Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies

Background

The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.

Methods

Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist''s report.

Results

15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.

Conclusions

This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.     

抗中性粒细胞胞浆抗体、抗核抗体联合检测对类风湿关节炎的诊断价值

目的：探讨抗中性粒细胞胞浆抗体（ANCA）与抗核抗体（ANA）联合检测对类风湿关节炎的临床意义。方法：采用IIF法对82例RA患者（RA组）、74例非RA自身免疫疾病患者（非RA组）和52例健康体检者（正常对照组）的血清ANCA和ANA谱进行了检测分析,并用ELISA法进行抗丝氨酸蛋白酶3（PR3）、抗髓过氧化物酶（MPO）、ANA谱的定量检测。结果：RA组82例患者中,64例ANCA阳性,阳性率为78．08％,其中核周型（PANCA）37例,阳性率为45．1％,胞浆型（CANCA）27例,阳性率为32．9％;非RA组74例患者中有7例ANCA阳性率分别为9．4％;正常对照组50例中没有一例ANCA阳性。利用Elisa法对患者血清进行检测,分别能够特异的检测到PR3、MPO、抗双链DNA抗体（抗ds—DNA抗体）、抗SS—A等抗体、抗ss—A抗体、抗PM—SCL抗体的存在。结论：联合ANCA、ANA检测有助于提高类风湿关节炎的诊断。     

Circulating antinuclear antibodies in patients with pelvic masses are associated with malignancy and decreased survival

Background

Circulating autoantibodies occur more frequently in cancer patients than in patients without cancer.

Methods and Findings

We examined sera from patients referred for pelvic mass symptoms to a tertiary university clinic. A total of 127 were diagnosed with epithelial ovarian cancer while 386 had a benign condition. A screen for IgG anti-nuclear antibodies (ANA) by indirect immunofluorescence on HEp-2 cells confirmed a highly significant overrepresentation of ANA in the cancer group where 40% had detectable (i.e., a titer ≥160) ANA compared with less than 12% in the benign group. The overrepresentation of ANA in the cancer group persisted (p<0.0001) after matching the age-profile of the benign group with the ovarian cancer group. Only 19 out of 127 patients in the age-matched benign subgroup were positive for ANA corresponding to an 85% specificity at 40% sensitivity of ANA as the only marker for malignancy. No correlation of ANA positivity in either group with specific bands in immunoblots could be demonstrated even though immunoblot positivity was clearly increased in the malignant group (41% vs. 3%). The presence, strength, and type of ANA did not correlate with serum CA-125 values or with staging, and ANA outcome did not contribute with independent diagnostic information. However, survival was significantly shorter in ANA-positive compared with ANA-negative cancer patients and patients with CA-125 below the median CA-125 value in the cancer group had a significantly decreased survival when positive for ANA. ANA status made no difference in the group with CA-125 values above the median. Also, there was a significant correlation between speckled ANA-strength and histological tumor grade.

Conclusions

Circulating antibodies are a promising source for new biomarkers in cancer. Characterization of epitope specificities and measurements of consecutive samples will be important for further elucidating the role of ANA in evaluating ovarian cancer patients.     

Interleukin-1 Antagonist Anakinra in Amyotrophic Lateral Sclerosis—A Pilot Study

Preclinical studies show that blocking Interleukin–1 (IL–1) retards the progression of Amyotrophic Lateral Sclerosis (ALS). We assessed the safety of Anakinra (ANA), an IL–1 receptor antagonist, in ALS patients. In a single arm pilot study we treated 17 ALS patients with ANA (100 mg) daily for one year. We selected patients with dominant or exclusive lower motor neuron degeneration (LMND) presentation, as peripheral nerves may be more accessible to the drug. Our primary endpoint was safety and tolerability. Secondary endpoints included measuring disease progression with the revised ALS functional rating scale (ALSFRSr). We also quantified serum inflammatory markers. For comparison, we generated a historical cohort of 47 patients that fit the criteria for enrolment, disease characteristics and rate of progression of the study group. Only mild adverse events occurred in ALS patients treated with ANA. Notably, we observed lower levels of cytokines and the inflammatory marker fibrinogen during the first 24 weeks of treatment. Despite of this, we could not detect a significant reduction in disease progression during the same period in patients treated with ANA compared to controls as measured by the ALSFRSr. In the second part of the treatment period we observed an increase in serum inflammatory markers. Sixteen out of the 17 patients (94%) developed antibodies against ANA. This study showed that blocking IL–1 is safe in patients with ALS. Further trials should test whether targeting IL–1 more efficiently can help treating this devastating disease.

