Effects of Factor XIII Deficiency on Thromboelastography. Thromboelastography with Calcium and Streptokinase Addition is more Sensitive than Solubility Tests.

BACKGROUND: Homozygous or double heterozygous factor XIII (FXIII) deficiency is characterized by soft tissue hematomas, intracranial and delayed spontaneous bleeding. Alterations of thromboelastography (TEG) parameters in these patients have been reported. The aim of the study was to show results of TEG, TEG Lysis (Lys 60) induced by subthreshold concentrations of streptokinase (SK), and to compare them to the clot solubility studies results in samples of a 1-year-old girl with homozygous or double heterozygous FXIII deficiency. CASE: A year one girl with a history of bleeding from the umbilical cord. During her first year of life, several hematomas appeared in soft upper limb tissue after punctures for vaccination and a gluteal hematoma. One additional sample of a heterozygous patient and three samples of acquired FXIII deficiency were also evaluated. MATERIALS AND METHODS: Clotting tests, von Willebrand factor (vWF) antigen and activity, plasma FXIII-A subunit (pFXIII-A) were measured by an immunoturbidimetric assay in a photo-optical coagulometer. Solubility tests were performed with Ca(2+)-5 M urea and thrombin-2% acetic acid. Basal and post-FXIII concentrate infusion samples were studied. TEG was performed with CaCl2 or CaCl2 + SK (3.2 U/mL) in a Thromboelastograph. RESULTS: Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, fibrinogen, factor VIIIc, vWF, and platelet aggregation were normal. Antigenic pFXIII-A subunit was < 2%. TEG, evaluated at diagnosis and post FXIII concentrate infusion (pFXIII-A= 37%), presented a normal reaction time (R), 8 min, prolonged k (14 and 11min respectively), a low Maximum-Amplitude (MA) ( 39 and 52 mm respectively), and Clot Lysis (Lys60) slightly increased (23 and 30% respectively). In the sample at diagnosis, clot solubility was abnormal, 50 and 45 min with Ca-Urea and thrombin-acetic acid, respectively, but normal (>16 hours) 1-day post-FXIII infusion. Analysis of FXIII deficient and normal plasma mixtures (< 2-102% of pFXIII-A), showed that Ca-urea solubility was abnormal at pFXIII-A < 9%, thrombin-acetic acid at pFXIII-A<18%, but TEG MA and elasticity at 23% and Lys60 with SK at pFXIII-A< 40%. CONCLUSIONS: TEG parameters MA and elasticity, and Lys 60 in TEG either with Ca(2+) or Ca(2+) and SK are more sensitive to low levels of pFXIII than solubility tests. The increased Lys60 induced by a subthreshold concentration of SK could probably reflect the clot characteristics "in vivo" in many patients with pFXIII levels between 5-40% and could be potentially considered as screening test.

Do PT and APTT sensitivities to factors' deficiencies calculated by the H47-A2 2008 CLSI guideline reflect the deficiencies found in plasmas from patients?

INTRODUCTION: Prothrombin time (PT) and activated partial thromboplastin time (APTT) sensitivity for detecting isolated factor deficiencies varies with different reagents and coagulometers. The Clinical and Laboratory Standards Institute (CLSI) H47A2 guideline proposed a method to calculate these sensitivities, but some inconsistency has been reported. This study aimed to calculate factor sensitivities using CLSI guideline and to compare them with those obtained from single factor-deficient patients' data. METHODS: Different mixtures of normal pooled and deficient plasmas were prepared (<1IU/dL to 100 IU/dL) according to the CLSI H47A2 guideline. PT with rabbit brain (RB) and human recombinant (HR) thromboplastins, APTT and factors' activities were measured in an ACL TOP coagulometer. Sensitivities (maximum factor concentration that produces PT or APTT values out of the reference range) were calculated from mixtures and from patients with single-factor deficiencies: 17 factor FV, 36 FVII, 19 FX, 39 FVIII, 15 FIX 15 FXI and 24 FXII. RESULTS: PT sensitivity with RB was as follows: FV 38 and 59, FVII 35 and 58, FX 56 and 64 IU/dL; PT sensitivity with HR was as follows: FV 39 and 45, FVII 51 and 50, FX 33 and 61 IU/dL; and APTT sensitivity was as follows: FV 39 and 45, FX 32 and 38, FVIII 47 and 60, FIX 35 and 44, FXI 33 and 43, FXII 37 and 46 IU/dL, respectively. CONCLUSIONS: Reagent-coagulometer combination has adequate sensitivities to factor deficiencies according to guideline recommendations (>30 IU/dL). These should not be considered as actual sensitivities because those obtained by analysing patients' plasmas with single-factor deficiencies were higher for most factors and could induce misinterpretation of the basic coagulation test results.

