Thiamine deficiency leads to reduced nitric oxide production and vascular dysfunction in rats.

BACKGROUND AND AIMS: Thiamine deficiency is a condition that is known to cause damage to the nervous and cardiovascular systems because it interferes with cellular metabolism. It is well known that the control of vascular function is highly dependent on the production of nitric oxide (NO) by NO synthases. Studies exploring the physiological relevance of NO signaling under conditions of thiamine deficiency are scarce. The present study sought to investigate whether chronic metabolic changes would cause alterations in vascular responsiveness. METHODS AND RESULTS: By removing thiamine from the diet, we observed a reduced acetylcholine-mediated relaxation and an increased phenylephrine-mediated vasoconstriction in the aortas containing functional endothelium. Removal of the endothelium or the pre-treatment of vessels with l-NAME restored the contractile responses to the level of controls. Conversely, indomethacin did not modify phenylephrine-mediated contractions. We also used carbon microsensors to continually measure NO production in situ while simultaneously measuring the vascular tone. The results revealed a significant decrease in NO production. Western blot analysis showed a decreased expression of the total eNOS in the thiamine-deficient aorta compared to the control. Concentration-response curves for phenylephrine indicated no difference between the control and deficient groups in the presence and absence of SOD or Tyron. The NO donor DEA-NONOate produced a concentration-dependent relaxation response in the endothelium-denuded vessels that did not differ between the control and thiamine-deficient rats. CONCLUSION: Thiamine deficiency modulates eNOS-dependent NO production, leading to a decreased vasorelaxation and an increased contractile response in the rat aorta.

Regional variation in prosthesis choice for aortic valve replacement in older patients.

BACKGROUND AND AIM OF THE STUDY: The study aim was to investigate regional practice patterns regarding aortic valve replacement (AVR) by comparing bioprosthetic versus mechanical valve usage in patients aged > or = 65 years, and to determine whether the choice of valve type for AVR in these patients varied by geographic region. METHODS: The details were acquired of all mechanical and bioprosthetic AVRs performed in patients aged > or = 65 years between 1999 and 2006, as contained in the Florida State Inpatient Database. By using a small area analysis, the patients' zip codes were aggregated into hospital referral regions based on where they were most likely to receive AVR. The regional rates of both mechanical and bioprosthetic AVR were then determined. RESULTS: Of 23,925 AVRs performed during this period, 15,368 involved a bioprosthetic aortic valve and 8,557 a mechanical aortic valve. Statewide, 64% of AVRs in these patients involved a bioprosthesis. Regional rates of mechanical AVRs varied widely, from 10% to 81%. CONCLUSION: Substantial regional differences were identified in practice patterns for AVR in patients aged > or = 65 years. This suggested that provider preference, in addition to patient pathology, would often determine the type of valve implanted.

Geography or pathology? Regional variation in atrial septal defect closure rates and techniques.

BACKGROUND: Since the introduction of percutaneous closure in the United States, rates of secundum atrial septal defect and patent foramen ovale closures have increased substantially. Whether or not closure rates are uniform or vary due to differences in regional practice patterns is unknown. We sought to investigate this by comparing regional rates of closure across Florida. METHODS: We identified all atrial septal defect closures from 2001 to 2006 in the Florida State Inpatient Database. Using small area analysis, zip codes were assigned to Hospital Referral Regions based on where patients were most likely to go for closure. We obtained population-normalised rates of overall, percutaneous, and surgical closure. RESULTS: Of 1830 atrial septal defect and patent foramen ovale closures from 2001 to 2006, 751 were surgical and 1004 were percutaneous. The statewide closure rate was 1.91 per 100,000 people per year; regional rates varied 3.8-fold from 0.78 to 2.94 per 100,000 people per year. Percutaneous rates varied sevenfold from 0.25 to 1.75 per 100,000 people per year, while surgical rates varied 2.71-fold from 0.53 to 1.44 per 100,000 people per year. CONCLUSIONS: Despite a consistent prevalence of atrial septal defects, and patent foramens ovale, rates of repair vary across regions, suggesting that closure is driven by provider practice patterns rather than patient pathology. Efforts should be directed towards increasing consensus regarding the appropriate, evidence-based indications for closure so as to avoid the costs and potential negative sequelae of over- or undertreatment.

Does Distance Among Colonies and Resource Availability Explain the Intercolonial Aggressiveness in Nasutitermes aff. coxipoensis?

