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R Horn

Publications and source records attributed to R Horn.

At least 55 records · Page 3Linked to original sources
PubMed1997

Anomalous effect of permeant ion concentration on peak open probability of cardiac Na+ channels.

Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. Decreasing extracellular permeant ion concentration decreases outward Na+ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na+, Li+, or hydrazinium) is substituted for a relatively impermeant cation (K+, Rb+, Cs+, N-methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na+ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to -140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.

Animals↗
PubMed1997

Effect of alkali metal cations on slow inactivation of cardiac Na+ channels.

Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. The kinetics of decaying outward Na+ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li+, Na+, K+, Rb+, and Cs+ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na+]zero from 10 to 150 mM increases the rate of recovery from slow inactivation at -140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na+]0 is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na+]0. However raising [Na+]0 decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na+]0, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.

Alkalies↗
PubMed1997

Gender-specific differences of serum leptin in obese and normal-weight adolescents: studies in type-I diabetes and Turner syndrome.

The influence of exogenous insulin and estrogen substitution on serum leptin-like immunoreactivity was studied longitudinally in patients with type-I diabetes and Turner syndrome using a specific radioimmunoassay. Prepubertal, pubertal and postpubertal samples of 17 patients (9 girls, 8 boys) with type-I diabetes mellitus developing obesity were compared to those of 17 normal-weight controls matched for gender, age and diabetes duration. Six obese and six normal-weight girls with Turner syndrome were studied without hormone substitution, with ethinylestradiol alone, and with cyclic estradiol/gestagen substitution. The mean leptin levels of the girls with diabetes were two times higher than boys at all times, while insulin doses and glycemic control had no influence. In Turner syndrome estrogen substitution led to increased leptin levels only in the obese group. This study revealed that both body weight above normal and female sex steroids seem to be necessary to elevate leptin concentrations, while exogenous insulin has no effect.

Adolescent↗
PubMed1997

UKPDS 20: plasma leptin, obesity, and plasma insulin in type 2 diabetic subjects.

We measured plasma leptin and insulin concentrations across a spectrum of obesity in 829 white Caucasian, 154 Afro-Caribbean, and 204 Asian type 2 diabetic subjects. Although the leptin concentrations covered a large range, there were no subgroups of diabetic subjects with very high or low leptin levels that would suggest mutations in the leptin gene or leptin receptor gene comparable to the obese diabetic ob/ob and db/db mice models respectively. In all three ethnic groups, leptin concentrations correlated with body mass index (BMI) in a similar manner to nondiabetic patients and were higher in females than males after adjustment for BMI, with no difference between ethnic groups. In a multivariate regression analysis, plasma leptin was associated with gender and BMI, (both P < 1 x 10(-17)) and with fasting plasma insulin concentrations (P = 5 x 10(-9)). Subjects treated with insulin had both raised insulin and leptin concentrations. When matched for different therapies, gender, and BMI, diabetic subjects with high leptin levels also had high insulin levels (P < 0.0009). High leptin concentrations may in part be influenced by hyperinsulinemia or impaired insulin sensitivity.

Aged↗
PubMed1996

Effects of chain length and sulphur position of thia fatty acids on their incorporation into phospholipids in 7800 C1 hepatoma cells and isolated rat hepatocytes, and their effects on fatty acid composition of phospholipids.

Incorporation of thia fatty acids and their effects on the fatty acid composition in phospholipids has been investigated in 7800 C1 hepatoma cells and cultured hepatocytes. 3-Thia fatty acids of chain lengths from dodecyl-to hexadecyl-thioacetic acid were incorporated into phospholipids during a 3-day incubation. Longer and shorter 3-thia fatty acids were barely detectable. Tetradecylthioacetic acid, 3-thia stearate, and their delta9- desaturated derivatives were maximally incorporated into whole-cell phospholipids. The amount of tetradecylthioacetic acid incorporated into phospholipids of hepatoma cells remained almost identical in cells cultured for 3 days or adapted over a period of 1 year. Delta9-desaturated metabolites of long chain thia fatty acids (C13-to C16-S-acetic acid) were identified by GC-MS in phospholipids. 3-Thia stearate appeared to be the best substrated for delta9 desaturase. Incubation of hepatoma cells with thia fatty acids led to alterations in the amount of normal fatty acids in total phospholipids. The amounts of 16:0 and 18:1 decreased and 18:2 (n-6) and 20:5 (n-3) increased. Changes in the normal fatty acid composition of phospholipids were seen both with thia acids incorporated into phospholipids and those not incorporated. These effects, therefore, may be only partially dependent on displacement of normal fatty acids by thia fatty acids. Morris 7800 C1 hepatoma cell acyl-CoA synthetase (ACS) and peroxisomal acyl-CpA oxidase (ACO) were induced by thia fatty acids of all chain lengths, and with the sulphur atom(s) in different positions. Control experiments with hepatocytes revealed a similar incorporation of thia fatty acids in these physiologically more normal cells.