Omental adipose tissue fibrosis and insulin resistance in severe obesity.

BACKGROUND/OBJECTIVES: The unresolved chronic inflammation of white adipose tissue (WAT) in obesity leads to interstitial deposition of fibrogenic proteins as reparative process. The contribution of omental adipose tissue (oWAT) fibrosis to obesity-related complications remains controversial. The aim of our study was to investigate whether oWAT fibrosis may be related to insulin resistance in severely obese population. SUBJECTS/METHODS: Forty obese subjects were studied by glucose clamp before undergoing bariatric surgery and thus stratified according to insulin resistance severity (M-value). From the first (Group B: n=13; M=1.9±0.7 mg kg(-1) min(-1)) and the highest (Group A: n=14; M=4.5±1.4 mg kg(-1) min(-1)) M-value tertiles, which were age-, waist- and body mass index-matched, oWAT samples were then obtained.Gene expression of collagen type I, III and VI, interleukin-6, profibrotic mediators (transforming growth factor (TGF)-ß1, activin A, connective tissue growth factor), hypoxia inducible factor-1α (HIF-1α) and macrophage (CD68, monocyte chemotactic protein (MCP)-1, CD86, CD206, CD150) markers were analyzed by quantitative reverse transcription PCR. Adipocyte size and total fibrosis were assessed by histomorphometry techniques. RESULTS: Fibrosis at morphological level resulted significantly greater in Group B compared with Group A, although collagens gene expression did not differ. Notably, collagen VI messenger RNA significantly correlated with collagen I, collagen III, HIF-1α, TGF-ß1, CD68, MCP-1 and CD86 transcription levels, supporting their relation with fibrosis development. CONCLUSIONS: In conclusion, we show for the first time that human oWAT fibrosis in severe obesity is consistent with a higher degree of insulin resistance measured by glucose clamp. Therefore, collagen deposition could represent a maladaptive mechanism contributing to obesity-related metabolic complications.

Iron status in obesity: An independent association with metabolic parameters and effect of weight loss.

BACKGROUND AND AIMS: Growing evidence has shown that ferritin concentrations are associated with obesity and insulin resistance, and with nonalcoholic fatty liver disease. However, it is unclear whether ferritin is simply an inflammatory marker, or it may directly contribute to the pathogenesis of obesity-related metabolic alterations. The aim of our study was to investigate the independent associations of ferritin levels with metabolic parameters in overweight/obese subjects before and after hypocaloric diet-induced weight changes. METHODS AND RESULTS: A sample study of 48 premenopausal, 39 postmenopausal women and 50 men was retrospectively analyzed. Clinical, bioimpedentiometry and biochemical data from baseline evaluations and after 3, 6 and 12 months of hypocaloric diet were collected. In the whole sample study, the baseline values of ferritin concentrations were positively correlated with body mass index (BMI) (r = 0.21, p < 0.05) and mass body fat (MBF) (r = 0.26, p < 0.05), whereas the serum iron level was negatively correlated with MBF (r = -0.29, p < 0.05). In premenopausal women, BMI-adjusted ferritin concentrations were negatively associated with high-density lipoprotein-cholesterol and positively related with triglycerides and aspartate aminotransferase. Moreover, the quantitative ferritin reduction at 12 months was positively associated with the relative reduction of BMI (r = 0.34, p < 0.05). Finally, the association between changes of alanine aminotransferase and ferritin levels at 12 months from baseline turned out to be independent of respective BMI changes (ß = 0.31, p < 0.05). CONCLUSION: In obesity, ferritin, putatively entailing increased iron storage, is independently associated with lipid derangements and transaminase levels, and the association with the latter persists after weight changes.