Aggressive behaviour can ensure animal access to local resources. To reduce constant costs in the defence of territories, species could save energy with conflicts avoiding aggression with neighbour or in situations with abundance of resources. In the present study, we analysed the effect of distance among colonies and resource availability on the aggression level and responses to chemical cues of Nasutitermes aff. coxipoensis (Holmgren) (Termitidae: Nasutitermitinae). Manipulation of resource offer was conducted in the field, where nests with different distances were kept without addition of baits (control), with addition of three or 16 sugarcane baits/nest. After 3 months, aggressiveness, linear and Y-shaped trail-following bioassays were carried out with all pairwise combinations of colonies in each treatment. Our results showed that aggressive index of N. aff. coxipoensis was affected by the resource availability. However, individuals from colonies with 0 and 3 baits/nest showed a higher number of fighting with neighbours than those from non-neighbours colonies. Termite workers from colonies without baits (control) followed shorter distance in the linear trails compared to those from colonies with addition of baits. In all treatments, there was no preference of workers in relation to the choice of chemical cues from own or other colonies. The response of intercolonial aggressiveness in N. aff. coxipoensis seems to be resource-dependent. These results may contribute to the comprehension of the use of space by N. aff. coxipoensis and could be useful to explain patterns of termite co-occurrence at different spatial scales, from local (inside the nest-e.g. cohabitation of nests by inquilines) to regional (e.g. around the nest).

Ants Associated with Turnera subulata (Turneraceae): Elaiosome Attraction, Seed Dispersion and Germination.

Symbiosis between plants and ants include examples in which the plant provides shelter and/or food for ants that, in turn, act in the defense or in the dispersion of seeds from the host plant. Although traditionally referred as mutualistic, the results of these interactions may vary with the ecological context in which patterns are involved. A range of species have facultative association with Turnera subulata (Turneraceae). Here, using behavioral bioassays, we investigated the effects of the most frequent ant species associated with T. subulata (Brachymyrmex sp.1, Camponotus blandus (Smith), Dorymyrmex sp.1, Crematogaster obscurata Emery, and Solenopsis invicta Buren) in the dispersion of plant host seeds and in the number of seedlings around the associated ant nests. We also evaluated the effects of these ant species in the germination of T. subulata seeds, in the consumption of elaiosome, and in the attractiveness to elaiosome odor. Our results showed that the ant species associated with T. subulata presented variation in the attraction by the odor and in the rate of consumption of the elaiosomes. However, none of the ant species studied contributed significantly to the increase of seed germination and seedling growth. Our results suggest that the consumption of the elaiosome by ant species is not a determinant factor to the success of germination of T. subulata. However, such species could contribute indirectly to seed germination by carrying seeds to sites more fertile to germination. In general, our results help to elucidate the results of ecological interactions involving ants and plants.

Is there a role for voltage-gated Na+ channels in the aggressiveness of breast cancer?

Breast cancer is the most common cancer among women and its metastatic potential is responsible for numerous deaths. Thus, the need to find new targets for improving treatment, and even finding the cure, becomes increasingly greater. Ion channels are known to participate in several physiological functions, such as muscle contraction, cell volume regulation, immune response and cell proliferation. In breast cancer, different types of ion channels have been associated with tumorigenesis. Recently, voltage-gated Na+ channels (VGSC) have been implicated in the processes that lead to increased tumor aggressiveness. To explain this relationship, different theories, associated with pH changes, gene expression and intracellular Ca2+, have been proposed in an attempt to better understand the role of these ion channels in breast cancer. However, these theories are having difficulty being accepted because most of the findings are contrary to the present scientific knowledge. Several studies have shown that VGSC are related to different types of cancer, making them a promising pharmacological target against this debilitating disease. Molecular biology and cell electrophysiology have been used to look for new forms of treatment aiming to reduce aggressiveness and the disease progress.

Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.

Mechanism of antitumor activity of a single-chain interleukin-12 IgG3 antibody fusion protein (mscIL-12.her2.IgG3).

We have constructed an antibody interleukin-12 (IL-12) fusion protein (mscIL-12.her2.IgG3) that demonstrates significant antitumor activity against the murine carcinoma CT26-expressing human HER2/neu. We now report that this antitumor activity is dose dependent and comparable to or better than recombinant murine IL-12 (rMuIL-12) using subcutaneous and metastatic models of disease. The antitumor activity of mscIL-12.her2.IgG3 is reduced in Rag2 knockout mice, suggesting that T cells play a role in tumor rejection. In SCID-beige mice, the antitumor activity is further reduced, suggesting that natural killer (NK) cells or macrophages or both are also important. The isotype of the antibody response to HER2/neu is consistent with a switch from a Th2 to a Th1 immune response and the infiltration of mononuclear cell in tumors from mice treated with mscIL-12.her2.IgG3. Immunohistochemistry reveals that mscIL-12.her2.IgG3 is antiangiogenic. Thus, the mechanism of the antitumor activity exhibited by mscIL-12.her2.IgG3 is highly complex and involves a combination of T and NK cell activity, a switch to a Th1 immune response, and antiantiogenic activity. This is the first study comparing the in vivo antitumor activity of an antibody-IL-12 fusion protein and free IL-12. Our results suggest that antibody-IL-12 fusion proteins may be useful for the treatment of human cancer.

