Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae.

The stress response promoter element (STRE) confers increased transcription to a set of genes following environmental or metabolic stress in Saccharomyces cerevisiae. A lambda gt11 library was screened to isolate clones encoding STRE-binding proteins, and one such gene was identified as MSN2, which encoded a zinc-finger transcriptional activator. Disruption of the MSN2 gene abolished an STRE-binding activity in crude extracts as judged by both gel mobility-shift and Southwestern blot experiments, and overexpression of MSN2 intensified this binding activity. Northern blot analysis demonstrated that for the known or suspected STRE-regulated genes DDR2, CTT1, HSP12, and TPS2, transcript induction was impaired following heat shock or DNA damage treatment in the msn2-disrupted strain and was constitutively activated in a strain overexpressing MSN2. Furthermore, heat shock induction of a STRE-driven reporter gene was reduced more than 6-fold in the msn2 strain relative to wild-type cells. Taken together, these data indicate that Msn2p is the transcription factor that activates STRE-regulated genes in response to stress. Whereas nearly 85% of STRE-mediated heat shock induction was MSN2 dependent, there was significant MSN2-independent expression. We present evidence that the MSN2 homolog, MSN4, can partially replace MSN2 for transcriptional activation following stress. Moreover, our data provides evidence for the involvement of additional transcription factors in the yeast multistress response.

Purification and cDNA cloning of a second apoptosis-related cysteine protease that cleaves and activates sterol regulatory element binding proteins.

We have purified from hamster liver a second cysteine protease that cleaves and activates sterol regulatory element binding proteins (SREBPs). cDNA cloning revealed that this enzyme is the hamster equivalent of Mch3, a human enzyme that is related to the interleukin 1beta converting enzyme. We call this enzyme Mch3/SCA-2. It is 54% identical to hamster CPP32/SCA-1, a cysteine protease that was earlier shown to cleave SREBPs at a conserved Asp between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. This cleavage liberates an NH2-terminal fragment of approximately 460 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. Mch3/SCA-2 and CPP32/SCA-I are synthesized as inactive 30-35 kDa precursors that are thought to be cleaved during apoptosis to generate active fragments of approximately 20 and approximately 10 kDa. The current data lend further support to the notion that SREBPs are cleaved and activated as part of the program in programmed cell death.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth.

VIP21/caveolin is a cholesterol-binding protein.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin.

Plakoglobin interacts with both classical and desmosomal cadherins. It is closely related to Drosophila aramadillo (arm) gene product; arm acts in the wingless (wg)-signaling pathway to establish segment polarity. In Xenopus, homologs of wg--i.e., wnts, can produce anterior axis duplications by inducing dorsal mesoderm. Studies in Drosophila suggest that wnt acts by increasing the level of cytoplasmic armadillo protein (arm). To test whether simply increasing the level of plakoglobin mimics the effects of exogenous wnts in Xenopus, we injected fertilized eggs with RNA encoding an epitope-tagged form of plakoglobin; this induced both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein was coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin were suppressed. Mutational analysis indicates that the central arm repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm.

Análise do papel da via miR156/SQUAMOSA Promoter-Binding Protein-Like (SPL) na organogênese in vitro a partir de raízes de Arabidopsis thaliana

Os microRNAs (miRNAs) são pequenos RNAs endógenos não codantes de 21-24 nucleotídeos (nt) que regulam a expressão gênica de genes-alvos. Eles estão envolvidos em diversos aspectos de desenvolvimento da planta, tanto na parte aérea, quanto no sistema radicular. Entre os miRNAs, o miRNA156 (miR156) regula a família de fatores de transcrição SQUAMOSA Promoter-Binding Protein-Like (SPL) afetando diferentes processos do desenvolvimento vegetal. Estudos recentes mostram que a via gênica miR156/SPL apresenta efeito positivo tanto no aumento da formação de raízes laterais, quanto no aumento de regeneração de brotos in vitro a partir de folhas e hipocótilos em Arabidopsis thaliana. Devido ao fato de que a origem da formação de raiz lateral e a regeneração in vitro de brotos a partir de raiz principal compartilham semelhanças anatômicas e moleculares, avaliou-se no presente estudo se a via miR156/SPL, da mesma forma que a partir de explantes aéreos, também é capaz de influenciar na regeneração de brotos in vitro a partir de explantes radiculares. Para tanto foram comparados taxa de regeneração, padrão de distribuição de auxina e citocinina, análises histológicas e histoquímicas das estruturas regeneradas em plantas com via miR156/SPL alterada, incluindo planta mutante hyl1, na qual a produção desse miRNA é severamente reduzida. Além disso, foi avaliado o padrão de expressão do miR156 e específicos genes SPL durante a regeneração de brotos in vitro a partir da raiz principal de Arabidopsis thaliana. No presente trabalho observou-se que a alteração da via gênica miR156/SPL é capaz de modular a capacidade de regeneração de brotos in vitro a partir de raiz principal de Arabidopsis thaliana e a distribuição de auxina e citocinina presente nas células e tecidos envolvidos no processo de regeneração. Plantas superexpressando o miR156 apresentaram redução no número de brotos regenerados, além de ter o plastochron reduzido quando comparado com plantas controle. Adicionalmente, plantas contento o gene SPL9 resistente à clivagem pelo miR156 (rSPL9) apresentaram severa redução na quantidade de brotos, além de terem o plastochron alongado. Interessantemente, plantas mutantes hyl1-2 e plantas rSPL10 não apresentaram regeneração de brotos ao longo da raiz principal, mas sim intensa formação de raízes laterais e protuberâncias, respectivamente, tendo essa última apresentado indícios de diferenciação celular precoce. Tomados em conjunto os dados sugerem que o miR156 apresenta importante papel no controle do processo de regeneração de brotos in vitro. Entretanto, esse efeito é mais complexo em regeneração in vitro a partir de raízes do que a partir de cotilédones ou hipocótilos.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Pro-inflammatory cytokines IL6, TNF-α, IL1β, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis

Objective: To evaluate the serum levels and diagnostic value of cytokines and acute phase proteins in patients with infective endocarditis (IE). Patients and methods: Serum samples from 63 patients diagnosed with IE and 71 control patients were analysed for the following markers: interleukin-6 (IL6), tumour necrosis factor-α (TNF-α), interleukin 1-β (IL1β), procalcitonin (PCT), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). Results: Serum levels of IL6, IL1β and CRP were significantly elevated in patients with IE as compared to controls. PCT, TNF-α and LBP were not elevated. Conclusion: Serum CRP and IL6 are elevated in IE. IL 6 may aid in establishing the diagnosis. There was no correlation between IL 6 levels and CRP, causative microorganism, echocardiographic features or outcome. © 2007 The British Infection Society.