Acyl-CoA Oxidase↗
PubMed1996

The CMS-associated 16 kDa protein encoded by orfH522 in the PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower.

In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3'-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3'-coding region of orfH522 to verify by immunological methods the identity of the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms which expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor.

Cytoplasm↗
PubMed1996

Molecular basis of charge movement in voltage-gated sodium channels.

Voltage-dependent movement of a sodium channel S4 segment was examined by cysteine scanning mutagenesis and testing accessibility of the residues to hydrophilic cysteine-modifying reagents. These experiments indicate that 2 charged S4 residues move completely from an internally accessible to an externally accessible location in response to depolarization by passage through a short "channel" in the protein. The energetic problems of S4 movement have thus been solved in the same way that may ion channels achieve highly selective and rapid ion permeation through an open pore, by restricting the contact region between the permion and its channel.

Amino Acid Sequence↗
PubMed1996

Interactions between a pore-blocking peptide and the voltage sensor of the sodium channel: an electrostatic approach to channel geometry.

Few experimental data illuminate the relationship between the molecular structures that mediate ion conduction through voltage-dependent ion channels and the structures responsible for sensing transmembrane voltage and controlling gating. To fill this void, we have used a strongly cationic, mutated mu-conotoxin peptide, which only partially blocks current through voltage-dependent sodium channels, to study voltage-dependent activation gating in both bound and unbound channels. When the peptide binds to the ion-conducting pore, it inhibit channel opening, necessitating stronger depolarization for channel activation. We show that this activation shift could result entirely from electrostatic inhibition of the movement of the voltage-sensing S4 charges and estimate the approximate physical distance through which the S4 charges move.

Animals↗
PubMed1996

Radioimmunoassay for the detection of leptin in human serum.

Human leptin, which is encoded by the obese (ob) gene, is secreted specifically from adipocytes and is involved in the regulation of satiety and energy consumption. We developed a radioimmunoassay for the determination of leptin in human serum using polyclonal antibodies generated in rabbits against a C-terminal fragment of leptin, leptin(126-140), coupled to hemocyanin. The sensitivity of the assay was app. 5 pmol/l leptin(126-140) equivalent to 0.5 fmol/tube. The intra-assay variation at 100 pmol/l was less than 4.8% and the interassay variation less than 8.3%. Dilution curves of serum samples containing high levels of leptin(126-140) were parallel to the standard curve. Following G-50 Sephadex chromatography a single specific peak was detected at app. 16 kd. The assay procedure compared well to a commercially available assay (Linco, St. Louis, USA) using polyclonal antibodies directed against the intact recombinant protein (R = 0.96; p < 0.0001). Serum levels were significantly higher than plasma levels (app.20%) over a wide range of the standard curve. Levels of serum leptin126-140 immunoreactivity were not altered by meals and no day-to-day variation was found. In a group of 148 healthy female and 108 healthy male subjects with a BMI between 18.2 and 40 kg/m2 there was a significant difference between sexes with higher circulating serum levels in female than in male subjects when tested for identical BMI (p < 0.001). Serum leptin levels in both male and female subjects were positively related to BMI (p < 0.001) when analysed for lean and obese subjects whereas in lean subjects this relation was not apparent. No relation of serum leptin levels and age was detectable in subjects with a BMI up to 30 kg/m2. These data support an important role of leptin in the regulation of body fat stores and BMI which is modulated by gender specific factors.

Adult↗
PubMed1996

Serum leptin levels following hypothalamic surgery.

To study a potential alteration of hypothalamic centers involved in the negative feedback action of leptin on body weight, serum leptin levels were measured in relation to BMI in 18 patients following surgery for a hypothalamic craniopharyngioma (Ctx), and were compared to levels found in 21 patients operated for a pituitary adenoma (Ptx) or in healthy control subjects. All subjects with Ptx received rhGH replacement therapy (0.5 to 2 IU/m2/d), and serum leptin levels were followed in 3 months intervals over 24 months. Serum leptin levels in patients with Ptx were comparable to controls, whereas 7 of the 18 patients with Ctx had higher than expected concentrations for their BMI. GH treatment in Ptx subjects did not alter serum leptin levels. In 5 Ctx patients where preoperative samples were available, weight gain in parallel to an increase in serum leptin levels was observed but only minimal changes in 4 others. Our data support the role of leptin as an important marker of body weight. The rapid increase in serum leptin levels observed in some Ctx subjects suggests that early postoperative measurement of serum leptin levels may help to identify patients at risk of weight gain following hypothalamic destruction.

