Characterization of a novel SCN5A mutation associated with Brugada syndrome reveals involvement of DIIIS4-S5 linker in slow inactivation

OBJECTIVE: Mutations in SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel (Na(v)1.5), have been associated with various inherited arrhythmia syndromes, including Brugada syndrome (BrS). Here, we report the functional consequences of a novel missense SCN5A mutation, G1319V, identified in a BrS patient. The G1319V mutation is located in the loop connecting transmembrane segments 4 and 5 in domain III (DIIIS4-S5), a region so far considered to be exclusively involved in fast inactivation. METHODS: Whole-cell mutant (G1319V) and wild-type (WT) sodium currents (I(Na)) were studied in the Human Embryonic Kidney cell line (HEK-293) transfected with Na(v)1.5 alpha-subunit cDNA (WT or mutant) together with hbeta(1)-subunit cDNA, using the patch-clamp technique. RESULTS: Maximal peak I(Na) and persistent sodium current were similar in WT and channel G1319V channels. The G1319V mutation shifted the potential of half-maximal (V(1/2)) activation towards more positive potentials (+3.7 mV), thereby increasing the degree of depolarization required for activation. The V(1/2) of inactivation of G1319V channels was shifted by -6.0 mV compared to WT, resulting in a reduced channel availability. The change in the steady-state inactivation was completely due to a negative shift (-6.8 mV) of the voltage-dependence of slow inactivation, while the voltage-dependence of fast inactivation was unaffected. The fast component of recovery from inactivation of G1319V channels was slowed down. Finally, the G1319V mutation caused a two-fold increase in the propensity of the channels to enter the slow inactivated state. Reduction in I(Na) peak amplitude on repetitive depolarizations at short interpulse intervals (40 ms) was significantly more pronounced in G1319V compared to WT. Accordingly, carriers of the G1319V mutation showed marked QRS widening upon increases in heart rate during exercise testing, pointing to enhancement of slow inactivation. CONCLUSIONS: We identified the DIIIS4-S5 linker as a new region involved in slow inactivation of Na(v)1.5. The biophysical alterations of the G1319V mutation all contribute to a reduction in I(Na), in line with the proposed mechanism underlying BrS.     

Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum

BACKGROUND: Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. AIM: To characterise the inherent pathomechanisms of G lamblia infection. METHODS: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short-circuit current (I(SC)) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining. RESULTS: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Omega cm(2); p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm(2)). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal I(SC) increased from 191 (20) in control to 261 (12) microA/h/cm(2) in giardiasis. The bumetanide-sensitive portion of I(SC) in giardiasis was also increased (51 (5) vs 20 (9) microA/h/cm(2) in control; p<0.05). Finally, phlorizin-sensitive Na(+)-glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) microA/h/cm(2)). CONCLUSIONS: G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na(+)-dependent d-glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components.     

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0)

An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G(1) fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G(0)/G(1) and S/G(2)/M. Interestingly, the G(0) cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle-associated change in in vivo stem cell homing, the cultured G(0)/G(1) and S/G(2)/M CD34(+) CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor beta(1) that increased the G(0)/G(1) fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle-associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo-manipulated grafts. (Blood. 2000;96:4185-4193)     

Squamous cutaneous epithelial cell carcinoma in two CML patients with progressive disease under imatinib treatment

Imatinib (glivec), formerly known as STI571) effectively blocks the ATP-binding site of the bcr/abl fusion protein thereby inactivating selectively the tyrosine kinase activity of bcr/abl. Therefore, it is a promising drug in Philadelphia chromosome positive chronic myeloid leukemia showing high hematologic and cytogenetic response rates combined with a mild toxicity profile. Here we report two cases of squamous cell carcinoma of the skin, which appeared in the photo-exposed areas in two elderly patients treated for advanced chronic myeloid leukemia with imatinib. The role of chemotherapy, chronic sun exposure and of possible additional risk factors such as human papillomavirus infection is discussed.     

Nucleolar staining cannot be used as a screening test for the scleroderma marker anti-RNA polymerase I/III antibodies

OBJECTIVE: Anti-RNA polymerase I/III (anti-RNAP I/III) antibodies are clinically useful markers of scleroderma, and their presence is associated with diffuse skin disease and an increased risk of cardiac and kidney involvement. Although RNAP I antibodies localize to the nucleolus, nucleolar staining by many anti-RNAP antibody-positive sera is not always observed. Nucleolar staining by anti-RNAP antibody-positive sera was examined by double staining with antifibrillarin antibodies to evaluate whether nucleolar staining can be used as a screening test for anti-RNAP I/III antibodies. In addition, the relationships between nucleolar staining and levels of anti-RNAP III antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) assay. METHODS: Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides, by anti-RNAP III ELISA, and by IP assay using (35)S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and by IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed. RESULTS: All 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44% of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining. CONCLUSION: Although some sera positive for anti-RNAP I/III antibodies clearly stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.     