Frequent false-positive results of lupus anticoagulant tests in plasmas of patients receiving the new oral anticoagulants and enoxaparin.

INTRODUCTION: Oral direct thrombin and Xa inhibitors are worldwide distributed for prevention and treatment of thrombosis. It is important to recognize their effects on lupus anticoagulant (LA) testing. The aim of the study is to describe the rate of false-positive results of LA tests on plasmas of patients with previous negative LA tests results that receive dabigatran etexilate (DAB) 110 mg/twice a day, rivaroxaban (RIV) 10 mg/day or 15 mg/twice a day, or enoxaparin 40 mg/day. METHODS: Blood was taken between 1.5 and 4 h post administration. Tests evaluated are as follows: prothrombin time, APTT, dilute Russell viper venom time (DRVVT) screen, APTT, and DRVVT mixing studies, index of circulating anticoagulant (ICA) with normal plasma, screen/confirm normalized ratio (NR) for DRVVT and silica clotting time (SCT). RESULTS: Plasmas from patients taking DAB (n = 22) presented 100% prolonged APTT and DRVVT with ICA above the cutoff point and 81.8% positive screen/confirm NR, 100% prolonged SCT screen, but 4.5% positive confirmatory NR. All patients receiving RIV at 15 mg/twice a day (n = 4) presented positive DRVVT screen, mixing, and confirmatory tests, 75% and 100% prolonged APTT and SCT screen, with negative screen/confirm NR. Those taking RIV 10 mg/day (n = 22) showed 81.8% prolonged DRVVT screen, 82.3% and 76.5% of them with positive mixing and confirmatory studies. Patients receiving enoxaparin also presented high prevalence of APTT and DRVVT false-positive results. CONCLUSION: Dabigatran etexilate, RIV, and enoxaparin affect tests for LA not only in screening and mixing, but also in confirmatory studies. We considered that LA testing should not to be performed when patients are taken these drugs, particularly if blood is collected at peak, in order to avoid false-positive results.

Effects of whole body vibration plus diet on insulin-resistance in middle-aged obese subjects.

We investigated the early effects of whole body vibration (WBV) added to hypocaloric diet on insulin-resistance and other parameters associated with glucose regulation in sedentary obese individuals. We randomly assigned 34 patients to WBV plus hypocaloric diet (WBV group) or diet alone (CON group) for 8 weeks. Fasting and post-load glucose, insulin, lipids, C-reactive protein, tumor necrosis factor-α, leptin, adiponectin were assessed. Insulin sensitivity index (ISI) was derived from oral-glucose-tolerance test. Body composition was evaluated with dual-energy X-ray absorptiometry. Both groups lost approximately 5% of weight, with greater reduction of body fat in WBV than in CON (-7.1±1.2 Kg vs. -5.3±1.0 Kg, p=0.003). Percent variation of ISI was more pronounced in WBV than in CON group (+35±4% vs. + 22±5%, p=0.002), accompanied by slight improvement in post-load glucose (-1.07±0.02 vs. - 0.12±0.01 mmol/l, p=0.031) but without changes in fasting levels. Adiponectin significantly increased in WBV group compared with CON (p=0.021 for comparison) whereas no differences in leptin and inflammatory markers were observed. In middle-aged sedentary obese subjects, WBV added to hypocaloric diet for 8 weeks improved body composition, insulin-resistance, glucose regulation and adiponectin levels to a greater extent compared with diet alone. Efficacy and feasibility of this approach in the long term need to be ascertained.

Genetic inactivation of Kcnj16 identifies Kir5.1 as an important determinant of neuronal PCO2/pH sensitivity.

