Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M(r) = 34,000 under reducing conditions and pI similar to 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (-70 to 37 degrees C), pH values (3-9) or in the presence of divalent metal ions (Ca(2+), Mg(2+), Zn(2+) and Mn(2+)). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom. (C) 2009 Elsevier Ltd. All rights reserved.

The prospects for immunotherapy in smoking cessation

Context Smoking is a major preventable cause of death and disability that is maintained by dependence on nicotine. Smoking cessation reduces mortality and morbidity. Although existing pharmacological aids to smoking cessation and relapse prevention (nicotine replacement therapy and bupropion) improve on unassisted quitting and behavioural methods, they are only modestly effective. More effective pharmacological methods are required that improve compliance, reduce side-effects, and can be used in combination with existing cessation methods. Starting point A nicotine vaccine is a promising immunotherapeutic approach to smoking cessation and relapse prevention. Such a vaccine would induce the immune system to form specific antibodies to nicotine to prevent it from crossing the blood-brain barrier to act on receptor sites in the central nervous system. Recent studies in rats provide proof of principle by showing that nicotine-specific antibodies can prevent the reinstatement of nicotine self-administration (N Lindblom et al, Respiration 2002; 69: 254–60) and block dopamine release in the shell of the nucleus accumbens (Sde Villiers et al, Respiration 2002; 69: 247–53). A phase 1 trial of a human cocaine vaccine has also recently been successfully completed (T Kosten et al, Vaccine 2002; 20: 1196–204). A safe and effective human nicotine vaccine would potentially have fewer side-effects and better compliance than existing smoking-cessation pharmacotherapies. It could also be used in combination with some of them (eg, bupropion). Where next? The most promising clinical application of a human nicotine vaccine is likely to be in relapse prevention in abstinent smokers. A vaccine may also have a role in preparing smokers to quit. Clinical trials of safety and efficacy in human smokers and ex-smokers are warranted. If a nicotine vaccine proves to be safe and effective, the health-care system will need to ensure that it is registered for clinical use and that the poorer members of the community (among whom smoking prevalence is now highest in developed countries) have access to the vaccine. The community will need to be appropriately informed about the role of a nicotine vaccine to ensure that it is not prematurely used for preventive purposes in children and adolescents.

Fault-tolerant quantum computation with cluster states

The one-way quantum computing model introduced by Raussendorf and Briegel [Phys. Rev. Lett. 86, 5188 (2001)] shows that it is possible to quantum compute using only a fixed entangled resource known as a cluster state, and adaptive single-qubit measurements. This model is the basis for several practical proposals for quantum computation, including a promising proposal for optical quantum computation based on cluster states [M. A. Nielsen, Phys. Rev. Lett. (to be published), quant-ph/0402005]. A significant open question is whether such proposals are scalable in the presence of physically realistic noise. In this paper we prove two threshold theorems which show that scalable fault-tolerant quantum computation may be achieved in implementations based on cluster states, provided the noise in the implementations is below some constant threshold value. Our first threshold theorem applies to a class of implementations in which entangling gates are applied deterministically, but with a small amount of noise. We expect this threshold to be applicable in a wide variety of physical systems. Our second threshold theorem is specifically adapted to proposals such as the optical cluster-state proposal, in which nondeterministic entangling gates are used. A critical technical component of our proofs is two powerful theorems which relate the properties of noisy unitary operations restricted to act on a subspace of state space to extensions of those operations acting on the entire state space. We expect these theorems to have a variety of applications in other areas of quantum-information science.

Australia’s Lily Brayton: Performer and theatre artist

Recognition is the aim of this account of an artist who is now remembered largely for her beauty, her wide iconic fame achieved by mass distribution of her image via photography and postcards, and her professional association with a internationally prominent producer who was also her husband. It is however a historically situated study, confining itself to readings of the kind of theatrical, social and cultural work performed by Brayton's presence in a rapidly-modernising Australia during the period between Federation in 1901 and the first World War, which event marks a disjuncture in the patterns of entertainment and cultural discourse in the new nation. Pre-war Australian theatre and vaudeville managements competed vigorously to secure the most acclaimed artists, seeing it as a kind of service and duty to boost their country's prestige along with their own coffers. Meanwhile, local playwrights and producers promoted a burgeoning repertoire of Australian dramas and films which played alongside the imported products in a complex network of cultural codes and affects.