Trial Registration

ClinicalTrials.gov NCT01277315     

The solution structure of double helical arabino nucleic acids (ANA and 2��F-ANA): effect of arabinoses in duplex-hairpin interconversion

We report here the first structure of double helical arabino nucleic acid (ANA), the C2′-stereoisomer of RNA, and the 2′-fluoro-ANA analogue (2′F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2′-deoxy-2′F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2′F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2′F-ANA and ANA) forming Watson–Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2′-endo conformation. In the ANA:ANA segment, steric interactions involving the 2′-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2′F-ANA:2′F-ANA regions of the duplex, where the –OH substituent is replaced by a smaller fluorine atom. 2′F-ANA nucleotides adopt the C2′-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2′F···H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2′F-ANA:RNA hybrids and in single 2′F-ANA nucleotides.     

Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.     

Quantification of bacterial polysaccharides by the purpald assay: measurement of periodate-generated formaldehyde from glycol in the repeating unit

Anandamide (ANA) and 2-arachidonoylglycerol (2-AG), two endogenous cannabinoids, can be generated by activated macrophages and platelets, respectively, in the context of endotoxic shock, and are proposed to play a crucial role in the induction of the shock-related hypotension. Taking advantage of our recently discovered function of polymyxin B (PMB) binding to ANA and 2-AG, we developed a new method for measuring ANA and 2-AG by applying PMB-immobilized beads to selectively adsorb them in biological fluids, instead of organic solvent extraction. The eluate from beads can be directly fractionated by reverse-phase high-performance liquid chromatography (HPLC), and the fractionations corresponding to authentic ANA and 2-AG are collected and derivatized with fluorogenic reagent and subsequently quantified by HPLC with fluorometric detection. The calibration graphs of ANA and 2-AG were linear over a range of 1 to 500 pmol/ml. The limits of detection for ANA and 2-AG were 20 and 50 fmol, respectively. Intraassay precision was 2.24-4.25 and 3.47-5.44%, and interassay was 4.05-6.14 and 4.92-7.28% for ANA and 2-AG, respectively. Using this method, we first determined a 4-fold and 3-fold higher level of ANA and 2-AG, respectively, in the sera of patients with endotoxic shock than in normal serum. This finding should help in elucidating the role of the endogenous cannabinoids in the hypotension of human endotoxic shock. This method is rapid, sensitive, and reliable for simultaneously quantifying ANA and 2-AG in biological fluids, and has potential for clinical usage.     

Variable modulation by cytokines and thiazolidinediones of the prototype Th1 chemokine CXCL10 in anaplastic thyroid cancer

Until now, no data are present in literature about the prototype Th1 chemokine (C-X-C motif) ligand 10 (CXCL10) in anaplastic thyroid cancer (ATC). This study aimed to test in "primary human ATC cells" (ANA) vs "normal thyroid follicular cells" (TFC): (a) CXCL10 secretion basally and after interferon (IFN)-γ and/or tumor necrosis factor (TNF)-α stimulation; (b) peroxisome proliferator-activated receptor (PPAR)-γ activation by thiazolidinediones, rosiglitazone or pioglitazone, on CXCL10 secretion, on proliferation and apoptosis in ANA. We demonstrate that: (a) ANA, but not TFC, produced basally CXCL10, and did so in half of cases; (b) IFN-γ stimulated dose-dependently CXCL10, in ANA and TFC; (c) TNF-α did not induce CXCL10 secretion, in ANA and TFC; (d) IFN-γ+TNF-α induced a synergistic but variable release of CXCL10 in the different ANA preparations, while it was more reproducible in TFC; (e) rosiglitazone action on CXCL10 in ANA was inhibitory in 2/6, stimulatory in 1/6 and nil in 3/6, whereas it was inhibitory in TFC; (f) rosiglitazone inhibition of proliferation in ANA was not associated with the effect on CXCL10; (g) nuclear factor-κB and ERK1/2 were basally activated in ANA, increased by IFN-γ+TNF-α, and rosiglitazone inhibited that activation. On the whole, the present data first show that ANA cells are able to produce CXCL10, basally and under the influence of cytokines. However, the pattern of modulation by IFN-γ, TNF-α or thiazolidinediones is extremely variable, suggesting that the intracellular pathways involved in the chemokine modulation in ATC have different types of deregulation.     