The molecular identity of ion channels which confer PCO(2)/pH sensitivity in the brain is unclear. Heteromeric Kir4.1/Kir5.1 channels are highly sensitive to inhibition by intracellular pH and are widely expressed in several brainstem nuclei involved in cardiorespiratory control, including the locus coeruleus. This has therefore led to a proposed role for these channels in neuronal CO(2) chemosensitivity. To examine this, we generated mutant mice lacking the Kir5.1 (Kcnj16) gene. We show that although locus coeruleus neurons from Kcnj16((+/+)) mice rapidly respond to cytoplasmic alkalinization and acidification, those from Kcnj16((-/-)) mice display a dramatically reduced and delayed response. These results identify Kir5.1 as an important determinant of PCO(2)/pH sensitivity in locus coeruleus neurons and suggest that Kir5.1 may be involved in the response to hypercapnic acidosis.

A novel KCNA1 mutation identified in an Italian family affected by episodic ataxia type 1.

Episodic ataxia type 1 (EA1) is a rare human neurological syndrome characterized by continuous myokymia and attacks of generalized ataxia that can be triggered by abrupt movements, emotional stress and fatigue. An Italian family has been identified where related members displayed continuous myokymia, episodes of ataxia, attacks characterized by myokymia only, and neuromyotonia. A novel missense mutation (F414C), in the C-terminal region of the K(+) channel Kv1.1, was identified in the affected individuals. The mutant homotetrameric channels were non-functional in Xenopus laevis oocytes. In addition, heteromeric channels resulting from the co-expression of wild-type Kv1.1 and Kv1.1(F414C), or wild-type Kv1.2 and Kv1.1(F414C) subunits displayed reduced current amplitudes and altered gating properties. This indicates that the pathogenic effect of this KCNA1 mutation is likely to be related to the defective functional properties we have identified.

Increased release and activity of matrix metalloproteinase-9 in patients with mandibuloacral dysplasia type A, a rare premature ageing syndrome.

Mandibuloacral dysplasia type A (MADA; OMIM 248370), a rare disorder caused by mutation in the LMNA gene, is characterized by post-natal growth retardation, craniofacial and skeletal anomalies (mandibular and clavicular hypoplasia, acroosteolysis, delayed closure of cranial sutures, low bone mass and joint contractures), cutaneous changes and partial lipodystrophy. Little is known about the molecular mechanisms by which LMNA mutations produce bone alterations. An altered bone extracellular matrix (ECM) remodelling could play a pivotal role in this disorder and influence part of the typical bone phenotype observed in patients. Therefore, we have focused our investigation on matrix metalloproteinases (MMPs), which are degradative enzymes involved in ECM degradation and ECM remodelling, thus likely contributing to the altered bone mineral density and bone metabolism values seen in five MADA patients. We evaluated the serum levels of several MMPs involved in bone development, remodelling and homeostasis, such as MMP-9, -2, -3, -8 and -13, and found that only the 82 kDa active enzyme forms of MMP-9 are significantly higher in MADA sera compared with healthy controls (n = 16). The serum level of MMP-3 was instead lower in all patients. No significant differences were observed between controls and MADA patients for the serum levels of MMP-2, -8 and -13 and of tissue inhibitor of metalloproteinase 2, a natural inhibitor of MMP-9. Similarly, normal serum levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta were detected. These data suggest a possible involvement of MMP-9 in MADA disease, underlying the potential use in diagnosis and therapy.

Identification of a heteromeric interaction that influences the rectification, gating, and pH sensitivity of Kir4.1/Kir5.1 potassium channels.

