Cathelicidin suppresses lipid accumulation and hepatic steatosis by inhibition of the CD36 receptor.

BACKGROUND AND OBJECTIVES: Obesity is a global epidemic which increases the risk of the metabolic syndrome. Cathelicidin (LL-37 and mCRAMP) is an antimicrobial peptide with an unknown role in obesity. We hypothesize that cathelicidin expression correlates with obesity and modulates fat mass and hepatic steatosis. MATERIALS AND METHODS: Male C57BL/6 J mice were fed a high-fat diet. Streptozotocin was injected into mice to induce diabetes. Experimental groups were injected with cathelicidin and CD36 overexpressing lentiviruses. Human mesenteric fat adipocytes, mouse 3T3-L1 differentiated adipocytes and human HepG2 hepatocytes were used in the in vitro experiments. Cathelicidin levels in non-diabetic, prediabetic and type II diabetic patients were measured by enzyme-linked immunosorbent assay. RESULTS: Lentiviral cathelicidin overexpression reduced hepatic steatosis and decreased the fat mass of high-fat diet-treated diabetic mice. Cathelicidin overexpression reduced mesenteric fat and hepatic fatty acid translocase (CD36) expression that was reversed by lentiviral CD36 overexpression. Exposure of adipocytes and hepatocytes to cathelicidin significantly inhibited CD36 expression and reduced lipid accumulation. Serum cathelicidin protein levels were significantly increased in non-diabetic and prediabetic patients with obesity, compared with non-diabetic patients with normal body mass index (BMI) values. Prediabetic patients had lower serum cathelicidin protein levels than non-diabetic subjects. CONCLUSIONS: Cathelicidin inhibits the CD36 fat receptor and lipid accumulation in adipocytes and hepatocytes, leading to a reduction of fat mass and hepatic steatosis in vivo. Circulating cathelicidin levels are associated with increased BMI. Our results demonstrate that cathelicidin modulates the development of obesity.

Phosphorylation of glycogen synthase kinase-3ß in metabolically abnormal obesity affects immune stimulation-induced cytokine production.

BACKGROUND: Obesity is a severe health problem worldwide, which leads to multiple comorbidities including type 2 diabetes mellitus and cardiovascular diseases. Inflammation has been found to be an important characteristic of adipose tissue in obese subjects. However, obesity is also associated with compromised immune responses to infections and the impact of obesity on immune function has not been fully understood. SUBJECTS/METHODS: To clarify the role of obesity in the immune responses, we investigated the Toll-like receptor (TLR)-induced cytokine secretion by leukocytes from obese and lean subjects. We also investigated the relationship between insulin-induced intracellular signaling and cytokine production using peripheral blood mononuclear cells (PBMCs) and a monocytic cell line THP-1. RESULTS: We found decreased TLR-induced interferon-γ, interleukin-6 (IL-6) and tumor necrosis factor-α secretions and elevated IL-10 secretion by leukocytes from obese subjects when compared with lean controls. PBMCs from obese subjects showed enhanced basal Akt and glycogen synthase kinase-3ß (GSK-3ß) phosphorylation, which did not further increase with insulin and lipopolysaccharide (LPS) stimulation. We also found that LPS-induced IκBα degradation was inhibited in PBMCs from obese subjects. By using THP-1 cells with GSK-3ß knockdown or cells treated with hyperinsulinemic and high-fatty acid conditions, we found that LPS-induced nuclear factor κB (NF-κB) activation was inhibited and cyclic adenosine monophosphate response element-binding protein (CREB) activation was enhanced. CONCLUSIONS: These findings indicate that GSK-3ß is important in the regulation of NF-κB and CREB activation in leukocytes under the metabolic condition of obesity. Our study revealed a key mechanism through which metabolic abnormalities compromise leukocyte functions in people with obesity.

The metabolome profiling and pathway analysis in metabolic healthy and abnormal obesity.

