A neurochemical analysis of the interaction of afferent signals at the spinal cord level]

The antinociceptive effect of electrical nerve stimulation with low (2-4 V, 100 Hz, 5 min) and high (45 V, 1 Hz, 1 min) intensity in the unanesthetized rats with the transsected spinal cord was determined by microelectrode recording in the ventrolateral tracts. The action of low intensity stimulation was reduced by yohimbine or during antinociceptive effect of clonidine. It was potentiated by baclofen and physostigmine. Prazosin, bicuculline, methysergid, strychnin, atropine, muscimole, THIP did not influence the effect of low-intensity stimulation. Metysergide partially reduced the effect of high-intensity stimulation. The data suggest that 2-adrenoreceptors are involved in the action of low-intensity stimulation and 5-HT-receptors are involved in the action of high-intensity one. For potentiating the effect of low-intensity stimulation on the segmental level it is possible to use baclofen and physostigmine.     

DNA binding chelates for nonviral gene delivery imaging

Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys(4)-Lys-(N-epsilon-[4-(psoralen-8-yloxy)]butyrate)-NH(2) has been characterized and labeled with reduced (99m)Tc pertechnetate (oxotechnetate). The psoralen moiety (a DNA binding group of PBC) allowed linking to double-stranded DNA upon short-term irradiation with the near UV range light (>320 nm). Approximately 30-40% of added (99m)Tc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of "naked" DNA and DNA complexes with lipid-based transfection reagents ("lipoplexes") has been performed after systemic or local administration of (99m)Tc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution using (99m)Tc-PBC and (32)P-labeled DNA and lipoplexes. A markedly different biodistribution of (99m)Tc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung. (99m)Tc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of "naked" (99m)Tc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging, (99m)Tc PBC-DNA lipoplexes showed a slower elimination from the site of injection than (99m)Tc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.     

Longitudinal ventricular function: normal values of atrioventricular annular and myocardial velocities measured with quantitative two-dimensional color Doppler tissue imaging.

OBJECTIVE: Quantitative 2-dimensional color Doppler tissue imaging is a new method to reveal impairment of left ventricular (LV) and right ventricular (RV) longitudinal function, which is a potential marker of early myocardial disease. The aim of this study was to obtain normal values for atrioventricular annular and regional myocardial velocities using this method. METHODS: A total of 123 healthy patients (age range: 22 to 89 years) underwent echocardiography including color Doppler tissue imaging using a scanner (Vivid 5, GE Vingmed, Horten, Norway) with postprocessing analysis (Echopac 6.3, GE Vingmed). Regional myocardial velocities were measured at 12 LV segments in 3 apical views and 2 segments of the free RV wall. Mitral annular velocities from 6 sites, and tricuspid annular velocities at its lateral site, were also assessed. At each site, systolic (S(m)), early diastolic (E(m)), and late diastolic (A(m)) velocities were measured, and the E(m)/A(m) ratio was calculated. RESULTS: Patients were classified into 4 groups aged 20 to 39, 40 to 59, 60 to 79, and >/=80 years. Mitral annular velocity and regional LV myocardial S(m) and E(m) progressively decreased with age. A(m), whereas low in the youngest age group, increased significantly in patients more than 40 years of age. The E(m)/A(m) ratio gradually declined with aging. There were no differences between age groups in S(m) measured at the tricuspid annulus and free RV wall, but the pattern of age-related changes of diastolic velocities and E(m)/A(m) ratio was the same as in the LV. Slight but significant sex-related differences were observed in middle-aged groups. The intraobserver and interobserver reproducibility was highest for atrioventricular annular velocities. CONCLUSIONS: A progressive decrease in S(m) reveals a decline in longitudinal systolic LV function with age, whereas systolic RV function remains unaffected. Atrioventricular annular velocity and regional E(m) decrease with aging in both ventricles, suggesting a deterioration in the diastolic properties of the myocardium, whereas A(m) increases from middle age implying a compensatory augmentation of atrial function. The study results can be used as reference data for the quantitative assessment of longitudinal LV and RV function in patients with cardiac disease.     

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Background

Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky.

Design and Methods

Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed.

Results

In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO₃-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na⁺/K⁺ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K⁺ uptake. Unidirectional K⁺ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K⁺,Cl⁻ co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors.

Conclusions

These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.     

Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases and mitogen activated kinase phosphatase‐1 in microglial cells

Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK‐3/6, MKK‐1/2 and MKK‐4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP‐1. Exposure of N9 microglia to Mn (250 µm ), LPS (100 ng ml^?1) or Mn + LPS increased MKK‐3/6 and MKK‐4 activity at 1 h; the effect of Mn + LPS on MKK‐4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK‐3/6 and MKK‐1/2 phosphorylation, whereas MKK‐4 was activated only by Mn and Mn + LPS. Besides activating MKK‐4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK‐4's phosphorylation on Ser80, which negatively regulates MKK‐4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP‐1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF‐α, IL‐6 and Cox‐2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK‐1/2, MKK‐3/6 and MKK‐4, are activated by Mn suggests that Mn‐induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn‐induced activation was persistent at least for 4 h, indicating a long‐term effect. Copyright © 2010 John Wiley & Sons, Ltd.