Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K-0.5 values for Na+ with minor alterations in K-0.5 values for K+ and N-H-4(+), causing a decrease in maximal velocities under saturating Na+, K+ and NH4+ concentrations. Phosphorylation measurements in the presence of 20 mu M ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na+, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na+, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na+ at the Na+-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments. (c) 2008 Elsevier Inc. All rights reserved.

Dendrimer intercalation in layered double hydroxides

New organic/inorganic (O/I) hybrid assemblies based on Layered Double Hydroxide (LDH) with polyamide amine dendrimer (PAMAM, generation -0.5 and generation +0.5) were prepared by two different routes using either the direct coprecipitation at constant pH or the anion exchange procedure in double surfactant S(+)S(-) phases. The obtained materials were characterized by means of powder X-ray diffraction, thermal gravimetric analysis associated with mass spectrometry, and Fourier-transform infrared spectroscopy. X-ray powder diffraction pattern of the O/I LDH assembly exhibit characteristic profiles of LDH-based materials with basal spacing depending on the nature of the dendrimer. Indeed, for both synthetic procedures, interleaved PAMAM -0.5 gives rise to an interlayer space in agreement with a perpendicular molecular arrangement against the layer of the host structure. For PAMAM+0.5, considering its spherical dimension, a much smaller basal spacing was observed. This observation was interpreted as shrinkage of the molecule to accommodate the interlayer LDH gap, which was rendered possible by the bond angle twisting within PAMAM-0.5. FTIR spectra confirm the presence of both moieties inside both Zn(2)Al/PAMAM G-0.5 and Zn(2)Al/PAMAM G+0.5 assemblies. Finally, thermal analysis associated with mass spectrometry confirm this composition, and in situ temperature XRD data reveal that the highly constrained arrangement for the generation +0.5 is not accompanied by a gain in thermal structural stability; in fact, the assembly prepared from PAMAM -0.5 is more stable. Both O/I PAMAM LDH assemblies constitute well-defined materials which are candidate for catalytic applications.

Temperature effect on the sorption of cholate anions on calcined LDH

Layered Double Hydroxides are a class of materials that can be described as positively charged layers of divalent and trivalent cations in the centre of edge-sharing octahedra. Cholesterol derivatives such as cholic acid are substances that play an important role in the digestion of fat components by the organism. This work presents a study on the intercalation of cholate anions in calcined MgAl-CO(3)-HDL. Isotherm experiments were performed at three different temperatures to evaluate the capacity of anion removal by sorption in the calcined LDH. The plateau was reached in all conditions. Increasing temperature results in decreasing cholate sorption. Characteristic peaks of LDH regenerated with OH(-) anions were observed at lower cholate concentrations. A peak in 2 theta equals to 7.5 degrees and peaks between 15 degrees and 20 degrees are observed. Those peaks are the same as the ones observed in the pure sodium cholate PXRD. At higher cholate concentrations the sorbed solids present PXRD related to an additional layered phase, which is related to intercalation of cholate anions with basal spacing equal to 34.3 angstrom. Thus, the cholate anions are also intercalated with a bilayer molecular arrangement at equilibrium concentrations at the isotherms plateau. (C) 2009 Elsevier Ltd. All rights reserved.

Microdialysis study of striatal dopamine in MPTP-hemilesioned rats challenged with apomorphine and amphetamine

Motor impairments of Parkinson`s disease (PD) appear only after the loss of more than 70% of the DAergic neurons of the substantia nigra pars compacta (SNc). An earlier phase of this disease can be modeled in rats that received a unilateral infusion of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) into the SNc. Though these animals do not present gross motor impairments, they rotate towards the lesioned side when challenged with DAergic drugs, like amphetamine and apomorphine. The present study aimed to test whether these effects occur because the drugs disrupt compensatory mechanisms that keep extracellular levels of dopamine in the striatum (DA(E)) unchanged. This hypothesis was tested by an in vivo microdialysis study in awake rats with two probes implanted in the right and left striatum. Undrugged rats did not present turning behaviour and their basal DA(E) did not differ between the lesioned and sham-lesioned sides. However, after apomorphine treatment, DA(E) decreased in both sides, but to a larger extent in the lesioned side at the time the animals started ipsiversive turning behaviour. After amphetamine challenge, DA(E) increased in both sides, becoming significantly higher in the non-lesioned side at the time the animals started ipsiversive turning behaviour. These results are in agreement with the hypothesis that absence of gross motor impairments in this rat model of early phase PD depends on maintenance of extracellular DA by mechanisms that may be disrupted by events demanding its alteration to higher or lower levels. (C) 2010 Elsevier B.V. All rights reserved.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Varicella zoster virus in bell`s palsy: a prospective study