Heteromultimerization between different potassium channel subunits can generate channels with novel functional properties and thus contributes to the rich functional diversity of this gene family. The inwardly rectifying potassium channel subunit Kir5.1 exhibits highly selective heteromultimerization with Kir4.1 to generate heteromeric Kir4.1/Kir5.1 channels with unique rectification and kinetic properties. These novel channels are also inhibited by intracellular pH within the physiological range and are thought to play a key role in linking K+ and H+ homeostasis by the kidney. However, the mechanisms that control heteromeric K+ channel assembly and the structural elements that generate their unique functional properties are poorly understood. In this study we identify residues at an intersubunit interface between the cytoplasmic domains of Kir5.1 and Kir4.1 that influence the novel rectification and gating properties of heteromeric Kir4.1/Kir5.1 channels and that also contribute to their pH sensitivity. Furthermore, this interaction presents a structural mechanism for the functional coupling of these properties and explains how specific heteromeric interactions can contribute to the novel functional properties observed in heteromeric Kir channels. The highly conserved nature of this structural association between Kir subunits also has implications for understanding the general mechanisms of Kir channel gating and their regulation by intracellular pH.

Expression in E. coli and purification of recombinant fragments of wild type and mutant human prion protein.

Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrP(C)) into an altered isoform (the prion scrapie, PrP(Sc)), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrP(Sc). We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an alpha-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200K mutant PrP fragment.

Differential pH sensitivity of Kir4.1 and Kir4.2 potassium channels and their modulation by heteropolymerisation with Kir5.1.

1. The inwardly rectifying potassium channel Kir5.1 appears to form functional channels only by coexpression with either Kir4.1 or Kir4.2. Kir4.1-Kir5.1 heteromeric channels have been shown to exist in vivo in renal tubular epithelia. However, Kir5.1 is expressed in many other tissues where Kir4.1 is not found. Using Kir5.1-specific antibodies we have localised Kir5.1 expression in the pancreas, a tissue where Kir4.2 is also highly expressed. 2. Heteromeric Kir5.1-Kir4.1 channels are significantly more sensitive to intracellular acidification than Kir4.1 currents. We demonstrate that this increased sensitivity is primarily due to modulation of the intrinsic Kir4.1 pH sensitivity by Kir5.1. 3. Kir4.2 was found to be significantly more pH sensitive (pK(a) = 7.1) than Kir4.1 (pK(a) = 5.99) due to an additional pH-sensing mechanism involving the C-terminus. As a result, coexpression with Kir5.1 does not cause a major shift in the pH sensitivity of the heteromeric Kir4.2-Kir5.1 channel. 4. Cell-attached single channel analysis of Kir4.2 revealed a channel with a high open probability (P(o) > 0.9) and single channel conductance of approximately 25 pS, whilst coexpression with Kir5.1 produced novel bursting channels (P(o) < 0.3) and a principal conductance of approximately 54 pS with several subconductance states. 5. These results indicate that Kir5.1 may form heteromeric channels with Kir4.2 in tissues where Kir4.1 is not expressed (e.g. pancreas) and that these novel channels are likely to be regulated by changes in intracellular pH. In addition, the extreme pH sensitivity of Kir4.2 has implications for the role of this subunit as a homotetrameric channel.

Experimental arterial thrombosis in genetically or diet induced hyperlipidemia in rats--role of vitamin K-dependent clotting factors and prevention by low-intensity oral anticoagulation.

To investigate the relationship among lipids, coagulation and thrombosis in the absence of atherosclerosis, spontaneous or dietary-induced hyperlipidemic (FHL) rats were studied. FHL showed higher levels of coagulation factors VII, IX, X, VIII and XII and a shortening of the occlusion time (OT) of an artificial arterial prosthesis as compared with normolipidemic (FNL) animals. Damage of abdominal aorta of FHL was followed by increased fibrin deposition in the vascular intima as compared to FNL. After 5 months of cholesterol-rich diet FNL showed increased cholesterol, triglycerides and factor II, VII, IX, X, XII levels. A significant shortening of the OT and increased fibrin deposition was also observed. Two-month diet withdrawal restored the initial condition. Warfarin treatment, at a dose decreasing vitamin K-dependent factor to levels found in FNL, prolonged the OT and reduced fibrin deposition, without modifying F XII or changing lipid profile. An increase in the activated form of F VII was observed. In contrast, no difference was found in F VII clearance. High lipid levels favour the process of thrombus formation by increasing the activation of vitamin K-dependent coagulation factors. Low-dose warfarin treatment reverts the prothrombotic effect of hyperlipidemia.

pH dependence of the inwardly rectifying potassium channel, Kir5.1, and localization in renal tubular epithelia.