Endocrinologic response to vasopressin infusion in advanced vasodilatory shock

OBJECTIVES: To evaluate the endocrinologic response to a combined arginine vasopressin and norepinephrine (AVP/NE) infusion in advanced vasodilatory shock, and to examine the relationship between baseline plasma AVP concentrations and the hemodynamic response to AVP. DESIGN: Preliminary, prospective, randomized, controlled clinical study. SETTING: Twenty-three-bed general and surgical intensive care unit. PATIENTS: Thirty-eight patients with advanced vasodilatory shock. Hemodynamic and laboratory data of 34 patients have already been presented in a recently published prospective, randomized, controlled study. INTERVENTIONS: Continuous AVP (4 units/hr) and NE infusion in study patients; NE infusion only in control patients. MEASUREMENTS AND MAIN RESULTS: At baseline, 24 hrs, and 48 hrs after randomization, plasma concentrations of AVP, adrenocorticotropic hormone, cortisol, renin, angiotensin II, aldosterone, prolactin, endothelin I, and atrial natriuretic factor were determined. Hemodynamic variables were recorded at baseline and 1, 12, and 24 hrs after randomization. Linear mixed effects models were used to test for differences between groups. The relationship between AVP plasma concentrations and hemodynamic response to AVP was analyzed using linear regression analyses. AVP/NE patients exhibited significantly higher AVP (p <.001) and prolactin (p <.001) plasma concentrations during the study period; there were no significant differences in plasma concentrations of other hormones. No significant correlation was detected between plasma AVP concentrations and the increase in mean arterial pressure after 1 hr (Pearson's correlation coefficient =.134, p =.584) and after 24 hrs (Pearson's correlation coefficient = -.198, p =.417). There were further no correlations between AVP plasma concentrations and the 24-hr response to AVP therapy in heart rate (Pearson's correlation coefficient = -.065, p =.791), stroke volume index (Pearson's correlation coefficient = -.106, p =.687), and NE requirements (Pearson's correlation coefficient =.04, p =.869). CONCLUSIONS: The preliminary results of this study indicate that a combined AVP and NE infusion increases prolactin plasma concentrations in advanced vasodilatory shock. Hemodynamic effects of AVP infusion are independent of baseline plasma AVP concentrations.     

Bladder pain syndrome

Bladder pain syndrome is a deceptively intricate symptom complex that is diagnosed on the basis of chronic pelvic pain, pressure, or discomfort perceived to be related to the urinary bladder, accompanied by at least one other urinary symptom. It is a diagnosis of exclusion in a patient who has experienced the symptoms for at least 6 weeks in the absence of any confusable diseases that may give rise to the symptoms. Symptoms compatible with the diagnosis are now thought to affect up to 3% of the female population in the United States with a 5:1 female-to-male preponderance. Diagnosis and treatment can be challenging, and misdiagnosis as a psychological problem, overactive bladder, or chronic urinary infection has plagued patients with the problem.     

Nonideal Rayleigh–Taylor mixing

Rayleigh–Taylor mixing is a classical hydrodynamic instability that occurs when a light fluid pushes against a heavy fluid. The two main sources of nonideal behavior in Rayleigh–Taylor (RT) mixing are regularizations (physical and numerical), which produce deviations from a pure Euler equation, scale invariant formulation, and nonideal (i.e., experimental) initial conditions. The Kolmogorov theory of turbulence predicts stirring at all length scales for the Euler fluid equations without regularization. We interpret mathematical theories of existence and nonuniqueness in this context, and we provide numerical evidence for dependence of the RT mixing rate on nonideal regularizations; in other words, indeterminacy when modeled by Euler equations. Operationally, indeterminacy shows up as nonunique solutions for RT mixing, parametrized by Schmidt and Prandtl numbers, in the large Reynolds number (Euler equation) limit. Verification and validation evidence is presented for the large eddy simulation algorithm used here. Mesh convergence depends on breaking the nonuniqueness with explicit use of the laminar Schmidt and Prandtl numbers and their turbulent counterparts, defined in terms of subgrid scale models. The dependence of the mixing rate on the Schmidt and Prandtl numbers and other physical parameters will be illustrated. We demonstrate numerically the influence of initial conditions on the mixing rate. Both the dominant short wavelength initial conditions and long wavelength perturbations are observed to play a role. By examination of two classes of experiments, we observe the absence of a single universal explanation, with long and short wavelength initial conditions, and the various physical and numerical regularizations contributing in different proportions in these two different contexts.