Point mutation in the band 4.2 gene associated with autosomal recessively inherited erythrocyte band 4.2 deficiency

Abstract: A patient who represented acute hemolytic crisis was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of band 4.2. In the family, the sister of the patient who had been clinically normal was also shown to be deficient in band 4.2. Binding studies showed that the propositus' membranes were able to bind normal band 4.2 protein as much as control. It was suggested that the binding sites for the protein were prepared on the membrane. We analyzed the band 4.2 cDNA of the propositus and detected a mutation that changes a codon for alanine to one for threonine at residue 142. Band 4.2 exon III of genomic DNA which included the mutation site was amplified and sequenced directly in the family members, and it was revealed that only the homozygotes of the mutation allele manifested band 4.2 deficiency and the parents, who were heterozygotes, showed normal amounts of band 4.2. Recently, the same mutation was reported as Protein 4.2^NIPPON in another 4 cases (Bouhassira et al. Blood 1992: 79: 1846–1854). This study supports the hypothesis that this mutation is the pathogenetic cause of band 4.2 deficiency and not a polymorphism.     

新生儿溶血病的不典型表现(附8例报告)

本文报道了8例新生儿溶血病的不典型临床表现及其实验窒检查。其中ABO不合7例,Rh不合1例。不典型表现是黄疸均在生后48小时后出现,还可出现后期贫血、低钙性抽搐、迁延性黄疸、肝炎综合征等,对其机制进行了探讨。     

Transient outward current (I A) in clonal anterior pituitary cells: blockade by aminopyridine analogs

Summary Whole cell voltage-clamp recordings from GH₃ cells, a clonal cell line derived from a rat anterior pituitary tumor, demonstrated a rapidly activating and inactivating (transient) voltage-dependent outward current. This current, referred to as I _A, was elicited by step depolarization from holding potentials negative to –50 mV, showed strong outward rectification at potentials positive to –30 mV, and exhibited steady state inactivation with V _1/2 near –64 mV. The current rose to a peak within < 10–20 ms following depolarization and decayed in two exponential phases, I _Af and IA _AS with time constants of 30–50 and 500–700 ms, respectively. Both I _A components exhibited similar voltage dependencies for activation and inactivation. Aminopyridines (2 mol/l – –5 mmol/l) produced a dose dependent, reversible blockade of I _A (70% inhibition at 0.5 to 2 mmol/l) with the following rank order of potencies: 4-aminopyridine > 3,4-diaminopyridine = 3-aminopyridine > 2-aminopyridine. These drugs reduced the peak conductance of I _A, and produced complex effects on its time-dependent decay. With submaximal degrees of block, there was an increase in the inactivation rate, suggesting that open channels are preferentially blocked by the drugs. It is concluded that GH₃ pituitary cells possess an aminopyridine-sensitive transient outward current comparable to the A-current in neural cells. However, this cell line is unusual in that it expresses both rapidly and slowly decaying A-current components.Abbreviations n-AP n-aminopyridine - 3,4-DAP 3,4-diaminopyridine - TEA tetraethylammonium - EGTA ethylene glycol bis(-aminoethyl ether)N,N-tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Send offprint requests to M. A. Rogawski at the above address     

The depressing effect of tetracaine and ryanodine on the slow outward current correlated with that of contraction in voltage-clamped frog muscle fibres

The effects of tetracaine (10–50 M) and ryanodine (0.1–10 M) were tested on the slow outward K⁺ current (I _so) and the mechanical tension of isolated frog muscle fibres in a voltage-clamp device (double mannitol-gap) connected to a mechanoelectric transducer. In the concentration range tested, both drugs induced a simultaneous inhibition of tension and current. In all cases the effect on tension was twice that on current. The tetracaine-induced current and tension blocks were fully reversible and dose-dependent. In contrast the ryanodine effects on current and tension were not reversible and did not exhibit a dose dependence except for the delay before the onset of the response, which was shortened when the concentration was raised. Linear regression analysis of the time-dependent and dose-dependent effects of both drugs indicated a strong correlation between the decreases in tension and current. It is concluded that the slow outward current is partly under the control of the Ca²⁺ release from sarcoplasmic reticulum during contraction.     

The sustained inward current in sino-atrial node cells of guinea-pig heart

 Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current I _f , the delayed rectifier K⁺ current I _K and the L-type Ca²⁺ current I _Ca,L as well as by the absence of both the transient outward current I _to and the inward rectifier K⁺ current I _K,1. After blocking I _f and I _K, depolarizing pulses from –80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca²⁺] ([Ca²⁺]_o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na⁺]_o, differently from I _Ca,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of I _st described in the rabbit SA node pacemaker cells, and that I _st is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither I _f nor I _st. Received: 22 July 1996 / Received after revision: 30 September 1996 / Accepted: 9 October 1996     

Multiple effects of caffeine on calcium current in rat ventricular myocytes

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca²⁺ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N,N-tetraacetic acid (BAPTA) (a fast Ca²⁺ chelator), (2) a stimulation of peak I _Ca that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak I _Ca that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of I _Ca inactivation is due to caffeine's ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak I _Ca is probably due to phosphodiesterase inhibition, while caffeine's inhibitory effect on I _Ca is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.The experimental part of this work was published during the postdoctoral stay of I. Zahradník in the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555, USA     

