SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration (in press)

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

Proton pump inhibitor-responsive oesophageal eosinophilia: an entity challenging current diagnostic criteria for eosinophilic oesophagitis.

Consensus diagnostic recommendations to distinguish GORD from eosinophilic oesophagitis (EoE) by response to a trial of proton pump inhibitors (PPIs) unexpectedly uncovered an entity called 'PPI-responsive oesophageal eosinophilia' (PPI-REE). PPI-REE refers to patients with clinical and histological features of EoE that remit with PPI treatment. Recent and evolving evidence, mostly from adults, shows that patients with PPI-REE and patients with EoE at baseline are clinically, endoscopically and histologically indistinguishable and have a significant overlap in terms of features of Th2 immune-mediated inflammation and gene expression. Furthermore, PPI therapy restores oesophageal mucosal integrity, reduces Th2 inflammation and reverses the abnormal gene expression signature in patients with PPI-REE, similar to the effects of topical steroids in patients with EoE. Additionally, recent series have reported that patients with EoE responsive to diet/topical steroids may also achieve remission on PPI therapy. This mounting evidence supports the concept that PPI-REE represents a continuum of the same immunological mechanisms that underlie EoE. Accordingly, it seems counterintuitive to differentiate PPI-REE from EoE based on a differential response to PPI therapy when their phenotypic, molecular, mechanistic and therapeutic features cannot be reliably distinguished. For patients with symptoms and histological features of EoE, it is reasonable to consider PPI therapy not as a diagnostic test, but as a therapeutic agent. Due to its safety profile, ease of administration and high response rates (up to 50%), PPI can be considered a first-line treatment before diet and topical steroids. The reasons why some patients with EoE respond to PPI, while others do not, remain to be elucidated.

Novas estratégias terapêuticas para o tratamento da depressão: uma visão da química medicinal

Depression is a widespread humor disturbance promoted mainly by depletion of biogenic neurotransmitter amines involved in the CNS synapses. Effective drug treatments for depression have been available for more than forty years. Despite its remarkable structural diversity, this paper discuss under the medicinal chemistry point of view, all different classes of "monoamine based" antidepressant drugs, emphasizing the rational design, structure-activity relationships (SAR), biotransformation and physico-chemical properties related with antidepressant activity and molecular mechanism of action.

Brain Dopamine and Serotonin Receptors in the Perception of Pain. Positron Emission Tomography Studies in Healthy Subjects

The role of dopamine and serotonin in spinal pain regulation is well established. However, little is known concerning the role of brain dopamine and serotonin in the perception of pain in humans. The aim of this study was to assess the potential role of brain dopamine and serotonin in determining experimental pain sensitivity in humans using positron emission tomography (PET) and psychophysical methods. A total of 39 healthy subjects participated in the study, and PET imaging was performed to assess brain dopamine D2/D3 and serotonin 5-HT_1A receptor availability. In a separate session, sensitivity to pain and touch was assessed with traditional psychophysical methods, allowing the evaluation of potential associations between D2/D3 and 5-HT_1A binding and psychophysical responses. The subjects’ responses were also analyzed according to Signal Detection Theory, which enables separate assessment of the subject’s discriminative capacity (sensory factor) and response criterion (non-sensory factor). The study found that the D2/D3 receptor binding in the right putamen was inversely correlated with pain threshold and response criterion. 5-HT_1A binding in cingulate cortex, inferior temporal gyrus and medial prefrontal cortex was inversely correlated with discriminative capacity for touch. Additionally, the response criterion for pain and intensity rating of suprathreshold pain were inversely correlated with 5-HT_1A binding in multiple brain areas. The results suggest that brain D2/D3 receptors and 5-HT_1A receptors modulate sensitivity to pain and that the pain modulatory effects may, at least partly, be attributed to influences on the response criterion. 5-HT_1A receptors are also involved in the regulation of touch by having an effect on discriminative capacity.

Development and validation of spectrophotometric method for the determination of DPP-4 inhibitor, sitagliptin, in its pharmaceutical preparations

A simple, sensitive and reproducible spectrophotometric method was developed for the determination of sitagliptin phosphate in bulk and in pharmaceutical formulations. The proposed method is based on condensation of the primary amino group of sitagliptin phosphate with acetyl acetone and formaldehyde producing a yellow colored product, which is measured spectrophotometrically at 430nm. The color was stable for about 1 hour. Beer's law is obeyed over a concentration range of 5-25 µg/ml. The apparent molar absorptivity and Sandell sensitivity values are 1.067 x 10(4) Lmol-1cm-1 and 0.0471 µgcm-2 respectively. All the variables were studied to optimize the reaction conditions. No interference was observed in the presence of common pharmaceutical excipients. The validity of the method was tested by analyzing sitagliptin phosphate in its pharmaceutical preparations. Good recoveries were obtained. The developed method was successfully employed for the determination of sitagliptin phosphate in various pharmaceutical preparations.