OBJECTIVES: Mechanisms of the development of abnormal metabolic phenotypes among obese population are not yet clear. In this study, we aimed to screen metabolomes of both healthy and subjects with abnormal obesity to identify potential metabolic pathways that may regulate the different metabolic characteristics of obesity. METHODS: We recruited subjects with body mass index (BMI) over 25 from the weight-loss clinic of a central hospital in Taiwan. Metabolic healthy obesity (MHO) is defined as without having any form of hyperglycemia, hypertension and dyslipidemia, while metabolic abnormal obesity (MAO) is defined as having one or more abnormal metabolic indexes. Serum-based metabolomic profiling using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of 34 MHO and MAO individuals with matching age, sex and BMI was performed. Conditional logistic regression and partial least squares discriminant analysis were applied to identify significant metabolites between the two groups. Pathway enrichment and topology analyses were conducted to evaluate the regulated pathways. RESULTS: A differential metabolite panel was identified to be significantly differed in MHO and MAO groups, including L-kynurenine, glycerophosphocholine (GPC), glycerol 1-phosphate, glycolic acid, tagatose, methyl palmitate and uric acid. Moreover, several metabolic pathways were relevant in distinguishing MHO from MAO groups, including fatty acid biosynthesis, phenylalanine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation. CONCLUSION: Different metabolomic profiles and metabolic pathways are important for distinguishing between MHO and MAO groups. We have identified and discussed the key metabolites and pathways that may prove important in the regulation of metabolic traits among the obese, which could provide useful clues to study the underlying mechanisms of the development of abnormal metabolic phenotypes.

Two-minute versus 6-minute walk distances during 6-minute walk test in neuromuscular disease: Is the 2-minute walk test an effective alternative to a 6-minute walk test?

Functional tests such as Motor Function Measure-32 (MFM-32), supine to stand, ascend/descend stairs permit the assessment of task-specific motor function in neuromuscular disease (NMD). The 6-min walk test (6MWT), though functional, is primarily used to assess endurance and disease progression in children with neuromuscular disorders. Barriers to 6MWT administration, in this population, can include reduced attention span due to age and inability to tolerate test length due to weakness. We propose task-specific functional deficits are related to endurance. Additionally, the 2-min walk test (2MWT) could effectively replace the 6MWT in this population. Seventy-seven participants, ages 5-18, with a variety of neuromuscular disorders performed the 6MWT, timed functional tests (TFT), and the MFM-32. Correlation and paired t-test analyses were used to compare the distance walked in the first 2 min (2MWD) to the distance walked in the entire 6 min (6MWD) and to the functional outcome measures above. The 2MWD strongly correlated with 6MWD and the other outcome measures. Paired t-test analysis also showed that the 2MWD did not differ from the distance walked in the last 2 min of the 6MWT. Although equivalence testing could not reject the claim that this difference exceeded the upper practical limit of 9.5 m, it only showed a modest overestimation of the 4-6MWD compared with the 2MWD. Together, our results support the ability of the 2MWD to predict the 6MWD, specifically in the pediatric neuromuscular disease population.

K+ pore structure revealed by reporter cysteines at inner and outer surfaces.

The structure of the carboxyl half of the pore-forming region of Kv2.1 was studied by replacing each of 15 consecutive residues between positions 383 and 369 with a reporter cysteine residue. Extracellular application of charged, membrane-impermeant methanethiosulfonates irreversibly modified currents at four cysteine-substituted positions, K382, Y380, I379, and D378. Intracellular exposure to methanethiosulfonate ethyltrimethylammonium revealed another set of reactive mutants (V374, T373, T372, and T370). Our results indicate that positions 378 and 374 are exposed at outer and inner mouths of the channel, respectively, and immersed in the aqueous phase. In contrast to present topological models, the 383-369 region appears to span the pore mainly as a nonperiodic structure.

Segmental exchanges define 4-aminopyridine binding and the inner mouth of K+ pores.

4-Aminopyridine (4AP) blocks the intracellular mouth of voltage-gated K+ channels. We identified critical regions for 4AP binding with chimeric channels in which segments of a low affinity clone (Kv2.1, IC50 = 18 mM) were replaced with those of a high affinity clone (Kv3.1, IC50 = 0.1 mM). 4AP sensitivity was not transferred with the S5-S6 linker (pore or P region). Instead, a chimera of the cytoplasmic half of S6 increased block 20-fold, without affecting gating. A double chimera of the cytoplasmic halves of S5 and S6 fully transferred 4AP sensitivity. Because 4AP block was inhibited by tetrapentylammonium, we conclude that determinants of 4AP binding lie in the S6 segment that forms the cytoplasmic vestibule of the pore and that this site may overlap a quaternary ammonium site.

The functional insufficiency of human CD4+CD25 high T-regulatory cells in allergic asthma is subjected to TNF-alpha modulation.

