lactobacillus plantarum is part of the natural microbiota of many food fermentations as well as the human gastro-intestinal tract. The cytosolic fraction of the proteome of L. plantarum WCFS1, whose genome has been sequenced, was studied. 2-DE was used to investigate the proteins from the cytosolic fraction isolated from mid- and late-log, early- and late-stationary phase cells to generate reference maps of different growth conditions offering more knowledge of the metabolic behavior of this bacterium. From this fraction, a total of 200 protein spots were identified by MALDI-MS and a proteome production map was constructed to facilitate further studies such as detection of suitable biomarkers for specific growth conditions. More than half (57%) of the identified proteins were predicted to be involved in metabolic pathways of the bacterium. The protein profile changed during the growth of the bacteria such that 29% of the identified proteins involved in anabolic pathways were at least twofold up-regulated throughout the mid- and late-exponential and early-stationary phases. In the late-stationary phase, six proteins involved in stress or with a potential role for survival during starvation were up-regulated significantly.     

Clinical versus ultrasound examination in the evaluation of hepatosplenic schistosomiasis mansoni in endemic areas

The best way to appraise the size of abdominal organs remains undefined. Herein we compare the size of liver and spleen in hepatosplenic schistosomiasis using clinical and ultrasound (US) examination, and the size of the organs measured by US with their visualization below the costal margin ("palpable by US"). For this study, 411 individuals from an endemic area for schistosomiasis mansoni in Brazil have been selected. We found that palpable spleens and left liver lobes are larger than non palpable ones. Also, 23% of normal spleens measured by US were palpable on clinical examination, and 22% of spleens increased in size on US were non palpable. A total of 21% of normal spleens were "palpable by US". We also found 54% of normal sized right liver lobes palpable on clinical examination, whilst 54% of the increased livers, measured by US, were non palpable. About 76% of normal right liver lobes were "palpable by US". We conclude that the association of clinical, ultrasound and magnetic resonance imaging (MRI) examinations, in the near future, should give the investigators the necessary tools to perform a more accurate clinical diagnosis of hepatosplenic schistosomiasis mansoni.     

Measurement of the extent of electron transfer to the bacteriopheophytin in the M-subunit in reaction centers of Rhodopseudomonas viridis

We have measured the extent of flash-induced electron transfer from the bacteriochlorophyll dimer, P, to the bacteriopheophytin in the M-subunit, H_M, in reaction centers of Rhodopseudomonas viridis. This has been done by measuring the transient states produced by excitation of reaction centers trapped in the PH_L ^–H_M state at 90 K. Under these conditions the normal forward electron transfer to the bacteriopheophytin in the L-subunit, H_L, is blocked and the yield of transient P⁺H_M ^– can be estimated with respect to the lifetime of P^*. Under these conditions flash induced absorbance decreases of the bacteriochlorophyll dimer 990 nm band suggest that a transient P⁺ state is formed with a quantum yield of 0.09±0.06 compared to that formed during normal photochemistry. These transient measurements provide an upper limited on the yield of a transient P⁺ H_M ^– state. An estimate of 0.09 as the yield of the P⁺ H_M ^– state is consistent with all current observations. This estimate and the lifetime of P^* suggest that the electron transfer rate from P^* to H_M, k_M, is about 5 × 10⁹ sec^–1 (_M = 200ps). These measurements suggest that the a branching ratio k_L/k_M is on the order of 200. The large value of the branching ratio is remarkable in view of the structural symmetry of the reaction center. This measurement should be useful for electron transfer calculations based upon the reaction center structure.     

Immunogenicity and safety assessment of the Cuban recombinant hepatitis B vaccine in healthy adults.

Manufactures of biotechnological/biological products (including vaccines) frequently make changes to manufacturing processes of products both during development and after approval. In our case, a non-inferiority bridging study was carried out to demonstrate that changes in the production plant facilities of Cuban recombinant hepatitis B vaccine, Heberbiovac HB, did not affect the safety and immunogenicity of the vaccine. This controlled, randomized, doubled-blinded trial included 501 volunteers, aged between 20 and 64, who were given three doses of vaccine (20 microg HBsAg/mL) at month 0, 1, and 2. Four lots were evaluated (three corresponding to the new production facilities and a control one produced in the older facilities). One month after the third dose, were observed protective levels of anti-HBsAg in 97% of the subjects that concluded the study with a geometric mean antibody titer (GMT) of 931.18 IU/L. Normal values of body mass index (BMI), the younger ages, and being a female, were significantly related to a good antibody response. The vaccine was well tolerated. Pain at the injection site was the most commonly reported symptom. We conclude that Heberbiovac HB vaccine maintains its characteristics after the modifications carried out in the production plant facilities and both, lot obtained in previous facilities and in the new ones, are comparable in terms of safety and immunogenicity.     

Evidence for the sucrose-binding protein role in carbohydrate metabolism and transport at early developmental stage

Despite the large amount of data regarding sucrose-binding proteins (SBP), their functions remain largely unknown and controversial. In this investigation we performed a detailed temporal and spatial characterization of the phenotypes related to photosynthesis, sucrose exudation and carbohydrate metabolism in SBP antisense plants to gain insights into the physiological role of SBP. Significant reductions in net photosynthesis and in stomatal conductance were observed in the SBP antisense lines but were restricted to the vegetative phase, and persisted during a daily time course at this phase. Photosynthesis was saturated at a substantially lower irradiance in source leaves of the antisense lines, suggesting that light utilization is decreased in these plants. A slight reduction in soluble sugars was observed throughout the development of source leaves, partially overlapping a decrease in sucrose synthase activity (EC 2.4.1.13); whereas a transient increase in starch and adinosine diphosphate (ADP)-glucose pyrophosphorylase activity (EC 2.7.7.27) as well as decreased leaf sucrose exudation were detected in the beginning of the vegetative phase. These changes in source leaves were accompanied by reductions in sucrose and starch in sink leaves, hexoses and sucrose in roots and hexoses in shoot apex, which were observed before the occurrence of a significant reduction in height and in leaf number in the transgenic lines. These alterations in growth parameters did not persist throughout the development, but were associated with a delay in flowering time and leaf senescence in the SBP antisense lines. A likely involvement of SBP in sink strength is discussed.