Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress

Orphanin FQ (OFQ, Nociceptin) is a recently discovered 17-amino acid neuropeptide that is structurally related to the opioid peptides but does not bind opioid receptors. OFQ has been proposed to act as an anti-opioid peptide, but its widespread sites of action in the brain suggest that it may have more general functions. Here we show that OFQ plays an important role in higher brain functions because it can act as an anxiolytic to attenuate the behavioral inhibition of animals acutely exposed to stressful/anxiogenic environmental conditions. OFQ anxiolytic-like effects were consistent across several behavioral paradigms generating different types of anxiety states in animals (light-dark preference, elevated plus-maze, exploratory behavior of an unfamiliar environment, pharmacological anxiogenesis, operant conflict) and were observed at low nonsedating doses (0.1–3 nmol, intracerebroventricular). Like conventional anxiolytics, OFQ interfered with regular sensorimotor function at high doses (>3 nmol). Our results show that an important role of OFQ is to act as an endogenous regulator of acute anxiety responses. OFQ, probably in concert with other major neuropeptides, exerts a modulatory role on the central integration of stressful stimuli and, thereby, may modulate anxiety states generated by acute stress.

CD1c molecules broadly survey the endocytic system

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting! functional rumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable. (C) 2004 Elsevier Ltd. All rights reserved.

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

The source, cost and prevention of central venous catheter-related infection

Central venous catheters have become an integral part of patient management however they are associated with many complications including infection. Despite efforts being made to reduce the incidence of such infect ions the problem continues to increase and has resource implications for the Health Service. Studies relating to the source of microorganisms causing CVC-associated infection, the cost of such infections and the efficacy of an antimicrobial catheter have been undertaken. Thirty patients who required a CVC as part of their medical management and underwent cardiac surgery had the distal tips of their catheters sampled whilst in situ. Sampling took place within 1 h of catheter placement. Bacteria were isolated from 16% of the catheter distal tips sampled in situ. The guidewires used to insert the devices were also contaminated (50%). When CVC were inserted via a protective sheath, avoiding contact with the skin. the incidence of microbial contamination was reduced. These findings suggest that despite rigorous skin disinfection and strict aseptic technique, viable microorganisms are impacted onto the distal tip of CVC during the insertion procedure. Needleless intravascular access devices have been introduced in order to reduce the incidence of need1estick injury. However, it was unclear whether such connectors would act as a portal of entry for microorganisms to CVC. The efficacy of these devices was investigated. Within the controlled laboratory environment it was demonstrated that needleless devices, when challenged with microorganisms, did not allow the passage of microbes when flu id was injected. This therefore suggested that the devices should not increase the risk of catheter colonisation. When used in clinical practice however microbial contamination of the needleless connectors was 55 % in comparison to the routinely used luer connectors (23%). The cost of infections associated with CVC was determined. Twenty patients catheterised with a CVC designed for long term use who were admitted to hospital with a presumptive diagnosis of catheter-related infection were studied. The treatment given specifically for this infection was costed. The mean cost of such an infection was £ 1781.81. Throughout the UK this may amount to £1.565.906 per annum. The cost of infections associated with CVC designed for short term use was estimated to be between 5 and 7 million pounds per annum in the UK. In an attempt to reduce both the incidence and cost of catheter- related infection antimicrobial CVC have been developed. The efficacy of a novel polyurethane CVC impregnated on both the internal and external catheter surface with the quaternary ammonium compound benzalkonium chloride was investigated. Eighty eight patients received an antimicrobial catheter and 78 patients a conventional polyurethane CVC. The anti-microbial CVC resulted in a reduction in microbial colonisation of the external and internal polymer surfaces as compared to the control device. The observed reduction in microbial colonisation with the anti-microbial CVC may decrease the likelihood of subsequent infection offering a useful approach to the prevention of catheter-related infections.

NADPH oxidase signaling and cardiac myocyte function

The NADPH oxidase family of enzymes has emerged as a major source of reactive oxygen species (ROS) that is important in diverse cellular functions including anti-microbial defence, inflammation and redox signaling. Of the five known NADPH oxidase isoforms, several are expressed in cardiovascular cells where they are involved in physiological and pathological processes such as the regulation of vascular tone, cell growth, migration, proliferation, hypertrophy, apoptosis and matrix deposition. This article reviews current knowledge regarding the role of NADPH oxidases in cardiomyocyte function in health and disease. © 2009 Elsevier Inc. All rights reserved.

