"Peptabody": a new type of high avidity binding protein.

A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer.

Proteins located on the surface of the pathogenic malaria parasite Plasmodium falciparum are objects of intensive studies due to their important role in the invasion of human cells and the accessibility to host antibodies thus making these proteins attractive vaccine candidates. One of these proteins, merozoite surface protein 3 (MSP3) represents a leading component among vaccine candidates; however, little is known about its structure and function. Our biophysical studies suggest that the 40 residue C-terminal domain of MSP3 protein self-assembles into a four-stranded alpha-helical coiled coil structure where alpha-helices are packed "side-by-side". A bioinformatics analysis provides an extended list of known and putative proteins from different species of Plasmodium which have such MSP3-like C-terminal domains. This finding allowed us to extend some conclusions of our studies to a larger group of the malaria surface proteins. Possible structural and functional roles of these highly conserved oligomerization domains in the intact merozoite surface proteins are discussed.

Radiometric age constraints on mineral growth, metamorphism, and tectonism of the Gummfluh klippe, Brianconnais domain of the Prealpes, Switzerland

A combined Ar-40/Ar-39, K/Ar, Rb/Sr and stable isotope study has been made of white micas from the Gummfluh klippe (Brianconnais domain of the Prealpes), Switzerland. The klippe consists mainly of Mesozoic to early Tertiary carbonate rocks metamorphosed from anchizonal to epizonal conditions. At the base of the klippe is a 10-50 m thick, ductilely deformed marble mylonite containing deformed authigenic quartz segregations. Stable isotope measurements of the coexisting calcite (deltaO-18SMOW=24.5) and quartz (deltaO-18SMOW=28.4) from the mylonite indicate relatively low temperatures (< 300-degreesC) during mylonitization. Analyses of white mica separates of varying size fractions from the mylonitic rocks by K/Ar and Rb/Sr techniques yield ages between 57 and 103 Ma. This variation is correlated with two parameters, the size of the mineral fraction, and the proportion of 2M1 (more phengitic) to 1M (more muscovitic) polytype in the sample. The K/Ar and Rb/Sr ages are generally younger in the smaller size fractions, which also containless 2M1 phengite. High precision Ar-40/Ar-39 age spectra from different size fractions of these micas record three distinct components, a small Hercynian component (ca. 200-300 Ma), a significant Eoalpine component (64-80 Ma) forming Ar-40/Ar-39 age plateaus, and a very minor Tertiary component (ca. 20-40 Ma). Characterization of the samples by SEM indicates the presence of two white mica populations, a coarser grained, deformed, detrital mica that probably corresponds to the 2M1 phengite and a finer grained neoformed 1M mica. Collectively these observations suggest that the Gummfluh samples contain a mixture of detrital phengites of Hercynian age together with neocrystallized muscovites grown during the late Eoalpine metamorphic event followed by minor argon loss during the Tertiary. The main geologic episode recorded in the Ar-40/Ar-39 age spectra of white micas in the mylonite is of Late Cretaceous/Early Tertiary age (64-80 Ma), representing the first reliable Eoalpine ages ever to be reported from the Prealpes. Contrary to tectonic models, the marble mylonite at the base of the Gummfluh klippe appears to be a Cretaceous thrust plane and not the thrust surface formed during transport of the klippe into its present position from the Penninic Alps during the Tertiary. The late Cretaceous thrust developed during marine sedimentation at a depth of 800 m below the seafloor at temperatures of approximately 280-degrees-C, facilitated by warm fluids along the tectonic discontinuity.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Deep learning via semi-supervised embedding

We show how nonlinear embedding algorithms popular for use with shallow semi-supervised learning techniques such as kernel methods can be applied to deep multilayer architectures, either as a regularizer at the output layer, or on each layer of the architecture. This provides a simple alternative to existing approaches to deep learning whilst yielding competitive error rates compared to those methods, and existing shallow semi-supervised techniques.

Avaliação de um modelo físico de produção de bovinos no semi-árido integrando caatinga, capim-buffel e leucena.: I. fase de cria

O estudo teve como objetivo avaliar um modelo físico do sistema de cria de bovinos para as áreas mais secas do Semi-Árido brasileiro, associando a pastagem natural com pastos cultivados tolerantes à seca. A caatinga foi pastejada no período verde, e o capim buffel no restante do ano. A suplementação com leucena ocorreu no período seco, sob pastejo direto e feno no cocho. Parâmetros de desempenho, monitorados de novembro de 1991 a outubro de 1995, mostraram uma taxa média de parição da ordem de 72,8% ao ano e taxas de mortalidade praticamente nulas. O peso vivo médio dos bezerros aos 205 dias de idade foi de 153,4 kg e a produ- ção de bezerros desmamados foi de 109,5 kg/matriz exposta/ano e de 25,5 kg/hectare/ano. Os resultados são considerados bastante expressivos, considerando-se que mais da metade da área era ocupada com pastagens nativas de caatinga e que a oferta de leucena foi limitada pela ocorrência de uma forte estiagem.

