NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Increased potency of a novel complement factor 5a receptor antagonist in a rat model of inflammatory bowel disease

We have previously shown that complement factor 5a(C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective, orally active C5a antagonist AcF-[OP(D-Cha) WR]. This study tested the efficacy and potency of a new C5a antagonist, hydrocinnamate (HC)-[OP(D-Cha) WR], which has limited intestinal lumenal metabolism, in this model of colitis. Analogs of AcF-[OP(D-Cha) WR] were examined for their susceptibility to alimentary metabolism in the rat using intestinal mucosal washings. One metabolically stable analog, HC-[OP(D-Cha)WR], was then evaluated pharmacokinetically and investigated at a range of doses (0.03 - 10 mg/kg/ day p.o.) in the 8-day rat TNBS- colitis model, against the comparator drug AcF-[OP(D-Cha) WR]. Using various amino acid substitutions, it was determined that the AcF moiety of AcF-[OP(D-Cha) WR] was responsible for the metabolic instability of the compound in intestinal mucosal washings. The analog HC-[OP( D-Cha) WR], equiactive in vitro to AcF-[OP(D-Cha) WR], was resistant to intestinal metabolism, but it displayed similar oral bioavailability to AcF-[OP(D-Cha) WR]. However, in the rat TNBS- colitis model, HC-[OP(D-Cha) WR] was effective at reducing mortality, colon edema, colon macroscopic scores, and increasing food consumption and body weights, at 10- to 30- fold lower oral doses than AcF-[OP( D-Cha) WR]. These studies suggest that resistance to intestinal metabolism by HC-[OP(D-Cha) WR] may result in increased local concentrations of the drug in the colon, thus affording efficacy with markedly lower oral doses than AcF-[OP(D-Cha) WR] against TNBS-colitis. This large increase in potency and high efficacy of this compound makes it a potential candidate for clinical development against intestinal diseases such as inflammatory bowel disease.

BMS-354825: a novel drug with potential for the treatment of imatinib-resistant chronic myeloid leukaemia

The BCR-ABL tyrosine kinase inhibitor imatinib has greatly improved the outcome for patients with chronic myeloid leukaemia (CML). Unfortunately, mutations causing resistance to imatinib are leading to relapses in some patients. In addition to inhibiting the wild-type BCR-ABL, BMS-354825 inhibited 14 of 15 BCR-ABL mutants. BMS-354825 treatment of immunodeficient mice prevented the progression of the disease in mice treated with the most clinical common imatinib-resistant mutant Met351Thr. The safety and efficacy of BMS-354825 is presently being evaluated in a phase I/II clinical trial in CML patients with imatinib resistance. The frequency of clinical use of SMS-3548125 in CML patients will depend on its efficacy/safety profile in clinical trial.

Mechanisms of inducible resistance against Bacillus thuringiensis endotoxins in invertebrates

Abstract Resistance in insect pests against the endotoxin of Bacillus thuringiensis (Berliner) (Bt) is a major threat to the usefulness of this biopesticide, both used as traditional formulations and in transgenic crops. A crucial requirement for the development of successful resistance management strategies is a molecular understanding of the nature and inheritance of resistance mechanisms. This information can be used to design management strategies that will delay or counteract Bt resistance. The best known Bt resistance mechanism is inactivation of brush border membrane receptors. This type of resistance has a largely recessive mode of inheritance, which has enabled the design of resistance management approaches involving high dose and refuge strategies. Recent observations suggest that other resistance mechanisms are possible, including a mechanism that sequesters the toxin in the gut lumen through inducible immune reactions. The elevated immune status associated with tolerance to the toxin can be transmitted to subsequent generations by a maternal effect, which has implications for resistance management in the field. The high dose/refuge strategy may not be appropriate for the management of these alternative resistance mechanisms and other strategies have to be developed if inducible dominant resistance or tolerance mechanisms occur frequently in the field.

Platelet nitric oxide responsiveness - A novel prognostic marker in acute coronary syndromes

Objectives - Nitric oxide (NO) is critically important in the regulation of vascular tone and the inhibition of platelet aggregation. We have shown previously that patients with acute coronary syndromes (ACS) or stable angina pectoris have impaired platelet responses to NO donors when compared with normal subjects. We tested the hypotheses that platelet hyporesponsiveness to NO is a predictor of (1) cardiovascular readmission and/or death and (2) all-cause mortality in patients with ACS (unstable angina pectoris or non-Q-wave myocardial infarction). Methods and Results - Patients (n = 51) with ACS had evaluation of platelet aggregation within 24 hours of coronary care unit admission using impedance aggregometry. Patients were categorized as having normal (>= 32% inhibition of ADP-induced aggregation with the NO donor sodium nitroprusside; 10 mu mol/L; n = 18) or impaired (>= 32% inhibition of ADP-induced aggregation; n = 33) NO responses. We then compared the incidence of cardiovascular readmission and death during a median of 7 years of follow-up in these 2 groups. Using a Cox proportional hazards model adjusting for age, sex, index event, postdischarge medical treatment, revascularization status, left ventricular systolic dysfunction, concurrent disease states, and cardiac risk factors, impaired NO responsiveness was associated with an increased risk of the combination of cardiovascular readmission and/or death (relative risk, 2.7; 95% CI, 1.03 to 7.10; P = 0.041) and all-cause mortality (relative risk, 6.3; 95% CI, 1.09 to 36.7; P = 0.033). Conclusions - Impaired platelet NO responsiveness is a novel, independent predictor of increased mortality and cardiovascular morbidity in patients with high-risk ACS.