Generation and Characterization of Knockout Mice and Analysis of Patient Material Demonstrates a Role for Tissue Inhibitor of Metalloproteinases 4 (Timp4) in the Cardiovascular System and Tumor Metastasis

This thesis focuses on tissue inhibitor of metalloproteinases 4 (TIMP4) which is the newest member of a small gene and protein family of four closely related endogenous inhibitors of extracellular matrix (ECM) degrading enzymes. Existing data on TIMP4 suggested that it exhibits a more restricted expression pattern than the other TIMPs with high expression levels in heart, brain, ovary and skeletal muscle. These observations and the fact that the ECM is of special importance to provide the cardiovascular system with structural strength combined with elasticity and distensibility, prompted the present molecular biologic investigation on TIMP4. In the first part of the study the murine Timp4 gene was cloned and characterized in detail. The structure of murine Timp4 genomic locus resembles that in other species and of the other Timps. The highest Timp4 expression was detected in heart, ovary and brain. As the expression pattern of Timp4 gives only limited information about its role in physiology and pathology, Timp4 knockout mice were generated next. The analysis of Timp4 knockout mice revealed that Timp4 deficiency has no obvious effect on the development, growth or fertility of mice. Therefore, Timp4 deficient mice were challenged using available cardiovascular models, i.e. experimental cardiac pressure overload and myocardial infarction. In the former model, Timp4 deficiency was found to be compensated by Timp2 overexpression, whereas in the myocardial infarct model, Timp4 deficiency resulted in increased mortality due to increased susceptibility for cardiac rupture. In the wound healing model, Timp4 deficiency was shown to result in transient retardation of re-epithelialization of cutaneous wounds. Melanoma tumor growth was similar in Timp4 deficient and control mice. Despite of this, lung metastasis of melanoma cells was significantly increased in Timp4 null mice. In an attempt to translate the current findings to patient material, TIMP4 expression was studied in human specimens representing different inflammatory cardiovascular pathologies, i.e. giant cell arteritis, atherosclerotic coronary arteries and heart allografts exhibiting signs of chronic rejection. The results showed that cardiovascular expression of TIMP4 is elevated particularly in areas exhibiting inflammation. The results of the present studies suggest that TIMP4 has a special role in the regulation of tissue repair processes in the heart, and also in healing wounds and metastases. Furthermore, evidence is provided suggesting the usefulness of TIMP4 as a novel systemic marker for vascular inflammation.

Identification and characterization of CIP2A as a novel oncogenic inhibitor of PP2A

Inhibition of the tumor suppressor protein phosphatase 2A (PP2A) activity has been identified as one of the five key alterations required for human cell transformation. Regardless of this crucial role in human cancer development, the detailed mechanisms by which PP2A inhibition occurs in human cancers remain largely uncharacterized. PP2A regulates a plethora of cellular signaling cascades. One of the targets of PP2A is Myc oncoprotein, which is destabilized and degraded in response to PP2A-mediated dephosphorylation of Myc serine 62. In this study we identify Cancerous Inhibitor of PP2A (CIP2A) as a previously uncharacterized endogenous inhibitor of PP2A in human cancer cells. CIP2A inhibits PP2A activity leading to subsequent stabilization of the Myc protein. CIP2A promotes malignant growth of cancer cells in vitro and xenograft tumor formation in vivo and is overexpressed in cancer. Moreover, we explored the effect of CIP2A on global transcriptional profiles and validated a CIP2A-dependent transcriptional signature. Analysis of the CIP2A signature revealed both Myc-dependent and -independent functions for CIP2A. Importantly, we demonstrate that the CIP2A signature has clinical relevance in human breast cancer subtypes. Finally, we identify the genes potentially mediating the long-term growth suppression in CIP2A depleted cancer cells. Taken together, this work identifies CIP2A as a novel human oncoprotein and describes its function in cancer cells. These results may open novel possibilities for patient stratification and therapeutic intervention of cancer.

