Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.     

Signaling role of PDE isozymes in pathobiology of glomerular mesangial cells. Studies in vitro and in vivo

Mesangial cells (MC) of renal glomeruli respond to immune-inflammatory injury by accelerated proliferation and generation of reactive oxygen metabolites (ROM). We studied in vivo and in vitro roles of cAMP-protein kinase A (PKA) signaling in modulation of these pathobiologic processes with focus on PDE isozymes. Mitogenic synthesis of DNA in mesangial cells grown in primary culture was blocked by forskolin and dibutyryl cyAMP. Incubation of MC with PDE-3 inhibitors, cilostamide and lixazinone, inhibited (> 50%) mitogenesis, whereas inhibitors of PDE-4, rolipram and denbufylline, caused little or no inhibition. Conversely, inhibitors of PDE-4 suppressed generation of ROM in MC, whereas inhibitors of PDE-3 had no effect. Incubation of mesangial cells with cilostamide or with rolipram increased in situ activity of PKA, and effects of the two inhibitors were additive. PDE inhibitors also decreased activity of mitogen-activated protein kinase. The efficacy of PDE isozyme inhibitors (IC50) to suppress mitogenesis or ROM generation paralleled IC50 for inhibition of cAMP hydrolysis by extracts from mesangial cells. Administration of lixazinone or lixazinone in combination with rolipram to rats with mesangial proliferative glomerulonephritis induced by antithymic serum suppressed proliferation of mesangial cells and also reduced other histopathologic manifestations of the disease. Based on these observations, we propose that in MC, a cAMP pool that is hydrolyzed by PDE-3 inhibits by negative crosstalk via activation of PKA, mitogen-activated protein kinase (MAPK) pathway, and mitogenesis; whereas cAMP pool linked to PDE-4 inhibits, also via activation of PKA, ROM generation in mesangial cells. Results also suggest that PDE isozyme inhibitors, in particular inhibitors of PDE-3, should be investigated for potential use for "signal transduction pharmacotherapy" of glomerulonephritis.     

Binding and lysing of blood clots using MRX-408

RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.     

Anion modulation of taste responses in sodium-sensitive neurons of the hamster chorda tympani nerve

Beidler's work in the 1950s showed that anions can strongly influence gustatory responses to sodium salts. We have demonstrated "anion inhibition" in the hamster by showing that the chorda tympani nerve responds more strongly to NaCl than to Na acetate over a wide range of concentrations. Iontophoretic presentation of Cl- and acetate to the anterior tongue elicited no response in the chorda tympani, suggesting that these anions are not directly stimulatory. Drugs (0.01, 1.0, and 100 microM anthracene-9-carboxylate, diphenylamine-2-carboxylate, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate, and furosemide) that interfere with movements of Cl- across epithelial cells were ineffective in altering chorda tympani responses to 0.03 M of either NaCl or Na acetate. Anion inhibition related to movements of anions across epithelial membranes therefore seems unlikely. The chorda tympani contains a population of nerve fibers highly selective for Na+ (N fibers) and another population sensitive to Na+ as well as other salts and acids (H fibers). We found that N fibers respond similarly to NaCl and Na acetate, with spiking activity increasing with increasing stimulus concentration (0.01-1.0 M). H fibers, however, respond more strongly to NaCl than to Na acetate. Furthermore, H fibers increase spiking with increases in NaCl concentration, but generally decrease their responses to increasing concentrations of Na acetate. It appears that anion inhibition applies to taste cells innervated by H fibers but not by N fibers. Taste cells innervated by N fibers use an apical Na+ channel, whereas those innervated by H fibers may use a paracellularly mediated, basolateral site of excitation.     