The physiological role of the inwardly rectifying potassium channel, Kir5.1, is poorly understood, as is the molecular identity of many renal potassium channels. In this study we have used Kir5.1-specific antibodies to reveal abundant expression of Kir5.1 in renal tubular epithelial cells, where Kir4.1 is also expressed. Moreover, we also show that Kir5.1/Kir4.1 heteromeric channel activity is extremely sensitive to inhibition by intracellular acidification and that this novel property is conferred predominantly by the Kir5.1 subunit. These findings suggest that Kir5.1/Kir4.1 heteromeric channels are likely to exist in vivo and implicate an important and novel functional role for the Kir5.1 subunit.

High insulin levels do not influence PC-1 gene expression and protein content in human muscle tissue and hepatoma cells.

BACKGROUND: To verify whether insulin levels influence PC-1 tissue content, we studied PC-1 gene expression and protein content in skeletal muscle of patients with insulinoma, a model of primary hyperinsulinemia. Data were compared with those obtained in matched insulin sensitive or resistant healthy subjects. In addition, the effect of high insulin concentration on PC-1 protein content was studied in HepG2 cells. METHODS: The following measurements were performed: insulin sensitivity by euglycemic clamp; PC-1 protein content and insulin receptor autophosphorylation by specific ELISAs; PC-1 gene expression by competitive polymerase chain reaction (PCR); phosphatidyl-inositol-3 kinase by immunoprecipitation and thin layer chromatography; glycogen synthesis by (14)C-glucose incorporation. RESULTS: Muscle PC-1 content was similar in the insulinoma patients and in insulin sensitive controls but higher (p<0.01) in insulin resistant controls (21.9+/-4.6 ng/mg protein, 23.8+/-3.9, 48.0+/-8.7, respectively). PC-1 protein content was inversely correlated with insulin sensitivity (r=-0.5, p<0.015) but with neither plasma insulin nor glucose levels. PC-1 protein content was correlated with PC-1 gene expression (r=0.53, p<0.05, n=14). Exposure to high insulin (100 nmol/l for 16 h) caused a significant (p<0.05-0.01) impairment of insulin receptor autophosphorylation, phosphatidyl-inositol-3 kinase activity and glycogen synthesis, but not of PC-1 protein content (114+/-3 vs 102+/-14 ng/mg protein) in HepG2 cells. CONCLUSION: These findings suggest that chronic high insulin levels do not influence PC-1 expression.

Role of receptor protein tyrosine phosphatase alpha (RPTPalpha) and tyrosine phosphorylation in the serotonergic inhibition of voltage-dependent potassium channels.

The activity of voltage-gated potassium (Kv) channels can be dynamically modulated by several events, including neurotransmitter-stimulated biochemical cascades mediated by G-protein-coupled receptors. By using a heterologous expression system, we show that activating the 5-HT2C receptor inhibits both Kv1.1 and Kv1.2 channels through a tyrosine phosphorylation mechanism. The major molecular determinants of channel inhibition were identified as two tyrosine residues located in the N-terminal region of the Kv channel subunit. Furthermore, we demonstrate that receptor protein tyrosine phosphatase alpha (RPTPalpha), a receptor protein tyrosine phosphatase, co-ordinates the inhibition process mediated via 5-HT2C receptors. We therefore propose that the serotonergic regulation of human Kv1.1 and Kv1.2 channel activity by the 5-HT2C receptor involves the dual coordination of both RPTPalpha and specific tyrosine kinases coupled to this receptor.

A soluble PC-1 circulates in human plasma: relationship with insulin resistance and associated abnormalities.