BACKGROUND: Natural CD4(+)CD25(high)Foxp3(+) regulatory T (nTreg) cells are important in maintaining immunologic tolerance, but their role in the pathogenesis of allergic asthma is unclear. We studied the function of nTreg cells in allergic asthmatic children and assessed the factors which may relate to the functional insufficiency of nTreg cells. METHODS: The percentage of CD4(+)CD25(high) Treg cells, the expression of Foxp3, and the cell-induced suppressive activity of nTreg cells isolated from nonatopic controls, allergic asthmatics, and allergen-specific immunotherapy (AIT)-treated asthmatic patients were studied. RESULTS: Although the percentage of nTreg in peripheral blood mononuclear cells was increased, the expression of Foxp3 and its cell-induced suppressive activity were significantly lower in Dermatophagoides pteronyssinus (Der p)-sensitive asthmatic children when compared to nonatopic controls. In contrast, the expression of Foxp3 and the functional activity of nTreg cells were reversed in allergic asthmatics who received AIT. The addition of recombinant tumor necrosis factor (TNF)-alpha directly downregulated Foxp3 expression and abrogated the cell-induced suppressive function of Treg cells. The anti-TNF-alpha reagent, etanercept, restored the functional activity and Foxp3 expression of CD4(+)CD25(high) Treg derived from allergic asthmatics. CONCLUSIONS: The functional insufficiency of nTreg cells in patients with allergic asthma may be related to the enhanced production of TNF-alpha and its effect on the Foxp3 expression. These results may explain, in part, the effectiveness of anti-TNF-alpha therapy in the treatment of allergic asthma.

Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(gamma-glutamic acid) by RSM.

Poly(gamma-glutamic acid) (gamma-PGA) is a material of polymer. Immobilization of Candida rugosa lipase (Lipase AY-30) by covalent binding on gamma-PGA led to a markedly improved performance of the enzyme. Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were employed to evaluate the effects of immobilization parameters, such as immobilization time (2-6h), immobilization temperature (0-26 degrees C), and enzyme/support ratio (0.1-0.5, w/w). Based on the analysis of ridge max, the optimum immobilization conditions were as follows: immobilization time 2.3h, immobilization temperature 13.3 degrees C, and enzyme/support ratio 0.41 (w/w); the highest lipase activity obtained was 1196 U/mg-protein.

Intervention strategies for preventing excessive gestational weight gain: systematic review and meta-analysis.

BACKGROUND: Interventions relevant to energy intake to prevent excessive gestational weight gain in pregnant overweight and obese women are important but scarce. This review synthesized healthy eating and physical activity strategies and their effects on excessive gestational weight gain prevention. METHODS: Twenty-three randomized controlled trials that included healthy eating and/or physical activity as an intervention in healthy pregnant overweight or obese adult women and gestational weight gain as a primary or secondary outcome were reviewed. FINDINGS: Heathy eating and/or physical activity (21 studies, n = 6,920 subjects) demonstrated 1.81 kg (95% CI: -3.47, -0.16) of gestational weight gain reduction favouring intervention. Healthy eating (-5.77 kg, 95% CI: -9.34, -2.21, p = 0.02) had a larger effect size than combined healthy eating/physical activity (-0.82 kg, 95% CI: -1.28, -0.36, p = 0.0005) in limiting gestational weight gain. Physical activity did not show a significant pooled effect. Healthy eating with prescribed daily calorie and macronutrient goals significantly limited gestational weight gain by 4.28 kg and 4.23 kg, respectively. CONCLUSION: Healthy eating and/or physical activity are effective in gestational weight gain control. Healthy eating with calorie and macronutrient goals are especially effective in limiting excessive gestational weight gain among pregnant overweight and obese women.

P2X7-related modulation of pathological nociception in rats.

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

A-272651, a nonpeptidic blocker of large-conductance Ca2+-activated K+ channels, modulates bladder smooth muscle contractility and neuronal action potentials.