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

Haloperidol and apomorphine differentially affect neuropeptidase activity

The effects of lipid peroxidation and the antioxidant vitamin E contained in LDL isolated from control plasma (LDL--) and from plasma preincubated with 0.5 mmol/ml alpha-tocopherol (LDL+) on the proliferation of estrogen-receptor positive (ER+ : ZR-75, T-47-D, MCF-7) and negative (ER--: HBL-100, MDA-MB-231) human breast cancer cells were studied. Human skin fibroblasts served as controls. Incubation of plasma with 0.5 mmol/ml alpha-tocopherol resulted in a 3-fold increase of its content and a significant reduction in lipid hydroperoxides and conjugated dienes in LDL. Incubation of fibroblasts or ER+ tumor cells with LDL- or LDL+ had an effect on neither cell proliferation nor on the cellular levels of peroxidation products as compared to control incubations in the absence of LDL. In ER- cells, however, LDL+ stimulated the proliferation, whereas LDL- yielded a cytotoxic effect. Moreover, LDL- supplementation resulted in an increase in the content of hydroperoxides and conjugated dienes. LDL+ supplemented cells exhibited hydroperoxide levels in these tumor cells comparable to the basal levels measured in the absence of LDL. Our data suggested that peroxidation products in LDL are cytotoxic to estrogen-receptor negative breast tumor cells and vitamin E counteracts this effect.     

Correlation of functional magnetic source imaging with intraoperative cortical stimulation in neurosurgical patients

This study compared noninvasive preoperative functional imaging by using magnetoencephalography (MEG) with intraoperative direct cortical stimulation in ten patients undergoing neurosurgery. The goal was to assess the accuracy and reliability of MEG-based functional imaging in these patients as a possible replacement or adjunct for direct cortical stimulation with electrocorticography. Objective comparison of intraoperative mapping with preoperative MEG procedures was achieved by intraoperative recording of mapped cortical locations for motor responses using an interactive image-guided surgical device, the ISG viewing wand, with which mapping points could be marked on a previously acquired (MRI) set. In all ten patients, at least one stimulation site elicited a response during both MEG and intraoperative mapping. The central sulcus ipsilateral to the lesion was only directly visible on high-resolution MRIs in 3/10 cases and equivocally in 2/10. Coregistered with MRI to form magnetic source images (MSIs), MEG predictions of the postcentral gyrus were possible in all 10 cases. In all 10 cases, these were in agreement with intraoperative estimation of the precentral gyrus. Functional mapping of somatosensory cortex was achieved noninvasively in surgical patients by using MSI. The accuracy, compared with cortical stimulation, was always sufficient to define motor and somatosensory strips.     

Applying the realized probability of dying to cancer survival

Age comparisons of survival in cancer cohorts generally utilize relative survival rates, which are based on indicators of the probability of survival for a given number of years after diagnosis. Cancer relative survival rates for the same number of years tend to decline as age at diagnosis increases. However, the same number of years of survival reflects higher relative longevity at older ages than at younger ages. The realized probability of dying (RPD) is a survival measure that expresses individual survival time after diagnosis relative to the survival distribution of an age-, race-, and sex-specific reference population, in effect weighing individual survival time more heavily as age at diagnosis increases. The purpose of this study was to apply the RPD as a survival measure in cancer epidemiology. Two cohorts of cancer patients, white males with prostate cancer and white females with breast cancer, aged 55 years and over at diagnosis, were followed for 15 years. Although older subjects survived less time after diagnosis than younger subjects, they achieved more favorable RPD values. We present survival analysis methods for analyzing the RPD in this population, an approach not previously used with this measure. The implications for use of the RPD in cancer epidemiology are discussed.     

Saccade-related omnivectoral pause neurons in the primate zona incerta

The zona incerta contains GABAergic neurons projecting directly on to collicular efferent neurons. This study examined the physiological characteristics of incertal neurons and their relationship to saccades. Cells in the zona incerta have moderate rates of activity and pause during saccades. These pauses begin prior to the start of saccades and activity resumes after the end of saccades in all directions and for all amplitudes. This perisaccadic pause is positively correlated with the duration of the saccade such that the longer the saccade is, the longer a pause is observed. These physiological data, in conjunction with anatomical observations, suggest that the zona incerta might normally function by inhibiting the superior colliculus and then by disinhibiting the superior colliculus for saccades.