An increased tissue content of PC-1, an inhibitor of insulin receptor signaling, may play a role in insulin resistance. Large scale prospective studies to test this hypothesis are difficult to carry out because of the need for tissue biopsies. The aim of this study was to investigate whether PC-1 is measurable in human plasma and whether its concentration is related to insulin sensitivity. A soluble PC-1, with mol wt and enzymatic activity similar to those of tissue PC-1, was measurable in human plasma by a specific enzyme-linked immunosorbent assay and was inversely correlated to skeletal muscle PC-1 content (r = -0.5; P < 0.01). The plasma PC-1 concentration was decreased (P < 0.05) in insulin-resistant (22.7 +/- 3.0 ng/mL; n = 25) compared to insulin-sensitive (36.7 +/- 4.5; n = 25) nondiabetic subjects and was correlated negatively with the waist/hip ratio (r = -0.48; P < 0.001) and mean blood pressure (r = -0.3; P < 0.05) and positively with high density lipoprotein/total cholesterol (r = 0.38; P < 0.01) and both the M value and the plasma free fatty acid level decrement at clamp studies (r = 0.28; n = 50; P = 0.05 and r = 0.43; n = 22; P < 0.05, respectively). A plasma PC-1 concentration of 19 ng/mL or less identified a cluster of insulin resistance-related alterations with 75% accuracy. In conclusion, PC-1 circulates in human plasma, and its concentration is related to insulin sensitivity. This may help to plan studies aimed at understanding the role of PC-1 in insulin resistance.

Mutations in the KCNA1 gene associated with episodic ataxia type-1 syndrome impair heteromeric voltage-gated K(+) channel function.

Episodic ataxia type-1 syndrome (EA-1) is an autosomal dominant neurological disorder that manifests itself during infancy and results from point mutations in the voltage-gated potassium channel gene hKv1.1. The hallmark of the disease is continuous myokymia and episodic attacks of spastic contractions of the skeletal muscles, which cause permanent disability. Coexpression of hKv1.1 and hKv1.2 subunits produces heteromeric potassium channels with biophysical and pharmacological properties intermediate between the respective homomers. By using tandemly linked subunits, we demonstrate that hKv1.1 subunits bearing the EA-1 mutations V408A and E325D combine with hKv1.2 to produce channels with altered kinetics of activation, deactivation, C-type inactivation, and voltage dependence. Moreover, hKv1.1V408A single-channel analysis reveals a approximately threefold reduction of the mean open duration of the channel compared with the wild-type, and this mutation alters the open-state stability of both homomeric and heteromeric channels. The results demonstrate that human Kv1.2 and Kv1.1 subunits coassemble to form a novel channel with distinct gating properties that are altered profoundly by EA-1 mutations, thus uncovering novel physiopathogenetic mechanisms of episodic ataxia type-1 myokymia syndrome.

Localization and age-dependent expression of the inward rectifier K+ channel subunit Kir 5.1 in a mammalian reproductive system.

Kir 5.1 is a member of the inward rectifier potassium channel superfamily which does not form functional channels when expressed by itself in Xenopus laevis oocytes. rt-PCR reveals high levels of Kir 5.1 mRNA expression in testis but the function of this channel remains unknown. To determine the cell-specific expression of this channel in the testis we raised a polyclonal antibody against an external epitope of Kir 5.1 and tested its specificity in Xenopus oocytes expressing several cloned Kir subunits. Strong immunoreactivity for Kir 5.1 was found in seminiferous tubules of rat testis and, particularly, in spermatogonia, primary and secondary spermatocytes, spermatids and in the head and body of spermatozoa. The intensity of Kir 5.1 immunofluorescence, quantified using laser scanning microscopy, increased with age at every stage in the development of sperm from spermatogonia and reached a peak in 60-day-old rats. In contrast, the immunofluorescence decreased in 90-day-old animals and was detected mostly in spermatozoa. The results demonstrate that Kir 5.1 expression in the testis is localised to cells involved in spermatogenesis, showing a temporal pattern of expression during sexual maturity.