Although Bell`s palsy is the major cause of acute peripheral facial palsy, its pathogenesis remains unknown. Reactivation of the varicella zoster virus has been implicated as one of the main causes of Bell`s palsy, however, studies which investigate the varicella zoster virus reactivation in Bell`s palsy patients are mostly Japanese and, therefore, personal and geographic characteristics are quite different from our population. Aims: To determine varicella zoster virus frequency in saliva samples from patients with Bell`s palsy, using PCR. Material and Method: One hundred seventy one patients with acute peripheral facial palsy were prospectively enrolled in this study. One hundred twenty were clinically diagnosed with Bell`s palsy, within one week of onset of the disease and no previous anti-viral therapy. We had 20 healthy adults as controls. Three saliva samples were collected from patients and controls at initial examination and at one and two weeks later. The detection of the varicella zoster virus DNA was performed using PCR. Results: Varicella zoster virus was detected in two patients (1.7%). The virus was not identified in saliva samples from the controls. Conclusions: Varicella zoster virus was detected in 1.7% of saliva samples from patients with Bell`s palsy, using PCR.

Selective Vagal Denervation of the Sinus and Atrioventricular Nodes, Guided by Vagal Reflexes Induced by High Frequency Stimulation, to Treat Refractory Neurally Mediated Syncope

Vagal Denervation and Neurally Mediated Syncope. A 15-year-old female patient presented with frequent episodes of vasovagal syncope refractory to non-pharmacological and pharmacological measures. Two tilt-table tests performed before and after conventional therapy were positive and reproduced the patient`s clinical symptoms. Selective vagal denervation, guided by HFS, was performed. Six radiofrequency pulses were applied on the left and right sides of the interatrial septum, abolishing vagal responses at these locations. Basal sinus node and Wenckebach cycle lengths changed significantly following ablation. A tilt test performed after denervation was negative and revealed autonomic tone modification. The patient reported significant improvement in quality of life and remained asymptomatic for 9 months after denervation. After this period, three episodes of NMS occurred during a 4-month interval and a tilt test performed 11 months after the procedure demonstrated vagal activity recovery. (J Cardiovasc Electrophysiol, Vol. 20, pp. 558-563, May 2009).

Effects of maternal glycemia on fetal heart rate in pregnancies complicated by pregestational diabetes mellitus

Objective: To investigate the influence of maternal glycemia on fetal heart rate (FHR) parameters analyzed by computerized cardiotocography in fetuses of diabetic mothers in the third trimester. Study design: Thirty-nine pregnant women with pregestational diabetes mellitus were studied prospectively. The inclusion criteria were a diagnosis of pregestational diabetes, singleton pregnancy between 36 and 40 weeks, and absence of fetal abnormalities. Computerized cardiotocography (System 8002) was performed over a period of 60 min and capillary glycemia was measured immediately before and 30 and 60 min after the beginning of the exam. The evaluations were done 2 h after lunch. Results: Nineteen patients (48.7%) presented mean glycemia >= 120 mg/dL The mean basal FHR was 136.7 +/- 10.0 bpm in the group with glycemia <120 mg/dL and 144.8 +/- 9.4 bpm in the group with glycemia >= 120 mg/dL (p = 0.013, Student`s t test). There was a significant positive correlation (Pearson`s test, p = 0.0001, r = 0.57) between basal FHR and mean glycemia. A significant negative correlation was observed between short-term variation and mean glycemia (Pearson`s test, p = 0.003, r = -0.47). No significant differences were observed between the other indices evaluated by computerized cardiotocography and glycemia. Conclusions: Maternal hyperglycemia at the time of cardiotocography is associated with elevated FHR. It seems to be important to understand how FHR parameters are influenced by maternal glycemic status at the time of fetal assessment in pregnancies complicated by diabetes. (C) 2009 Published by Elsevier Ireland Ltd.