BACKGROUND AND PURPOSE: The large-conductance Ca(2+)-activated K(+) channel (BK(Ca), K(Ca)1.1) links membrane excitability with intracellular Ca(2+) signaling and plays important roles in smooth muscle contraction, neuronal firing, and neuroendocrine secretion. This study reports the characterization of a novel BK(Ca) channel blocker, 2,4-dimethoxy-N-naphthalen-2-yl-benzamide (A-272651). EXPERIMENTAL APPROACH: (86)Rb(+) efflux in HEK-293 cells expressing BK(Ca) was measured. Effects of A-272651 on BK(Ca) alpha- and BK(Ca) alphabeta1-mediated currents were evaluated by patch-clamp. Effects on contractility were assessed using low-frequency electrical field stimulated pig detrusor and spontaneously contracting guinea pig detrusor. Effects of A-272651 on neuronal activity were determined in rat small diameter dorsal root ganglia (DRG). KEY RESULTS: A-272651 (10 microM) inhibited (86)Rb(+) efflux evoked by NS-1608 in HEK-293 cells expressing BK(Ca) currents. A-272651 concentration-dependently inhibited BK(Ca) currents with IC(50) values of 4.59 microM (Hill coefficient 1.04, measured at +40 mV), and 2.82 microM (Hill coefficient 0.89), respectively, for BK(Ca) alpha and BK(Ca) alphabeta1-mediated currents. Like iberiotoxin, A-272651 enhanced field stimulated twitch responses in pig detrusor and spontaneous contractions in guinea pig detrusor with EC(50) values of 4.05+/-0.05 and 37.95+/-0.12 microM, respectively. In capsaicin-sensitive DRG neurons, application of A-272651 increased action potential firing and prolonged action potential duration. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that A-272651 modulates smooth muscle contractility and neuronal firing properties. Unlike previously reported peptide BK(Ca) blockers, A-272651 represents one of the first small molecule BK(Ca) channel blockers that could serve as a useful tool for further characterization of BK(Ca) channels in physiological and pathological states.

Characterization of a novel ATP-sensitive K+ channel opener, A-251179, on urinary bladder relaxation and cystometric parameters.

BACKGROUND AND PURPOSE: ATP-sensitive K(+) channels (K(ATP)) play a pivotal role in contractility of urinary bladder smooth muscle. This study reports the characterization of 4-methyl-N-(2,2,2-trichloro-1-(3-pyridin-3-ylthioureido)ethyl)benzamide (A-251179) as a K(ATP) channel opener. EXPERIMENTAL APPROACH: Glyburide-sensitive membrane potential, patch clamp and tension assays were employed to study the effect of A-251179 in vitro. The in vivo efficacy of A-251179 was characterized by suppression of spontaneous contractions in obstructed rat bladder and by measuring urodynamic function of urethane-anesthetized rat models. KEY RESULTS: A-251179 was about 4-fold more selective in activating SUR2B-Kir6.2 derived K(ATP) channels compared to those derived from SUR2A-Kir6.2. In pig bladder smooth muscle strips, A-251179 suppressed spontaneous contractions, about 27- and 71-fold more potently compared to suppression of contractions evoked by low-frequency electrical stimulation and carbachol, respectively. In vivo, A-251179 suppressed spontaneous non-voiding bladder contractions from partial outlet-obstructed rats. Interestingly, in the neurogenic model where isovolumetric contractions were measured by continuous transvesical cystometry, A-251179 at a dose of 0.3 micromol kg(-1), but not higher, was found to increase bladder capacity without affecting either the voiding efficiency or changes in mean arterial blood pressure. CONCLUSIONS AND IMPLICATIONS: The thioureabenzamide analog, A-251179 is a potent novel K(ATP) channel opener with selectivity for SUR2B/Kir6.2 containing K(ATP) channels relative to pinacidil. The pharmacological profile of A-251179 is to increase bladder capacity and to prolong the time between voids without affecting voiding efficiency and represents an interesting characteristic to be explored for further investigations of K(ATP) channel openers for the treatment of overactive bladder.

Live volumetric (4D) visualization and guidance of in vivo human ophthalmic surgery with intraoperative optical coherence tomography.

Minimally-invasive microsurgery has resulted in improved outcomes for patients. However, operating through a microscope limits depth perception and fixes the visual perspective, which result in a steep learning curve to achieve microsurgical proficiency. We introduce a surgical imaging system employing four-dimensional (live volumetric imaging through time) microscope-integrated optical coherence tomography (4D MIOCT) capable of imaging at up to 10 volumes per second to visualize human microsurgery. A custom stereoscopic heads-up display provides real-time interactive volumetric feedback to the surgeon. We report that 4D MIOCT enhanced suturing accuracy and control of instrument positioning in mock surgical trials involving 17 ophthalmic surgeons. Additionally, 4D MIOCT imaging was performed in 48 human eye surgeries and was demonstrated to successfully visualize the pathology of interest in concordance with preoperative diagnosis in 93% of retinal surgeries and the surgical site of interest in 100% of anterior segment surgeries. In vivo 4D MIOCT imaging revealed sub-surface pathologic structures and instrument-induced lesions that were invisible through the operating microscope during standard surgical maneuvers. In select cases, 4D MIOCT guidance was necessary to resolve such lesions and prevent post-operative complications. Our novel surgical visualization platform achieves surgeon-interactive 4D visualization of live surgery which could expand the surgeon's capabilities.