Uses of also in oral semi-informal German

In recent grammars and dictionaries also (`therefore, so, well¿) continues to be preferably presented as an adverb with a conclusive-consecutive connective function that essentially corresponds to its use in formal written German. Its function as a modal particle is documented, however, since the beginnings of what is known as Partikelforschung, though not all its uses have been systematically investigated contrasting oral and written German, either in mode or concept. In this article we analyse the uses of also in semi-informal oral interactions on the basis of empirical data (from a subsample of the VARCOM corpus). Specifically, we will analyse the presence and frequency of also at the beginning of a sentence or sequence, the functions it serves as a logical-semantic connector or discourse and interaction marker and the interrelations between these functions, in order to contrast these results with the description of also provided by current reference works.

SUPLEMENTAÇÃO ALIMENTAR DE CABRAS ANGLO-NUBIANAS NA ÉPOCA CHUVOSA, NA REGIÃO SEMI-ÁRIDA DO NORDESTE BRASILEIRO

Foi realizado um experimento para avaliar a influência da suplementação alimentar na produção de leite de cabras da raça Anglo-nubiana, durante a época chuvosa, na região semi-árida do Nordeste brasileiro. Vinte e quatro cabras foram mantidas em pastagem nativa rebaixada, distribuídas ao acaso, por tipo de parto e produção de leite, em três níveis de suplementação: N0 - sem suplementação, N1 -150 g de concentrado/animal/dia e N2 - 300 g de concentrado/animal/dia. Não houve diferença significativa (p>0,05) entre os níveis de suplementação. A produção de leite foi descrita por uma regressão múltipla em função do período de lactação em semana. A interação semana x níveis de suplementação foi significativa (p<0,01). Os resultados revelam que a suplementação com concentrado, na dose de 300 g/animal/dia, foi a melhor opção para se produzir leite com cabras Anglo-nubianas, durante a época chuvosa.

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.

Efeito da calagem e inoculação de sabiá em solo da mata úmida e do semi-árido de Pernambuco

Realizou-se um experimento, usando solo álico da Zona da Mata e do Semi-Árido do Estado de Pernambuco, com o objetivo de avaliar o efeito da calagem (0, 3 e 6 t ha-1) e da inoculação das estirpes de rizóbio: NFB 539, NFB 577 e NFB 578, previamente selecionadas para sabiá (Mimosa caesalpiniaefolia) em meio de cultura ácido. Adicionaram-se tratamentos sem inoculação, sem e com adição de N mineral (100 kg ha-1), para fins comparativos. O experimento seguiu o esquema fatorial 2x3x5, no delineamento em blocos ao acaso, com quatro repetições. As plantas foram colhidas 110 dias após a emergência (DAE). A inoculação de rizóbio, em todos os níveis de calagem, mostrou efeito significativo nos parâmetros avaliados. No nível de 3 t ha-1 não houve efeito da calagem, porém a adição de 6 t ha-1 de calcário reduziu o peso de matéria seca, o N total na parte aérea, a nodulação e a atividade da nitrogenase. O baixo pH e Al3+ do solo não prejudicou a fixação do N2, e o crescimento das plantas que receberam o inóculo. Portanto, torna-se desnecessária a calagem no cultivo de sabiá em solos ácidos, quando usadas estirpes selecionadas visando à resistência a acidez.

Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036.

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis.

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65(-/-) mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

Efeito de inoculante ectomicorrízico produzido por fermentação semi-sólida sobre o crescimento de Eucalyptus dunnii Maiden

O crescimento de Eucalyptus é favorecido por fungos ectomicorrízicos (FECM). De ocorrência comum nas plantações florestais, os FECM diferem, entretanto, em infectividade e eficiência. O objetivo deste estudo foi testar a compatibilidade e a eficiência do inoculante ectomicorrízico produzido por fermentação semi-sólida, em relação a Eucalyptus dunnii Maiden. Neste sentido, o inoculante de Pisolithus sp., produzido por fermentação semi-sólida em vermiculita-solução nutritiva, foi aplicado ao substrato turfa-vermiculita (30:70, v/v) nas concentrações de 0, 1, 3, 5 e 10% (v/v) que foi semeado com E. dunnii, e mantido em casa de vegetação. A inoculação proporcionou colonização radicular e aumento da matéria seca da parte aérea e do conteúdo de P em razão da concentração do inoculante. O peso da matéria seca das plantas com 1% de inoculante não diferiu do peso das testemunhas. A 3% e 5%, a matéria seca aumentou 62%. O maior valor foi obtido com 10% de inoculante, onde o peso das plantas foi 73% superior ao das testemunhas, e o teor de P, 130%. Resultados semelhantes foram obtidos em relação à raiz. Altura e diâmetro foram positivamente afetados pela inoculação, mas não a relação raiz/parte aérea. As doses mais eficientes de inoculante foram aquelas de 3% ou superiores. Este método de produção deverá ser testado com outros FECM, e sua eficiência, avaliada em viveiro e no campo.

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.