Globe fringerush (Fimbristylis miliacea) cross resistance to als-inhibitor herbicides under field conditions in irrigated rice in the south of Brazil

ALS-inhibiting herbicides usually provide adequate weed control in irrigated rice fields. After consecutive years of use, the Cyperaceae species, globe fringerush (Fimbristylis miliacea) began to show resistance to ALS (acetolactate synthase) inhibitors. Globe fringerush is one of the most problematic herbicide-resistant weeds in irrigated rice in the state of Santa Catarina in the South of Brazil. The objective of this research was to examine cross resistance of globe fringerush to ALS inhibitors, under field conditions. Two experiments were conducted in a rice field naturally infested with ALS-resistant globe fringerush in Santa Catarina, in the 2008/09 and 2009/10 cropping seasons. The experimental units were arranged in randomized complete block design, with five replicates, consisting of two factors (herbicide and dose) in a 4 x 5 factorial arrangement. ALS herbicides included bispyribac-sodium, ethoxysulfuron, pyrazosulfuron-ethyl and penoxsulam. Six-leaf globe fringerush was sprayed with herbicide doses of 0, 0.5, 1, 2 and 4X the recommended doses in a spray volume of 200 L ha-1. The number of rice culm, filled and sterile grains, plant height, dry shoot biomass and grain yield were recorded. Globe fringerush control was evaluated 28 and 70 days after herbicide application (DAA); shoots were harvested at 13 weeks after herbicide application and dry weight recorded. Competition with globe fringerush reduced the number of culm and rice grain yield. The globe fringerush biotype in this field was resistant to all ALS herbicides tested. Penoxsulam had the highest level of activity among treatments at 28 and 70 DAA, but the control level was only 50% and 42%, respectively, in the second year of assessment. This was not enough to prevent rice yield loss. Alternative herbicides and weed control strategies are necessary to avoid yield losses in rice fields infested with ALS-resistant biotypes of globe fringerush.

Resistance of Amaranthus retroflexus to acetolactate synthase inhibitor herbicides in Brazil

When in competition with cotton, Amaranthus retroflexus can cause high yield losses. Due to the limited availability of selective herbicides registered for post emergence control of this weed, the same herbicides have been used repeated times over the last few years, which may have selected resistant biotypes. Biotypes of A. retroflexus collected from the main areas of cotton cultivation in Brazil were submitted to dose-response trials, by applying the herbicides trifloxysulfuron-sodium and pyrithiobac-sodium in doses equivalent to 0, ¼, ½, 1, 2 and 4 times the recommended rates. Resistance to ALS inhibitors was confirmed in biotypes of A. retroflexus. Biotype MS 2 from Mato Grosso do Sul, was cross-resistant to both trifloxysulfuron-sodium and pyrithiobac-sodium, while biotype MS 1 was resistant to trifloxysulfuron-sodium only. Likewise, singular and cross resistance was also confirmed in biotypes from Goiás (GO 3, GO 4 and GO 6), in relation to trifloxysulfuron­sodium and pyrithiobac-sodium. One biotype from Mato Grosso (MT 13) was not resistant to any of the ALS inhibitors evaluated in this work.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

The study of genetic polymorphisms related to serotonin in Alzheimer's disease: a new perspective in a heterogenic disorder

Genetic and environmental factors have been implicated in the development of Alzheimer's disease (AD), the most common form of dementia in the elderly. Mutations in 3 genes mapped on chromosomes 21, 14 and 1 are related to the rare early onset forms of AD while the e4 allele of the apolipoprotein E (APOE) gene (on chromosome 19) is the major susceptibility locus for the most common late onset AD (LOAD). Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter implicated in the control of mood, sleep, appetite and a variety of traits and behaviors. Recently, a polymorphism in the transcriptional control region upstream of the 5-HT transporter (5-HTT) gene has been studied in several psychiatric diseases and personality traits. It has been demonstrated that the short variant(s) of this 5-HTT gene-linked polymorphic region (5-HTTLPR) is associated with a different transcriptional efficiency of the 5-HTT gene promoter resulting in decreased 5-HTT expression and 5-HT uptake in lymphocytes. An increased frequency of this 5-HTTLPR short variant polymorphism in LOAD was recently reported. In addition, another common polymorphic variation in the 5-HT2A and 5-HT2C serotonin receptor genes previously analyzed in schizophrenic patients was associated with auditory and visual hallucinations in AD. These observations suggest that the involvement of the serotonin pathway might provide an explanation for some aspects of the affective symptoms commonly observed in AD patients. In summary, research on genetic polymorphisms related to AD and involved in receptors, transporter proteins and the enzymatic machinery of serotonin might enhance our understanding of this devastating neurodegenerative disorder.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110- 90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

Effects of a neuronal nitric oxide synthase inhibitor on lipopolysaccharide-induced fever

It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg/kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02). The coinjection of LPS and 7-NI was followed by a significant (P<0.02) hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 µl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 ± 1.54, MB = 18.5 ± 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 ± 1.97, MB = 33.77 ± 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.