Inhibition of neuronal high-voltage activated calcium channels by the omega-phoneutria nigriventer Tx3-3 peptide toxin.

We have investigated the effect of omega-PnTx3-3 (referred to in previous papers simply as Tx3-3), a peptide toxin from the venom of the spider Phoneutria nigriventer, on neuronal high-voltage activated (HVA) Ca(2+) channels, using whole-cell patch-clamp. omega-PnTx3-3 (120 nM) blocked 74+/-8% of the total HVA Ca(2+) currents of cerebellar granule neurones, without affecting the low-voltage activated (LVA) current. P/Q/R-type currents in cerebellar granule neurones, isolated using 4 microM nicardipine and 100 nM omega-conotoxin GVIA, were markedly (79+/-6%) inhibited by 60 nM omega-PnTx3-3. R-type currents, isolated either by additional application of 0.5-1 microM of omega-agatoxin IVA or by pre-incubation with 5 microM omega-conotoxin MVIIC were inhibited almost totally by 120 nM of omega-PnTx3-3. omega-PnTx3-3 reversibly altered the kinetics of the P/Q/R current, increasing the degree of inactivation that occurred during a 50 ms pulse from 20% to 40%. N-type currents, recorded from neuroblastoma N18 cells, were partially (34+/-2%) inhibited by 320 nM omega-PnTx3-3. L-type currents, recorded from GH3 cells, were partially (45+/-12%) inhibited by 80 nM omega-PnTx3-3. We conclude that omega-PnTx3-3 inhibits all known HVA Ca(2+) channels, and most effectively the P/Q- and R-type currents.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Regulation of the glutamate uptake by extracellular calcium.

To determine whether [Ca(2+)](e) modulates glutamate re-uptake, we studied the uptake mechanism into rat cerebrocortical synaptosomes. The removal of extracellular Ca(2+) caused a negative modulation in the uptake mechanism. The calculated K(50) value was 0.185 +/- 0.019 mM (n = 4). The Michaelis-Menten data analysis indicate that absence of Ca(2+) diminished the V(max) kinetic parameter by about 60% without changing significantly the K(m) suggesting a non-competitive mechanism. We also tested the involvement of intracellular Ca(2+) in this phenomenon by trapping BAPTA into the synaptosomal vesicles to control the Ca(2+) concentration. Our results suggest that intracellular Ca(2+) changes have a less predominant role on the glutamate uptake than do extracellular Ca(2+). These findings argue in favor of an important role of extracellular [Ca(2+)] in maintaining the L-glutamate re-uptake mechanism in the mammalian central nervous system.

Tension generation and increase in voltage-activated Na+ current by crotamine.

We performed the present experiments to study the action of crotamine, a toxin isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus, on macroscopic Na+ currents in frog skeletal muscle by using the loose patch clamp technique. Crotamine at 50 microM increased the peak Na+ current by 50% (P < 0.05). In addition, the voltage dependence of inactivation was shifted by +8 mV. Other parameters of Na+ currents (reversal potential, voltage-dependence of activation and time courses of inactivation, of activation and of removal of inactivation) were not significantly affected. We suggest that crotamine inhibits the direct transition of channels from closed to inactivated states, thereby forcing their transition through the open states.

Tityustoxin effect on nerve compound action potentials requires extracellular sodium.

Previous studies have demonstrated that Li(+) ions can substitute for Na(+) in a variety of functional systems. Using the single sucrose-gap recording technique, we measured the nerve compound action potential to study the effects of tityustoxin (an alpha-scorpion toxin that selectively inhibits fast Na(+) channel inactivation) upon removal of extracellular Na(+). Our results suggest that tityustoxin requires the presence of extracellular Na(+) to produce its typical pharmacological effect on Na(+) channel inactivation kinetics, but not to bind to its site.

Glutamate transport in rat cerebellar granule cells is impaired by inorganic epileptogenic agents.

There is evidence that extracellular glutamate levels are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. There appears to be a correlation between the capacity of removing released glutamate and the genesis of epileptiform activity. Some models make use of metals, such as Co(2+) and Ni(2+), to induce epilepsy. We used patch-clamp recordings to measure the electrogenic glutamate transport in neuronal cells. The present results indicate that Co(2+) (1 mM) and Ni(2+) (5 mM) blocked glutamate transport by 17.6+/-3.9% (n=5, P<0.05) and by 31.8+/-6.2% (n=7, P<0.05), respectively. Ni(2+) inhibited glutamate uptake in a dose-dependent manner. The IC(50) value obtained was 66.6 microM and the maximum inhibition was 40%. We conclude that one mechanism that may explain the seizures induced by exposure to those divalent cations is inhibition of the glutamate transporter.