Structure-reactivity probes for active site shapes of cholesterol esterase by carbamate inhibitors.

4,4'-Biphenyl-di-N-butylcarbamate (1), (S)-1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (S-2), (S)-1, 1'-bi-2-naphthyl-2-N-butylcarbamate-2'-butyrate (S-3), 2, 2'-biphenyl-di-N-butylcarbamate (4), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-octylcarbamate (5), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-phenylcarbamate (6), 2, 2'-biphenyl-2-N-butylcarbamate-2'-butyrate (7), 2, 2'-biphenyl-2-N-butylcarbamate-2'-ol (8), 2, 2'-biphenyl-2-N-octylcarbamate-2'-ol (9), (R)-1, 1'-bi-2-N-naphthyl-2-butylcarbamate-2'-ol (R-10), and glyceryl-1,2, 3-tri-N-butylcarbamate (11) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1-3 and 7-10 are the first alkyl chain and esteratic binding site-directed irreversible inhibitors due to the fact that the reactivity of the enzyme is protected by the irreversible inhibitor, trifluoroacetophenone in the presence of these carbamates. Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one of the carbamyl groups of the inhibitor molecule covalently interacts with the first alkyl chain binding site of the enzyme while the other carbamyl group of the inhibitor molecule exposes outside the active site. With near orthogonal conformations at the pivot bond of biaryl groups, one carbamyl group of carbamates S-2, S-3, and R-10 covalently binds to the first alkyl chain binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site probably due to the orthogonal conformations. Carbamates 4-9 and 11 are very potent inhibitors for the enzyme probably due to the fact that all these molecules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains binding sites of the enzyme. Although, carbamates 4-6 and 11 are irreversible inhibitors of cholesterol esterase, the enzyme is not protected but further inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4-6 and 11 covalently bind to the first alkyl chain binding site of the enzyme by one of the carbamyl groups and may also bind to the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable pi-pi interaction between Phe 324 at the second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8-10 are more potent than carbamates S-2, 4, and 5 probably due to the fact that the inhibitor molecules interact with the second alkyl chain binding site of the enzyme through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site.

Decreased sodium and increased transient outward potassium currents in iron-loaded cardiac myocytes. Implications for the arrhythmogenesis of human siderotic heart disease.

BACKGROUND: Patients with chronic iron overload may develop a cardiomyopathy manifested by ventricular arrhythmias and heart failure. We hypothesized that iron-loaded cardiomyocytes may have abnormal excitability. METHODS AND RESULTS: We examined a new model of human iron overload, the Mongolian gerbil given repeated injections of iron dextran. In ventricular myocytes, we measured iron concentration and distribution, action potential, sodium and potassium currents, and sodium channel protein. We showed for the first time that (1) the iron content of gerbil ventricular cardiomyocytes was increased to amounts similar to those of patients with iron-induced cardiomyopathy; (2) the overshoot and duration of the cardiac action potential decreased; (3) sodium current was reduced, steady-state inactivation was enhanced, and single-channel currents were unchanged; and (4) transient outward potassium current was increased, but inwardly rectifying potassium current was unchanged. Neonatal rat cardiomyocytes incubated with iron for 1 to 3 days showed similar changes, and levels of cardiac sodium channel proteins were unchanged. CONCLUSIONS: Abnormal excitability and heterogeneous cardiac iron deposition may cause the arrhythmogenesis of human siderotic heart disease.

Role of transmembrane segment S5 on gating of voltage-dependent K+ channels.