Synthesis and biophysical properties of arabinonucleic acids (ANA): circular dichroic spectra, melting temperatures, and ribonuclease H susceptibility of ANA.RNA hybrid duplexes

Arabinonucleic acid (ANA), the 2'-epimer of RNA, was synthesized from arabinonucleoside building blocks by conventional solid-phase phosphoramidite synthesis. In addition, the biochemical and physicochemical properties of ANA strands of mixed base composition were evaluated for the first time. ANA exhibit certain characteristics desirable for use as antisense agents. They form duplexes with complementary RNA, direct RNase H degradation of target RNA molecules, and display resistance to 3'-exonucleases. Since RNA does not elicit RNase H activity, our findings establish that the stereochemistry at C2' (ANA versus RNA) is a key determinant in the activation of the enzyme RNase H. Inversion of stereochemistry at C2' is most likely accompanied by a conformational change in the furanose sugar pucker from C3'-endo (RNA) to C2'-endo ("DNA-like") pucker (ANA) [Noronha and Damha (1998) Nucleic Acids Res. 26, 2665-2671; Venkateswarlu and Ferguson (1999) J. Am. Chem. Soc. 121, 5609-5610]. This produces ANA/RNA hybrids whose CD spectra (i.e., helical conformation) are more similar to the native DNA/RNA substrates than to those of the pure RNA/RNA duplex. These features, combined with the fact that ara-2'OH groups project into the major groove of the helix (where they should not interfere with RNase H binding), help to explain the RNase H activity of ANA/RNA hybrids.     

Formation of the compact confomer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity.

Full-length Drosophila kinesin heavy chain from position 1 to 975 was expressed in Escherichia coil (DKH975) and is a dimer. The sedimentation coefficient of DKH975 shifts from 5.4 S at 1 M NaCl to approximately 6.9 S at <0.2 M NaCl. This transition of DKH975 between extended and compact conformations is essentially identical to that for the heavy chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D., and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic property of the heavy chains and requires neither light chains nor eukaryotic post-translational modification. DKH960 undergoes a similar transition, indicating that the extreme COOH-terminal region is not required. More extensive deletions from the COOH-terminal (DKH945 and DKH937) result in a shift in the midpoint for the transition to lower salt concentrations. DKH927 and shorter constructs remaining extended even in the absence of added salt. Thus the COOH-terminal approximately 50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH2-terminal head/neck segments interact in a salt-dependent manner that is consistent with the compact conformer being produced by the interaction of domains from these regions of the heavy chain dimer. The microtubule-stimulated ATPase rate of DKH975 in the compact conformer is strongly inhibited compared with the rate of extended DKH894 (4 s-1 and 35 s-1, respectively, for kcat at saturating microtubules).     

Naphthylphthalamic acid is enzymatically hydrolyzed at the hypocotyl-root transition zone and other tissues of Arabidopsis thaliana seedlings

During prolonged storage, solid naphthylphthalamic acid (NPA) turns purple due to the formation of the dye, 1,1'-azonaphthylene (ANA). ANA forms oxidatively from two molecules of the NPA degradation product, α-naphthylamine (αNA). At concentrations ≥ 30 μM, solutions of `purple NPA' stained Arabidopsis thaliana seedlings specifically at the hypocotyl-root transition zone and other regions. Staining was caused by the aggregation of ANA and could be reconstituted using undegraded NPA and either αNA or ANA. Studies with [³H]-NPA confirmed that NPA is localized at sites of staining. Genestein, curcumin and quercitin inhibited the staining reaction. Liquid chromatography-mass spectral (LC-MS) analysis of the ANA in the stained tissue indicated that ANA is synthesized at sites of staining. It was postulated that the amide bond of NPA is enzymatically cleaved, producing αNA. The αNA combines to form ANA, which aggregates to yield an insoluble precipitate. Consistent with this hypothesis, NPA amidase activity was detected in purified plasma membranes. The NPA amidase activity was activated by 500 μM MnCl₂ and inhibited by phloretin, genestein, quercitin, bestatin and EDTA.     