Cloning of cDNAS encoding neurotoxic peptides from the spider Phoneutria nigriventer.

A cDNA library made from venom glands of the spider Phoneutria nigriventer was constructed and used to clone neurotoxic peptides. A cDNA of about 360 nucleotides encoding the precursor for the toxin Tx2-1 active on mammals has been isolated. The deduced amino acid sequence for the mature polypeptide confirms the polypeptide sequence previously published. In addition, two new putative toxins called Pn2-1A and Pn2-5A have been characterized and their complete amino acid sequence show 92% similarity to Tx2-1 and 94% similarity to Tx2-5 respectively. The cDNAs revealed that the precursors contain signal peptides characterized by a very hydrophobic core and a propeptide interposed between the signal sequence and the peptide toxin.

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer).

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.

Antibody-cytokine fusion proteins: innovative weapons in the war against cancer.

Cancer patients who are administered therapeutic doses of cytokines (e.g., interleukin-2, granulocyte macrophage colony stimulating factor, interleukin-12, and tumor necrosis factor-alpha) frequently develop devastating toxic side effects that can lead to discontinuation of therapy. This problem has compelled numerous investigators to design innovative strategies that will reduce prolonged systemic cytokine exposure and promote cytokine accumulation at the site of the tumor. One such strategy involves the use of antibody-cytokine fusion proteins consisting of immunoenhancing cytokines genetically fused to antibodies that are able to target specific antigens exclusively expressed or overexpressed on the surface of tumor cells. Preclinical studies examining their therapeutic efficacy demonstrate that they posses potent tumoricidal activity, suggesting that they may be clinically useful as novel cancer therapeutic agents.

Utilization of O-phthalaldehyde-sulphuric acid as a spray reagent in thin-layer chromatographic detection of some indolealkylamines and application to cutaneous secretion extracts of toad species.

An expansion of the utilisation of o-phthalaldehyde in sulphuric acid medium as spray reagent was carried out when tryptophan and some tryptophan-derived indole alkylamines such as tryptamine, serotonin, bufotenine, dehydrobufotenine and bufotenidine were examined by thin-layer chromatography. Rf-values and limits of detection ranging from 20 (serotonin) to 100 (dehydrobufotenine) ng per spot were found. Application of this reagent for the detection of some of these compounds was carried out, using either methanolic extracts or column chromatographic fractions of the skin secretion of the toads Bufo ictericus and Odontophrynus cultripes.

A recombinant IgG3-(IL-2) fusion protein for the treatment of human HER2/neu expressing tumors.

Anti-HER2/neu therapy of human HER2/neu expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu human IgG3 fusion protein containing interleukin-2 (IL-2) fused at its carboxyl terminus. Anti-Her2/neu IgG3-(IL-2) retained antibody and cytokine related activity. Treatment of immunocompentent mice with this antibody fusion protein resulted in significant retardation in the subcutaneous (s.c.) growth of CT26-HER2/neu tumors suggesting that anti-HER2/neu IgG3-(IL-2) fusion protein will be useful in the treatment of HER2/neu expressing tumors. We also found that fusing IL-2 to human IgG3 results in a significant enhancement of the murine anti-human antibody (MAHA) response.

Partial purification and pharmacological characterization of a neurotoxic fraction isolated from the venom of the spider Lycosa erythrognatha.

The effect of the venom of the spider Lycosa erythrognatha on the frog sciatic nerve was investigated with the single sucrose-gap method. Solutions containing the crude venom (40 micrograms protein/ml) markedly increased the duration of compound action potentials and caused the appearance of long-lasting depolarizing post-potentials. These effects were only partially (20%) reversed by extensive washing with control solution. The active material was sensitive to proteolytic treatments with pronase or trypsin and was separated with 20% acetonitrile and 0.1% trifluoroacetic acid by reverse phase chromatography. The effect of this fraction (LycIV) on the post-potential amplitude was concentration-dependent, and was fitted with a quadratic hyperbola having a half maximal effect of 0.9 microgram protein/ml. SDS-polyacrylamide gel electrophoresis of LycIV showed an enriched polypeptide band with apparent molecular weight of approximately 8 kDa. The observed effects were similar to those of toxins that inhibit sodium channel inactivation and different from the effects of potassium channel blockers. Pore formation or membrane disruption could be ruled out. It was concluded that the venom contains a neurotoxic polypeptide that alters the repolarization of action potentials, probably by inhibiting sodium channel inactivation.