Antinociception induced by epidural motor cortex stimulation in naive conscious rats is mediated by the opioid system

Epidural motor cortex stimulation (MCS) has been used for treating patients with neuropathic pain resistant to other therapeutic approaches. Experimental evidence suggests that the motor cortex is also involved in the modulation of normal nociceptive response, but the underlying mechanisms of pain control have not been clarified yet. The aim of this study was to investigate the effects of epidural electrical MCS on the nociceptive threshold of naive rats. Electrodes were placed on epidural motor cortex, over the hind paw area, according to the functional mapping accomplished in this study. Nociceptive threshold and general activity were evaluated under 15-min electrical stimulating sessions. When rats were evaluated by the paw pressure test, MCS induced selective antinociception in the paw contralateral to the stimulated cortex, but no changes were noticed in the ipsilateral paw. When the nociceptive test was repeated 15 min after cessation of electrical stimulation, the nociceptive threshold returned to basal levels. On the other hand, no changes in the nociceptive threshold were observed in rats evaluated by the tail-flick test. Additionally, no behavioral or motor impairment were noticed in the course of stimulation session at the open-field test. Stimulation of posterior parietal or somatosensory cortices did not elicit any changes in the general activity or nociceptive response. Opioid receptors blockade by naloxone abolished the increase in nociceptive threshold induced by MCS. Data shown herein demonstrate that epidural electrical MCS elicits a substantial and selective antinociceptive effect, which is mediated by opioids. (C) 2008 Elsevier B.V. All rights reserved.

Superior discriminating value of ACTH-stimulated serum 21-deoxycortisol in identifying heterozygote carriers for 21-hydroxylase deficiency

P>Background Congenital adrenal hyperplasia caused by classic 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder with a high prevalence of asymptomatic heterozygote carriers (HTZ) in the general population, making case detection desirable by routine methodology. HTZ for classic and nonclassic (NC) forms have basal and ACTH-stimulated values of 17-hydroxyprogesterone (17OHP) that fail to discriminate them from the general population. 21-Deoxycortisol (21DF), an 11-hydroxylated derivative of 17OHP, is an alternative approach to identify 21OHD HTZ. Objective To determine the discriminating value of basal and ACTH-stimulated serum levels of 21DF in comparison with 17OHP in a population of HTZ for 21OHD (n = 60), as well as in NC patients (n = 16) and in genotypically normal control subjects (CS, n = 30), using fourth generation tandem mass spectrometry after HPLC separation (LC-MS/MS). Results Basal 21DF levels were not different between HTZ and CS, but stimulated values were increased in the former and virtually nonresponsive in CS. Only 17 center dot 7% of the ACTH-stimulated 21DF levels overlapped with CS, when compared to 46 center dot 8% for 17OHP. For 100% specificity, the sensitivities achieved for ACTH-stimulated 21DF, 17OHP and the quotient [(21DF + 17OHP)/F] were 82 center dot 3%, 53 center dot 2% and 87%, using cut-offs of 40, 300 ng/dl and 46 (unitless), respectively. Similar to 17OHP, ACTH-stimulated 21DF levels did not overlap between HTZ and NC patients. A positive and highly significant correlation (r = 0 center dot 846; P < 0 center dot 001) was observed between 21DF and 17OHP pairs of values from NC and HTZ. Conclusion This study confirms the superiority of ACTH-stimulated 21DF, when compared to 17OHP, both measured by LC-MS/MS, in identifying carriers for 21OHD. Serum 21DF is a useful tool in genetic counselling to screen carriers among relatives in families with affected subjects, giving support to molecular results.

