First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from S?o Vicente, state of S?o Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.     

Comparative analysis of peripheral and localised cytokine secretion in glioblastoma patients

BACKGROUND: Malignant gliomas are the most common primary brain tumours of both children and adults. The unique aspects of their biology and anatomic site render them refractory to conventional therapeutic strategies such as surgery and chemotherapy. Significant attention has been given, recently, to immunotherapy which, although promising in preclinical studies, has not yet enhanced the survival of patients with glioblastomas. METHODS: To further understand the immunobiology of glioblastomas in clinical settings, we examined the secretion of four main cytokines in the peripheral blood and in primary cell cultures of 33 human glioblastoma patients. An ELISPOT methodology was used for the first time to examine Th1, and Th2 cytokine secretion from both peripheral lymphocytes and glioma tumour cells. RESULTS: Th1 cytokines (tumour necrosis factor (TNF-alpha), interferon (IFN-gamma) were markedly reduced compared to control levels (P=0.01 and P<0.001, respectively), whereas in contrast, Th2 (interleukin (IL)-4 and IL-10) were strongly expressed in both peripheral lymphocytes and glioma cell cultures (P=0.05 and P<0.001, respectively). CONCLUSION: This pattern indicates an 'immunosuppressive status' in glioblastomas which is related to their origination and the evasion of glioma cells from immune surveillance and could account for the failure of immunotherapy in such tumours. Furthermore, ELISPOT methodology can be used for monitoring of cytokine secretion from tumour cells, in addition to the well-established peripheral cytokine secretion.     

Breast cancer clinical and translational research: analogies and implications for prostate cancer

Breast and prostate cancer, respectively, are the most common cancers in women and in men in the United States. The management of locally advanced prostate cancer involves a multidisciplinary approach, bearing similarity to the therapeutic approach to breast cancer. Better understanding of the molecular biology of these cancers and the identification of the role played by the cancer stem cells and the tumor microenvironment may translate into better clinical decision making regarding risk classification and treatment allocation. A systematic assessment is presented of the many parallel evolutions in defining and treating high-risk breast cancer as they pertain to prostate cancer.     

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA<Subscript>2</Subscript> (F6a) from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> Snake Venom

A new crotoxin B isoform PLA₂ (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA₂ (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA₂ (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA₂ (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA₂s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA₂s. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA₂ (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA₂ (F6a) was clearly cytotoxic to these cells.     

UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors

1. Download high-res image (232KB)
2. Download full-size image

     

Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana

Thylakoid rhodanase‐like protein (TROL) is a nuclear‐encoded protein of thylakoid membranes required for tethering of ferredoxin:nicotinamide adenine dinucleotide phosphate (NADPH) oxydoreductase (FNR). It has been proposed that the dynamic interaction of TROL with flavoenzyme FNR, influenced by environmental light conditions, regulates the fate of photosynthetic electrons, directing them either to NADPH synthesis or to other acceptors, including reactive oxygen species detoxification pathways. Inside the chloroplasts, TROL has a dual localization: an inner membrane precursor form and a thylakoid membrane mature form, which has been confirmed by several large‐scale chloroplast proteomics studies, as well as protein import experiments. Unlike the localization, the topology of TROL in the membranes, which is a prerequisite for further studies of its properties and function, has not been experimentally confirmed yet. Thermolysin was proven to be a valuable protease to probe the surface of chloroplasts and membranes in general. By treating the total chloroplast membranes using increasing protease concentration, sequential degradation of TROL was observed, indicating protected polypeptides of TROL and possible domain orientation. To further substantiate the obtained results, TROL‐overexpressing Arabidopsis line (OX) and line in which the central rhodanase‐like domain (RHO) has been partially deleted (ΔRHO), were used as well. While OX line showed the same degradation pattern of TROL as the wild‐type, surprisingly, TROL from ΔRHO membranes was not detectable even at the lowest protease concentration applied, indicating the importance of this domain to the integrity of TROL. In conclusion, TROL is a polytopic protein with a stroma‐exposed C‐terminal FNR‐binding region, and the thylakoid lumen‐located RHO domain.     

Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG

The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two‐gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon’s function. Interestingly, influx of the fluorescent dyes BCECF‐AM and calcein‐AM in de‐energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.     

Structural and functional characterization of a novel T cell receptor co-regulatory protein complex, CD97-CD55

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.     

Fever-like hyperthermia controls T Lymphocyte persistence by inducing degradation of cellular FLIPshort

Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIP(S)) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIP(S), the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIP(S). Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.     

A glycosylphosphatidylinositol-based treatment alleviates trypanosomiasis-associated immunopathology

The GPI-anchored trypanosome variant surface glycoprotein (VSG) triggers macrophages to produce TNF, involved in trypanosomiasis-associated inflammation and the clinical manifestation of sleeping sickness. Aiming at inhibiting immunopathology during experimental Trypanosoma brucei infections, a VSG-derived GPI-based treatment approach was developed. To achieve this, mice were exposed to the GPI before an infectious trypanosome challenge. This GPI-based strategy resulted in a significant prolonged survival and a substantial protection against infection-associated weight loss, liver damage, acidosis, and anemia; the latter was shown to be Ab-independent and correlated with reduced macrophage-mediated RBC clearance. In addition, GPI-based treatment resulted in reduced circulating serum levels of the inflammatory cytokines TNF and IL-6, abrogation of infection-induced LPS hypersensitivity, and an increase in circulating IL-10. At the level of trypanosomiasis-associated macrophage activation, the GPI-based treatment resulted in an impaired secretion of TNF by VSG and LPS pulsed macrophages, a reduced expression of the inflammatory cytokine genes TNF, IL-6, and IL-12, and an increased expression of the anti-inflammatory cytokine gene IL-10. In addition, this change in cytokine pattern upon GPI-based treatment was associated with the expression of alternatively activated macrophage markers. Finally, the GPI-based treatment also reduced the infection-associated pathology in Trypanosoma congolense and Trypanosoma evansi model systems as well as in tsetse fly challenge experiments, indicating potential field applicability for this intervention strategy.