The integration of ProxiMAX randomisation with CIS display for the production of novel peptides

Saturation mutagenesis is a powerful tool in modern protein engineering, which permits key residues within a protein to be targeted in order to potentially enhance specific functionalities. However, the creation of large libraries using conventional saturation mutagenesis with degenerate codons (NNN or NNK/S) has inherent redundancy and consequent disparities in codon representation. Therefore, both chemical (trinucleotide phosphoramidites) and biological methods (sequential, enzymatic single codon additions) of non-degenerate saturation mutagenesis have been developed in order to combat these issues and so improve library quality. Large libraries with multiple saturated positions can be limited by the method used to screen them. Although the traditional screening method of choice, cell-dependent methods, such as phage display, are limited by the need for transformation. A number of cell-free screening methods, such as CIS display, which link the screened phenotype with the encoded genotype, have the capability of screening libraries with up to 1014 members. This thesis describes the further development of ProxiMAX technology to reduce library codon bias and its integration with CIS display to screen the resulting library. Synthetic MAX oligonucleotides are ligated to an acceptor base sequence, amplified, and digested, subsequently adding a randomised codon to the acceptor, which forms an iterative cycle using the digested product of the previous cycle as the base sequence for the next. Initial use of ProxiMAX highlighted areas of the process where changes could be implemented in order to improve the codon representation in the final library. The refined process was used to construct a monomeric anti-NGF peptide library, based on two proprietary dimeric peptides (Isogenica) that bind NGF. The resulting library showed greatly improved codon representation that equated to a theoretical diversity of ~69%. The library was subsequently screened using CIS display and the discovered peptides assessed for NGF-TrkA inhibition by ELISA. Despite binding to TrkA, these peptides showed lower levels of inhibition of the NGF-TrkA interaction than the parental dimeric peptides, highlighting the importance of dimerization for inhibition of NGF-TrkA binding.

Survey of veterinery drug residues, environmental contaminants and mycotoxins of cultured shirmp in Iran

Economical achievement of optimal growth in developing countries may lead to sustainable poverty reduction. Agricultural activities play an important role in economy and human being welfare, which leads to establishment of food security and quality. Aquaculture products in developing countries share 51.4 percent of total agricultural production and 241 percent in developed countries. Therefore undoubtedly food production by means of quality and quantity has to be increased .The history of shrimp production goes back to 500 years ago. Today 50 countries of the world produce shrimp. In Islamic Republic of Iran shrimp production started since 1992 in the coastal region of Persian Gulf. The shrimp culture farms can be classified in to 4 different categories; extensive, semi-extensive, intensive and super intensive. Global ecological maintenance is one of the major concerns of authorities Human manipulation of nature is the most destructive activity. Industrial sewage leakage in to the rivers and water sources is a big issue that causes reduction in the aquatic population. Heavy metals have an inhibitory effect in the production and growth of sea life. Human intake of food treated with anti-microbial cause's allergy, hypersensitivity and develops microbial resistance. Organochlorine compounds contamination may found in hepatopancreatic tissue of aquatic products, Arsenic may transfer to man via plant and animal product contamination.

Antimicrobial and bond strength properties of a dental adhesive containing zinc oxide nanoparticles

Aim: To assess the effect of adding zinc oxide nanoparticles to dental adhesives on their anti-microbial and bond strength properties. Methods: 45 human premolars were cut at the cement enamel junction (CEJ) and the crowns were sliced into buccal and lingual halves. The specimens were classified into three groups, etched with 37% phosphoric acid for 15 s and rinsed for 30 s. Single Bond, Single Bond+5% zinc oxide and Single Bond+10% zinc oxide were used in the first, second and third groups. A cylinder of Z250 composite was bonded and cured for 40 s. For anti-bacterial testing, 10 samples of each group were assessed by direct contact test; 10 μL of bacterial suspension was transferred into tubes containing adhesives and incubated for one hour; 300 μL of brain heart infusion (BHI) broth was added to each tube and after 12 h, 50 μL of bacteria and broth were spread on blood agar plates and incubated for 24 h. Results: The colony count decreased significantly in the second and third groups compared to the first. Conclusions: Incorporation of zinc oxide nanoparticles into dental adhesives increases their anti-microbial properties without affecting their bond strength.

Antibacterial activity of the venom of Heterometrus xanthopus

Heterometrus xanthopus (Scorpion) is one of the most venomous and ancient arthropods. Its venom contains anti-microbial peptides like hadrurin, scorpine, Pandinin 1, and Pandinin 2 that are able to effectively kill multidrug-resistant pathogens. The present study was conducted to evaluate the anti-bacterial activity of H. xanthopus venom. Six Gram-positive and Gram-negative bacterial strains were tested against 1/100, 1/10, and 1/1 fractions of distilled water diluted and crude venom. 1/100 and 1/10 dilutions were not successful in any of the six bacterial strains studied while the 1/1 dilution was effective on Bacillus subtilis ATCC 6633, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 27853 with highest zone of inhibition were obtained on B. subtilis. Crude venom was effective against Enterococcus faecalis ATCC 14506, B. subtilis, S. typhimurium, and P. aeruginosa. The most effective results were observed on B. subtilis.

Recognition of Membrane-Bound Fusion-Peptide/MPER Complexes by the HIV-1 Neutralizing 2F5 Antibody : Implications for Anti-2F5 Immunogenicity

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Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-I-127 iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 mu g/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10626 +/- 2886 mu g/L and 528 +/- 191 mu g/L, and the terminal elimination half-lives (T,12) were 6.3 +/- 0.9 h and 5.5 +/- 1.0 h, respectively. After sc, T-max was 0.25-2 h, and the absolute bioavailability was 49% +/- 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 mu g/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T-1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.

Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.