Watkins and the Pragmatic Problem of Induction

Watkins proposes a neo-Popperian solution to the pragmatic problem of induction. He asserts that evidence can be used non-inductively to prefer the principle that corroboration is more successful over all human history than that, say, counter-corroboration is more successful either over this same period or in the future. Watkins's argument for rejecting the first counter-corroborationist alternative is beside the point. However, as whatever is the best strategy over all human history is irrelevant to the pragmatic problem of induction since we are not required to act in the past, and his argument for rejecting the second presupposes induction.

Traditional healers as tuberculosis treatment supervisors: precedent and potential

SETTING: Hlabisa, South Africa. OBJECTIVE: To determine precedent and potential for traditional healers to act as tuberculosis (TB) treatment supervisors. METHODS: Literature review to describe precedent for the involvement of traditional healers in TB treatment supervision. Interviews with 100 TB patients to determine use of healers and their acceptability as supervisors. Interviews with 24 healers in the project sub-district to determine willingness to act as supervisors. RESULTS: Despite extensive literature on the interaction between traditional healers and conventional health services, including descriptions of traditional understandings of TB, no published work was identified that reported supervision of TB patients by traditional healers. Of 100 patients interviewed, only 10% had used a healer as the first health provider for their illness, but 40% had attended a healer at some time prior to diagnosis. Although only 4% believe healers can cure TB, 84% would consider choosing a healer as a treatment supervisor. Of the 24 healers, 15 (63%) distinguished between two types of diagnosis made among patients with. symptoms suggestive of TB: TB and idliso. Idliso is poisoning or bewitching, and is said to be best cured by healers, while TB is infectious and cannot be cured by healers. Most healers (88%) reported having referred patients with possible TB to hospital in the past; all were keen to negotiate collaboration with health services, and 92% were willing to act as treatment supervisors. CONCLUSIONS: While there is little reported precedent for traditional healers to interact formally with tuberculosis treatment services, the potential for collaboration seems to be high, at least in our setting.

GABA receptors inhibited by benzodiazepines mediate fast inhibitory transmission in the central amygdala

The amygdala is intimately involved in emotional behavior, and its role in the generation of anxiety and conditioned fear is well known. Benzodiazepines, which are commonly used for the relief of anxiety, are thought to act by enhancing the action of the inhibitory transmitter GABA. We have examined the properties of GABA-mediated inhibition in the amygdala. Whole-cell recordings were made from neurons in the lateral division of the central amygdala. Application of GABA evoked a current that reversed at the chloride equilibrium potential. Application of the GABA antagonists bicuculline or SR95531 inhibited the GABA-evoked current in a manner consistent with two binding sites. Stimulation of afferents to neurons in the central amygdala evoked an IPSC that was mediated by the release of GABA. The GABA(A) receptor antagonists bicuculline and picrotoxin failed to completely block the IPSC. The bicuculline-resistant IPSC was chloride-selective and was unaffected by GABA(B)-receptor antagonists. Furthermore, this current was insensitive to modulation by general anesthetics or barbiturates. In contrast to their actions at GABA(A) receptors, diazepam and flurazepam inhibited the bicuculline-resistant IPSC in a concentration-dependent manner. These effects were fully antagonized by the benzodiazepine site antagonist Ro15-1788. We conclude that a new type of ionotropic GABA receptor mediates fast inhibitory transmission in the central amygdala. This receptor may be a potential target for the development of new therapeutic strategies for anxiety disorders.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Coordinated plant defense responses in Arabidopsis revealed by microarray analysis

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2.375 selected genes were examined simultaneously by cDNA microarray analysis in Arabidopsis thaliana after inoculation with an incompatible fungal pathogen Alternaria brassicicola or treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene, Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology, In leaf tissue inoculated with A. brassicicola, the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.