Emulsion strategies in the microencapsulation of cells: Pathways to thin coherent membranes

Microencapsulation of cell spheroids in an immunoselective, highly biocompatible, biomembrane offers a way to create viable implantation options in the treatment of insulin-dependent diabetes mellitus (IDDM). Traditionally the encapsulation process has been achieved through the injection/extrusion of alginate/cell mixtures into a calcium chloride solution to produce calcium alginate capsules around the cells. A novel alternative is explored here through a procedure using an emulsion process to produce thin adherent calcium alginate membranes around cell spheroids. In this study, a thorough investigation has been used to establish the emulsion process parameters that are critical to the formation of a coherent alginate coat both on a model spheroid system and subsequently on cell spheroids. Optical and fluorescence microscopy are used to assess the morphology and coherence of the calcium alginate/ poly-L-ornithine/alginate (APA) capsules produced. (c) 2005 Wiley Periodicals, Inc.

A novel plant toxin, persin, with in vivo activity in the mammary gland, induces Bim-dependent apoptosis in human breast cancer cells

Phytochemicals have provided an abundant and effective source of therapeutics for the treatment of cancer. Here we describe the characterization of a novel plant toxin, persin, with in vivo activity in the mammary gland and a p53-, estrogen receptor-, and Bcl-2-independent mode of action. Persin was previously identified from avocado leaves as the toxic principle responsible for mammary gland-specific necrosis and apoptosis in lactating livestock. Here we used a lactating mouse model to confirm that persin has a similar cytotoxicity for the lactating mammary epithelium. Further in vitro studies in a panel of human breast cancer cell lines show that persin selectively induces a G(2)-M cell cycle arrest and caspase-dependent apoptosis in sensitive cells. The latter is dependent on expression of the BH3-only protein Bim. Bim is a sensor of cytoskeletal integrity, and there is evidence that unique structure of the compound, persin could represent a novel class of microtubule-targeting agent with potential specificity for breast cancers.

Cyclic MrIA: A stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter

Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the, beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

Transgenic gene silencing strategies for virus control

Co-suppression of transgenes and their homologous viral sequences by RNA silencing is a powerful strategy for achieving high-level virus resistance in plants. This review provides a brief overview of RNA silencing mechanisms in plants and discusses important transgene construct design features underpinning successful RNA silencing-mediated transgenic virus control. Application of those strategies to protect horticultural and field crops from virus infection and results of field tests are also provided. The effectiveness and stability of RNA-mediated transgenic resistance are assessed taking into account effects of viral, plant and environmental factors.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator

In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 +/- 1.4 and 2.9 +/- 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 +/- 6.3 and 93.3 +/- 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 +/- 3 h) compared with the human (3.5 +/- 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis.

Prevalence of drug-resistance mutations and non-subtype B strains among HIV-infected infants from New York State

Summary: Prevalence studies indicate that transmission of drug-resistant HIV has been rising in the adult population, but data from the perinatally infected pediatric population are limited. In this retrospective study, we sequenced the pol region of HIV from perinatally infected infants diagnosed in New York State in 2001-2002. Analyses of drug resistance, subtype diversity, and perinatal antiretroviral exposure were conducted, and the results were compared with those from a previous study of HIV-infected infants identified in 1998-1999. Eight of 42 infants (19.1%) had provirus carrying at least 1 drug-resistance mutation, an increase of 58% over the 1998-1999 results. Mutations conferring resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 7.1%, 11.9%, and 2.4% of specimens, respectively. Consistent with previous results, perinatal antiretroviral exposure was not associated with drug resistance (P = 0.70). Phylogenetic analysis indicated that 16.7% of infants were infected with a non-subtype B strain of HIV. It seems that drug-resistant and non-subtype B strains of HIV are becoming increasingly common in the perinatally infected population. Our results highlight the value of resistance testing for all HIV-infected infants upon diagnosis and the need to consider subtype diversity in diagnostic and treatment strategies.