Mutational and gene expression analysis of mtrDEF,omcA and mtrCAB during arsenate and iron reduction in Shewanella sp. ANA‐3

Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA‐3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA‐3) compared with wild‐type ANA‐3. However, the presence of arsenate adsorbed onto ferrihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild‐type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA‐3), arsenate was reduced similarly to wild‐type ANA‐3 indicating the Fe(III) reduction pathway is not required for ferrihydrite‐associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA‐3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre‐adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsenate can co‐induce operons (arr and mtr) implicated in arsenic mobilization.     

Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), 3H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). 3H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. 3H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.     

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels

Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.     

类风湿关节炎患者RF、ANA、CCP、Ig及炎症因子的水平测定及临床意义

目的:探讨类风湿关节炎(RA)患者血清类风湿性因子(RF)、抗核抗体(ANA)、抗环瓜氨酸肽(CCP)抗体、免疫球蛋白(Ig)、补体(C3、C4)以及炎症因子的水平及临床意义。方法:收集2016年9月至2017年4月我院收治的165例RA患者为RA组,其中RA活动期患者93例(RA活动组),RA缓解期患者72例(RA缓解组),并于同期随机选取30例健康体检者为对照组。采用免疫散射比浊法检测各组血清RF、IgM、IgG、IgA、C3、C4水平,采用酶联免疫吸附法(ELISA)检测各组血清CCP抗体,电化学发光法检测白介素-6(IL-6)、化学发光法检测白介素-8(IL-8)。免疫荧光法检测ANA。比较不同组别各检测指标水平,并分析RA患者RF、ANA、CCP抗体、Ig、C3、C4与炎症因子的相关性。结果:RA活动组、RA缓解组血清RF、ANA、CCP抗体、IgM、IgG、IgA、IL-6、IL-8水平高于对照组,且RA活动组血清RF、ANA、CCP抗体、IgM、IgG、IgA、IL-6、IL-8水平高于RA缓解组,差异均有统计学意义(P0.05)。RA活动组、RA缓解组血清C3、C4水平低于对照组,且RA活动组血清C3、C4水平低于RA缓解组,差异均有统计学意义(P0.05)。经Pearson积矩相关分析,RA活动期和缓解期患者血清RF、ANA、CCP抗体、IgM、IgG、IgA与炎症因子IL-6、IL-8呈正相关关系(P0.05),血清C3、C4与炎症因子IL-6、IL-8呈负相关关系(P0.05)。结论:RA患者体内RF、ANA、CCP抗体、Ig及IL-6、IL-8水平明显较高,C3,C4水平明显较低,活动期RA患者更为显著,联合检测可早期辅助诊断RA及判断病情进展,在临床上有重要的参考意义。     

CFTR inhibition by glibenclamide requires a positive charge in cytoplasmic loop three

The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR(inh)-172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.     

CFTR inhibition by glibenclamide requires a positive charge in cytoplasmic loop three

The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR_inh-172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.     

Effects of microgravity elicited by parabolic flight on abdominal aortic pressure and heart rate in rats.

Abdominal aortic pressure (AAP), heart rate (HR), and aortic nerve activity (ANA) during parabolic flight were measured by using a telemetry system to clarify the acute effect of microgravity (microG) on hemodynamics in rats. While the animals were conscious, AAP increased up to 119 +/- 3 mmHg on exposure to microG compared with the value at 1 G (95 +/- 3 mmHg; P < 0.001), whereas AAP decreased immediately on exposure to microG under urethane anesthesia (microG: 72 +/- 9 mmHg vs. 1 G: 78 +/- 8 mmHg; P < 0.05). HR also increased during microG in conscious animals (microG: 349 +/- 12 beats/min vs. 1 G: 324+9 beats/min; P < 0.01), although no change was observed under anesthesia. ANA, which was measured under anesthesia, decreased in response to acute microG exposure (microG: 33 +/- 7 counts/s vs. 1 G: 49 +/- 5 counts/s; P < 0.01). These results suggest that microG essentially induces a decrease of arterial pressure; however, emotional stress and body movements affect the responses of arterial pressure and HR during exposure to acute microG.     

Polymyxin B binds to anandamide and inhibits its cytotoxic effect

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.