Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

The functional and biological properties of whey proteins : prospects for the development of functional foods

Selostus: Heraproteiinit terveysvaikutteisten elintarvikkeiden kehittämisessä

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Rpe65 microarrays, from genes to phosphorylated proteins

Purpose:To describe a novel in silico method to gather and analyze data from high-throughput heterogeneous experimental procedures, i.e. gene and protein expression arrays. Methods:Each microarray is assigned to a database which handles common data (names, symbols, antibody codes, probe IDs, etc.). Links between informations are automatically generated from knowledge obtained in freely accessible databases (NCBI, Swissprot, etc). Requests can be made from any point of entry and the displayed result is fully customizable. Results:The initial database has been loaded with two sets of data: a first set of data originating from an Affymetrix-based retinal profiling performed in an RPE65 knock-out mouse model of Leber's congenital amaurosis. A second set of data generated from a Kinexus microarray experiment done on the retinas from the same mouse model has been added. Queries display wild type versus knock out expressions at several time points for both genes and proteins. Conclusions:This freely accessible database allows for easy consultation of data and facilitates data mining by integrating experimental data and biological pathways.

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs.

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.

Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.

The antiviral adaptor proteins Cardif and Trif are processed and inactivated by caspases

The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses

Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17.

The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

Patterns of calcium-binding proteins in human inferior colliculus: identification of subdivisions and evidence for putative parallel systems.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures.

p53 and Ki-ras as prognostic factors for Dukes' stage B colorectal cancer.

Mutations of the TP53 and Ki-ras genes have been reported to be of prognostic importance in colorectal carcinomas. An increased intracellular concentration of the p53 protein, although not identical to, is sometimes seen in tumours with TP53 mutation and has been correlated with poor prognosis in some tumour types. Previous colorectal cancer studies, addressing the prognostic importance of Ki-ras mutation and TP53 aberrations, yielded contradictory results. The aim of this study was to determine in a clinically and therapeutically homogeneous group of 122 sporadic Dukes' B colorectal carcinomas with a median follow-up of 67 months (3-144 months) whether or not p53 protein expression, TP53 mutation and K-ras mutation correlated with prognosis. p53 staining was performed by immunohistochemistry, using the monoclonal antibody DO7 on paraffin-embedded tissue. Mutations in exons 5-8 of the TP53 gene and in codons 12 and 13 of the K-ras gene were assayed in paraffin-embedded tissue by the single-strand conformation polymorphism (SSCP) assay. Nuclear p53 staining was found in 57 (47%) tumours. Aberrant migration patterns indicating mutation of the TP53 gene were found in 39 (32%) tumours. Forty-six carcinomas (38%) showed a mutation of the Ki-ras codons 12 or 13. In a univariate analysis, patients with wild-type TP53 status showed a trend towards better survival, compared with those with mutated TP53 (log-rank test, P = 0.051). Likewise, tumours immunohistochemically positive for p53 showed a worse prognosis than p53-negative tumours (P = 0.010). The presence or absence of mutations in Ki-ras did not correlate with prognosis (P = 0.703). In multivariate analysis, only p53 immunoreactivity emerged as an independent marker for prognosis hazard ratio (HR) = 2.16, 95% confidence interval (CI) 1.12-4.11, P = 0.02). Assessment of p53 protein expression is more discriminative than TP53 mutation to predict the outcome of Dukes' stage B tumours and could be a useful tool to identify patients who might benefit from adjuvant therapy.

Identifying knots in proteins.

Polypeptide chains form open knots in many proteins. How these knotted proteins fold and finding the evolutionary advantage provided by these knots are among some of the key questions currently being studied in the protein folding field. The detection and identification of protein knots are substantial challenges. Different methods and many variations of them have been employed, but they can give different results for the same protein. In the present article, we review the various knot identification algorithms and compare their relative strengths when applied to the study of knots in proteins. We show that the statistical approach based on the uniform closure method is advantageous in comparison with other methods used to characterize protein knots.

Controlled expression of recombinant proteins in Physcomitrella patens by a conditional heat-shock promoter: a tool for plant research and biotechnology.

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using beta-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 degrees C. In contrast, a short non-damaging heat-treatment at 38 degrees C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 degrees C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.

The emerging importance of the SPX domain-containing proteins in phosphate homeostasis

Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.