Sputum induction as a research tool for the study of human respiratory mucus

The study objectives were to compare in vitro transportability and physical properties of respiratory mucus, obtained invasively by direct collection (DC) right after endotracheal intubation and non-invasively by sputum induction with 3% hypertonic saline solution inhalation (SI) 24 h before the anesthesia. Twenty-two patients with no pulmonary disease scheduled for elective abdominal surgical procedures were studied. The parameters analyzed and the main results are as follows. (1) Transportability by cilia (MCT), SI was higher than DC (0.94+/-0.25 and 0.62+/-0.25; P<0.001). There was a significant correlation between the two methods and DC could be estimated by: DC=0.21+(0.44 SI) (r=0.44; P<0.001). (2) Transportability by cough (CC), SI was higher than DC (68.23+/-32.1 and 33.58+/-19.04 mm; P=0.002). (3) Contact angle (CA), SI was lower than DC (10+/-3 degrees and 22+/-14 degrees ; P=0.025). (4) Rheological properties (no significant difference obtained between SI and DC). These results indicated that SI changes mucus physical properties and transportability in non-expectorators.     

Decrease of potassium permeability by intracellular application of sodium ions in snail neurons

In the identified neurons B1, B2 and B3 of Helix pomatia an intracellular injection of Na+ induced an outward current in 10% and an inward current in 90% of the experiments. The outward current was associated with an increase and the inward current with a decrease of the membrane conductance. Both currents reversed at membrane potentials of between -60 and -70 mV. Inward currents were also elicited by intracellular Li+ or tris-[hydroxymethyl]-aminomethane (Tris+) injection. All inward currents were reduced by extracellular administration of tetraethylammonium or quinine. It is suggested that the outward current represents a calcium-activated potassium current and that the inward current is due to a blockade of potassium channels from the intracellular side.     

Development of a non-invasive treatment system for urinary incontinence using a functional continuous magnetic stimulator (FCMS)

A system composed of a functional continuous magnetic stimulator (FCMS) and a saddle-type coil has been developed for non-invasive treatment of urinary incontinence, especially stress incontinence and urge incontinence. The FCMS conditions were as follows: 2 kW maximum electrical power consumption, 800 V maximum capacitor voltage, 720 μs pulsewidth (180 μs rise time), and 5–30 Hz frequency. A frequency between 5 and 10 Hz is used to treat urge incontinence and a frequency between 25 Hz and 30 Hz is used to treat urge incontinence. The coil (120 mm long, 90 mm wide and 50 mm thick) fits the most suitable region for this treatment, the region from the anus to the perineum. The coil is cooled to maintain a coil temperature between 20 and 25°C so that it can be used efficiently and safely. In experiments with anaesthetised dogs, it was confirmed that the urethral pressure increased when the circumference of the perineum received continuous magnetic stimulation of 720 μs pulsewidth (180 μs rise time), 10Hz frequency and about 520 V capacitor voltage. This result suggests that magnetic stimulation can be effective as a urinary incontinence therapy.     

Primary sequence and functional expression of a novel β subunit of the P-ATPase gene family

The cortical collecting tubule (CCT) of the mammalian kidney reabsorbs sodium and potassium, processes that are mediated by Na/K-ATPase and H/K-ATPase. CCT is also an important site for proton secretion, which is driven, in part, by H/K-ATPase. Na/K-ATPase and H/K-ATPase are members of the ion-motive P-ATPase gene family. They are closely related plasma membrane proteins which consist of heterodimers. The urinary bladder of the toad Bufo marinus is the amphibian counterpart of mammalian CCT. We have previously characterized a ouabain-resistant Na/K-ATPase [see ref. 17], from TBM cells, a clonal cell line derived from the toad bladder, which expresses transepithelial sodium transport. In the present study, we report the primary sequence and functional expression of a novel subunit ( _bladder= _bl) isolated from a toad bladder epithelial cell cDNA library. The deduced polypeptide is 299 amino acids in length and has a predicted molecular mass of 33 kDa. The _bl protein exhibits 35% amino acid identity to the previously characterized ₁ of B. marinus Na/K-ATPase and 39% identity with ₃ of B. marinus Na/K-ATPase. It shares 38% identity with the mammalian gastric H/K-ATPase and 52% with the mammalian ₂ Na/K-ATPase. Northern blot analysis shows that a 1.4×10³-base mRNA is expressed at a high level in bladder epithelial cells and eye and at a trace level in kidney; it is not detectable in significant amounts in the stomach, colon and small intestine. The _bl subunit can associate with the ₁ subunit of B. marinus Na/K-ATPase to form a functional sodium pump in the Xenopus laevis oocyte. Our data indicate that, in addition to the known ₁ and ₃ isoforms, a third distinct isoform of the subunit is present in the bladder epithelium. This new isoform could be functionally associated with subunits of either Na/K- or H/K-ATPase.