Unraveling the molecular underpinnings of Philadelphia-negative myeloproliferative neoplasms: implications for future therapeutic strategies

The present thesis encompasses the two researches projects I conducted during my PhD program in Molecular Biology and Pathology. The common thread is represented by the analysis of the signaling pathways implicated in the pathophysiology of the two most aggressive Philadelphia-negative myeloproliferative neoplasms, namely, atypical chronic myeloid leukemia (aCML) and primary myelofibrosis (PMF). In the last decade, since the description of the JAK2V617F mutation in 2005, the field of the molecular characterization of Philadelphia-negative myeloproliferative neoplasms has experienced an astonishing implementation that led to the discovery of 16 new mutations involving signal transduction, epigenetic modifiers, cell cycle regulators. Nevertheless, their pathogenetic relevance and whether they could represent good “druggable” candidates have to be proved yet. In the first section I provide the first report of the signaling cascade down-stream the rare cytogenetic lesion t(8;9)(p22;p24)/PCM1-JAK2 associated with aCML, finding that it selectively activates the ERK1/2 signaling without affecting JAK/STAT phosphorylation. In the second part, I investigated the implication of the ε isoform of novel Protein kinase Cs (PKCs) in the pathophysiology of the aberrant megakaryocytopoiesis in PMF, concluding that the over-expression of PKCε detains a crucial relevance in the aberrant behavior of PMF megakaryocytes and its inhibition is capable to restore their normal differentiation and abrogate the anti-apoptotic signaling. Both results are discussed in the view of their therapeutic implications. In case PCM1/JAK2-related hematologic neoplasms, ERK-inhibitors rather than JAK-inhibitors (i.e. ruxolitinib) should be considered as a “tailored” drugs. In case of PMF, PKCε-inhibitors (i.e. εV1-2 peptide) configure as an appealing strategy to re-direct the megakaryocytic neoplastic clone.

An investigation into children’s inductive reasoning strategies:What drives the development of category induction?

In a series of studies, I investigated the developmental changes in children’s inductive reasoning strategy, methodological manipulations affecting the trajectory, and driving mechanisms behind the development of category induction. I systematically controlled the nature of the stimuli used, and employed a triad paradigm in which perceptual cues were directly pitted against category membership, to explore under which circumstances children used perceptual or category induction. My induction tasks were designed for children aged 3-9 years old using biologically plausible novel items. In Study 1, I tested 264 children. Using a wide age range allowed me to systematically investigate the developmental trajectory of induction. I also created two degrees of perceptual distractor – high and low – and explored whether the degree of perceptual similarity between target and test items altered children’s strategy preference. A further 52 children were tested in Study 2, to examine whether children showing a perceptual-bias were in fact basing their choice on maturation categories. A gradual transition was observed from perceptual to category induction. However, this transition could not be due to the inability to inhibit high perceptual distractors as children of all ages were equally distracted. Children were also not basing their strategy choices on maturation categories. In Study 3, I investigated category structure (featural vs. relational category rules) and domain (natural vs. artefact) on inductive preference. I tested 403 children. Each child was assigned to either the featural or relational condition, and completed both a natural kind and an artefact task. A further 98 children were tested in Study 4, on the effect of using stimuli labels during the tasks. I observed the same gradual transition from perceptual to category induction preference in Studies 3 and 4. This pattern was stable across domains, but children developed a category-bias one year later for relational categories, arguably due to the greater demands on executive function (EF) posed by these stimuli. Children who received labels during the task made significantly more category choices than those who did not receive labels, possibly due to priming effects. Having investigated influences affecting the developmental trajectory, I continued by exploring the driving mechanism behind the development of category induction. In Study 5, I tested 60 children on a battery of EF tasks as well as my induction task. None of the EF tasks were able to predict inductive variance, therefore EF development is unlikely to be the driving factor behind the transition. Finally in Study 6, I divided 252 children into either a comparison group or an intervention group. The intervention group took part in an interactive educational session at Twycross Zoo about animal adaptations. Both groups took part in four induction tasks, two before and two a week after the zoo visits. There was a significant increase in the number of category choices made in the intervention condition after the zoo visit, a result not observed in the comparison condition. This highlights the role of knowledge in supporting the transition from perceptual to category induction. I suggest that EF development may support induction development, but the driving mechanism behind the transition is an accumulation of knowledge, and an appreciation for the importance of category membership.

Drug delivery strategies for the treatment of Helicobacter pylori infections

Helicobacter pylori is one of the most common pathogenic bacterial infections, colonising an estimated half of all humans. It is associated with the development of serious gastroduodenal disease - including peptic ulcers, gastric lymphoma and acute chronic gastritis. Current recommended regimes are not wholly effective and patient compliance, side-effects and bacterial resistance can be problematic. Drug delivery to the site of residence in the gastric mucosa may improve efficacy of the current and emerging treatments. Gastric retentive delivery systems potentially allow increased penetration of the mucus layer and therefore increased drug concentration at the site of action. Proposed gastric retentive systems for the enhancement of local drug delivery include floating systems, expandable or swellable systems and bioadhesive systems. Generally, problems with these formulations are lack of specificity, limited to mucus turnover or failure to persist in the stomach. Gastric mucoadhesive systems are hailed as a promising technology to address this issue, penetrating the mucus layer and prolonging activity at the mucus-epithelial interface. This review appraises gastroretentive delivery strategies specifically with regard to their application as a delivery system to target Helicobacter. As drug-resistant strains emerge, the development of a vaccine to eradicate and prevent reinfection is an attractive proposition. Proposed prophylactic and therapeutic vaccines have been delivered using a number of mucosal routes using viral and non-viral vectors. The delivery form, inclusion of adjuvants, and delivery regime will influence the immune response generated. © 2005 Bentham Science Publishers Ltd.