Resident tissue macrophages within the normal rat iris lack immunosuppressive activity and are effective antigen-presenting cells

Despite extensive study of the numerous immunoregulatory mechanisms that contribute to the immune-privileged nature of the anterior chamber (AC) of the eye, little is known of the functional nature of antigen-presenting cells (APC) present in the tissues adjoining the AC. In the present study, we have compared the antigen-presenting capacity of dendritic cells (DC) and macrophages isolated from the normal rat iris. Whereas iris DC exhibited a potent ability to stimulate resting allogeneic T cells in MLR cultures (an in-vitro correlate of the ability to induce primary T cell responses), resident iris macrophages displayed negligible MLR-stimulatory capacity. Significantly, iris macrophages could efficiently elicit proliferation of primed antigen-specific T cells (an in-vitro correlate of the ability to act as local APC in secondary responses). This antigen-presenting activity was approximately half that of fully mature iris DC and considerably greater than that of freshly isolated iris DC. A key contributor to the effectiveness of resident iris macrophage antigen presentation was considered to be the absence of lymphocytostatic control of T cell proliferation exerted by these cells. The results indicate dichotomous but complementary roles for DC (immune surveillance) and macrophages (local antigen presentation in secondary responses) in this tissue.

Long-distance signalling and a mutational analysis of branching in pea

Four ramosus mutants with increased branching at basal and aerial nodes have been used to investigate the genetic regulation of bud outgrowth in Pisum sativum L. (garden pea). Studies of long-distance signalling, xylem sap cytokinin concentrations, shoot auxin level, auxin transport and auxin response are discussed. A model of branching control is presented that encompasses two graft-transmissible signals in addition to auxin and cytokinin. Mutants rms1 through rms4 are not deficient in indole-3-acetic acid (IAA) or in the basipetal transport of this hormone. Three of the four mutants, rms1, rms3 and rms4, have very reduced cytokinin concentrations in xylem sap from roots. This reduction in xylem sap cytokinin concentration appears to be caused by a property of the shoot and may be part of a feedback mechanism induced by an aspect of bud outgrowth. The shoot-to-root feedback signal is unlikely to be auxin itself, as auxin levels and transport are not correlated with xylem sap cytokinin concentrations in various intact and grafted mutant and wild-type plants. Rms1 and Rms2 act in shoot and rootstock to regulate the level or transport of graft-transmissible signals. Various grafting studies and double mutant analyses have associated Rms2 with the regulation of the shoot-to-root feedback signal. Rms1 is associated with a second unknown graft-transmissible signal that is postulated to move in the direction of root-to-shoot. Exogenous auxin appears to interact with both of the signals regulated by Rms1 and Rms2 in the inhibition of branching after decapitation. The action of Rms3 and Rms4 is less apparent at this stage, although both appear to act largely in the shoot.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican

BACKGROUND. Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS. Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS. The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65-95 kDa. The KS moiety was susceptible to digestion with keratanase 11 and peptide N-glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS. The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation. (C) 2003 Wiley-Liss, Inc.

Tripolyphosphate as precursor for REPO4: Eu3+ (RE = Y, La, Gd) by a polymeric method

A modification of the Pechini method was applied to obtain luminescent rare earth orthophosphates. The developed synthetic route is based on the ability of the tripolyphosphate anion (P3O105-) to act both as a complexing agent and as an orthophosphate precursor. Heating of aqueous solutions containing RE3+, Eu3+, P3O105-, citric acid, and ethylene glycol led to polymeric resins. The ignition of these resins at different temperatures yielded luminescent orthophosphates. The produced nanosized phosphors (YPO4:Eu3+, (Y,Gd)PO4:Eu3+, and LaPO4:Eu3+) were analyzed by infrared and luminescence spectroscopies, X-ray diffractometry, and scanning electron microscopy.