High Frequency of Lamivudine Resistance Mutations in Brazilian Patients Co-Infected With HIV and Hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co-infected with HIV and hepatitis B virus (HBV). A cross-sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an ""in-house"" real-time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty-eight patients with co-infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L+ rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sl195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481-1488, 2010. (C) 2010 Wiley-Liss, Inc.

In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas

Aims: To analyse the expression of three homeobox genes (HOXA7, PITX1 and PRRX1) in oral squanous cell carcinomas (OSCC) and the relationship of such expression to certain distinct histopathological features of OSCC and in comparison to adjacent non-neoplastic epithelium (NT). Methods and results: Digoxigenin-labelled riboprobes that are specific for each homeobox gene were generated and in situ hybridization was carried out on frozen sections. In NT samples, HOXA7 and PITX1 transcripts were found more frequently in all epithelial layers, while PRRX1 was expressed in the basal layer. With OSCC samples, expression of the three genes was associated with all histological features. However, the HOXA7 and PITX1 signals were more intense in sheets and nests and PRRX1 in small nests and isolated cells. Conclusion: HOXA7, PIXT1 and PRRX1 homeobox genes have different patterns of expression in OSCC depending on its histological features.

Botulinum Toxin Injection in Long-Standing Facial Paralysis Patients: Improvement of Facial Symmetry Observed up to 6 Months

Despite modern reanimation surgical techniques, facial paralysis presents with functional and aesthetic deficits. We evaluated facial symmetry after treating with botulinum toxin the healthy side of the face of 25 patients with long-standing facial paralysis who had previously been treated by surgical methods, with 6 months follow-up. Evaluation consisted of a clinical score, the two subscales of the Facial Disability Index, and surface electromyography. The mean botulinum toxin dose was 38 +/- A 5 U (range = 15-69 U). The clinical score showed significant reduction of asymmetry of 48.4% at 1 month and 16.8% after 6 months. The initial result was a consequence of reduced motion on the treated side combined with better motion on the paralyzed side. At 6 months, the treated side returned to basal scores. The residual effect seen in symmetry was due to an increase (18%) of motion in the paralyzed side. There was a significant decrease in the action potential of muscles on the nonparalyzed side 1 month post injection but completely reverted after 6 months. The Physical Function Index increased, but not significantly. The Social/Well-Being Function Index showed a significant increase at 6 months compared to pretreatment. The proposed treatment improved facial symmetry for up to 6 months. Even after the end of the clinical effect of the drug, the paralyzed side`s clinical score was 18% higher than pretreatment, with an increased quality of life.

Perineural Invasion in Aggressive Skin Carcinomas of the Head and Neck

Introduction: Perineural invasion is a well-recognized form of cancer dissemination. However, it has been reported only in few papers concerning cutaneous carcinomas ( basal cell, BCC, and squamous cell, SCC). Moreover, the incidence is considered to be very low. Niazi and Lambert [Br J Plast Surg 1993; 46: 156-157] reported only 0.18% of perineural invasion among 3,355 BCCs. It is associated with high-risk subtypes, as morphea-like, as well as with an increased risk of local recurrence. No paper was found in the literature looking for perineural invasion in very aggressive skin cancers with skull base extension, with immunohistochemical analysis. Methods: This is a retrospective review, including 35 very advanced skin carcinomas with skull base invasion (24 BCCs and 11 SCCs, operated on at a single institution from 1982 to 2000). Representative slides were immunohistochemically evaluated with antiprotein S-100, in order to enhance nerve fibers and to detect perineural invasion. The results were compared to 34 controls with tumors with a good outcome, treated in the same time frame at the same Institution. Results: Twelve (50.0%) of the BCCs with skull base invasion had proven perineural invasion, as opposed to only 1 (4.6%) of the controls, and this difference was statistically significant (p < 0.001). Regarding SCCs, 7 aggressive tumors (63.6%) showed perineural invasion compared to only 1 (10.0%) of the controls, but this difference did not reach significance (p=0.08), due to the small number of cases. Conclusions: In this series, it was demonstrated that immunohistochemically detected perineural invasion was very prevalent in advanced skin carcinomas. In addition, it was statistically associated with extremely aggressive BCCs with skull base invasion. Copyright (c) 2008 S. Karger AG, Basel