The cytoplasmic half of S5 (5'S5) has been identified as part of the inner mouth of the pore based on evidence that mutations in this region greatly alter single channel conductance, 4-aminopyridine (4-AP) block and the rate of channel closing upon repolarization (deactivation). The latter effect, suggestive of a role for 5'S5 in channel gating was investigated in the present study. The biophysical properties of chimeric channels, in which the 5'S5 regions were exchanged between two host channels (Kv2.1 and Kv3.1) that differ in 4-AP sensitivity and deactivation rate, were examined in a Xenopus oocyte expression system. Exchange of 5'S5 between Kv2.1 and Kv3.1 confers steady-state voltage dependence of activation and rates of channel deactivation similar to those of the donor channel. The involvement of voltage-dependent gating was confirmed by the observation that exchanging the 5'S5 segment of Kv2.1 with that of Kv3.1 confers a change from slow to fast deactivation kinetics by accelerating the decay of off-gating charge movement. We suggest that a conformational change that extends from the voltage-sensor in S4 to the region of the pore lined by S5 regulates the stability of the open state. Therefore, the cytoplasmic end of S5, in addition to forming part of the conduction pathway near the inner mouth of the pore, also participates in the conformational rearrangements associated with late steps in channel activation and early steps in deactivation.

Membrane anchoring of calnexin facilitates its interaction with its targets.

Calnexin, a chaperone that resides in the endoplasmic reticulum, participates in the quality control function of this compartment. Many glycoproteins in the process of folding associate transiently with this chaperone via interactions involving the recognition of their mono-glucosylated glycans. Some misfolded proteins which are retained in the endoplasmic reticulum exhibit prolonged association with calnexin. We have examined whether the transmembrane and cytoplasmic domains of calnexin influence the association of this chaperone with its targets. Interactions of wild type and truncated calnexin with a glycoprotein that is retained in the endoplasmic reticulum (the lymphocyte tyrosine kinase, Ltk), with membrane IgM heavy chains, and with the MHC class I heavy chain protein were investigated. A soluble calnexin molecule lacking the transmembrane domain and cytoplasmic tail does not associate with any of these proteins. When a heterologous transmembrane domain is fused to the lumenal portion of calnexin, this membrane-bound protein can bind Ltk, IgM heavy chains, and MHC class I heavy chain proteins. These results suggest that calnexin must be membrane-anchored in order to recognize its substrates.

Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas.

P-Glycoprotein (Pgp), product of the mdr-1 gene, is a 130- to 180-kDa plasma membrane phosphoglycoprotein which mediates multidrug resistance in cell culture by increasing efflux of the natural product chemotherapeutic agents. High levels of expression of mdr-1/Pgp are found in both the normal adrenal and adrenocortical cancers. By RNA in situ hybridization the expression in adrenocortical cancer is shown to be widely distributed. The present study demonstrates that decreased drug accumulation mediated by mdr-1/Pgp can be overcome by clinically achievable concentrations of mitotane (o,p'-DDD). The increase in drug accumulation with the addition of mitotane is due at least in part to a decrease in drug efflux and results in an increase in cytotoxicity when agents of the natural product class are used. This effect is observed in cells with a broad range of mdr-1/Pgp expression, including levels comparable to those found in most adrenocortical cancers. Similar increases in drug accumulation can be demonstrated in an unselected adrenocortical cancer cell line that expresses mdr-1/Pgp. The finding that multidrug resistance mediated by mdr-1/Pgp can be reversed by mitotane provides a rational basis for exploring the use of mitotane in combination with natural product chemotherapeutic agents in adrenocortical cancer.

Molecular characterization of human SUR2-containing K(ATP) channels.

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K(ATP) channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone. Quantification of RNA for SUR2 exon 17+ and SUR2 exon 17- splice variants using real-time Taqman PCR indicated differential levels of expression in brain, kidney, skeletal muscle, heart and small intestine. Interestingly, the SUR2 exon 17+ variant is the major species expressed in all tissues examined in this study. Each of the SUR2 splice variants transiently expressed with the inward rectifier Kir 6.2 formed functional K(ATP) channels in HEK 293 cells as assessed either by changes in DiBAC(4)(3) fluorescence responses or glyburide-sensitive whole cell currents. Collectively, our findings demonstrate that various SUR2 splice variants have distinct expression patterns and can form functional K(ATP) channels.

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons.

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and NS1619, a BK(Ca) channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 microM) increased opening activity of a Ca(2+)-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK(Ca) channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K(+) channel (A-current) blocker, in an IbTx-sensitive manner. These results indicate that IbTx-sensitive BK(Ca) channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK(Ca) channels. These results suggest that opening BK(Ca) channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.