Adenoma↗
PubMed1996

Paramyotonia congenita mutations reveal different roles for segments S3 and S4 of domain D4 in hSkM1 sodium channel gating.

Mutations in the gene encoding the voltage-gated sodium channel of skeletal muscle (SkMl) have been identified in a group of autosomal dominant diseases, characterized by abnormalities of the sarcolemmal excitability, that include paramyotonia congenita (PC) and hyperkalemic periodic paralysis (HYPP). We previously reported that PC mutations cause in common a slowing of inactivation in the human SkMl sodium channel. In this investigation, we examined the molecular mechanisms responsible for the effects of L1433R, located in D4/S3, on channel gating by creating a series of additional mutations at the 1433 site. Unlike the R1448C mutation, found in D4/S4, which produces its effects largely due to the loss of the positive charge, change of the hydropathy of the side chain rather than charge is the primary factor mediating the effects of L1433R. These two mutations also differ in their effects on recovery from inactivation, conditioned inactivation, and steady state inactivation of the hSkMl channels. We constructed a double mutation containing both L1433R and R1448C. The double mutation closely resembled R1448C with respect to alterations in the kinetics of inactivation during depolarization and voltage dependence, but was indistinguishable from L1433R in the kinetics of recovery from inactivation and steady state inactivation. No additive effects were seen, suggesting that these two segments interact during gating. In addition, we found that these mutations have different effects on the delay of recovery from inactivation and the kinetics of the tail currents, raising a question whether this delay is a reflection of the deactivation process. These results suggest that the S3 and S4 segments play distinct roles in different processes of hSkM1 channel gating: D4/S4 is critical for the deactivation and inactivation of the open channel while D4/S3 has a dominant role in the recovery of inactivated channels. However, these two segments interact during the entry to, and exit from, inactivation states.

Adult↗
PubMed1996

Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker.

A pair of conserved methionine residues, located on the cytoplasmic linker between segments S4 and S5 in the fourth domain of human heart Na channels (hH1), plays a role in the kinetics and voltage dependence of inactivation. Substitution of these residues by either glutamine (M1651M1652/QQ) or alanine (MM/AA) increases the inactivation time constant (tau) at depolarized voltages, shifts steady-state inactivation (h infinity) in a depolarized direction, and decreases the time constant for recovery from inactivation. The data indicate that the mutations affect the rate constants for both binding and unbinding of a hypothetical inactivation particle from its binding site. Cytoplasmic application of the pentapeptide KIFMK in Na channels mutated to remove inactivation produces current decays resembling inactivation (Eaholtz, G., T. Scheuer, and W.A. Catterall. 1994. Neuron. 12: 1041-1048.). KIFMK produces a concentration-dependent, voltage-independent increase in the decay rate of MM/QQ and MM/AA currents at positive membrane potentials (Ki approximately 30 microM), while producing only a small increase in the decay rate of wild-type currents at a concentration of 200 microM. Although MM/QQ inactivates approximately 2.5-fold faster than MM/AA in the absence of peptide, the estimated rate constants for peptide block and unblock do not differ in these mutants. External Na+ ions antagonize the block by cytoplasmic KIFMK of MM/AA channels, but not the inactivation kinetics of this mutant in the absence of peptide. The effect of external [Na+] is interpreted as a voltage-dependent knock-off mechanism. The data provide evidence that KIFMK can only block channels when they are open and that peptide block does not mimic the inactivation process.

Base Sequence↗
PubMed1996

A unique role for the S4 segment of domain 4 in the inactivation of sodium channels.

Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-S6). The highly charged S4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in S4 segments of each domain of the human heart Na+ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na+ current, consistent with a role of S4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (eo) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h infinity) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in S4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.

Animals↗
PubMed1996

Serum leptin and weight reduction in female obesity.

Leptin, an adipocyte-derived hormone, induces a decrease in food intake and increases energy expenditure via hypothalamic interactions. In animal models obesity can be caused by leptin deficiency or by a dysfunction of the hypothalamic leptin receptor. Using a radioimmunoassay for the determination of leptin in human serum, we measured serum leptin levels in 227 otherwise healthy normal weight (N = 78; body mass index = 16.1-27.7 kg/m2) or obese women (N = 149; body mass index = 27.8-56.7 kg/m2). Fifty-three subjects were followed over a period of 12 weeks under weight reduction (800 kcal/day) and a subgroup of 33 for another 13 weeks after termination of the diet. Body mass index and serum leptin concentrations were measured longitudinally and compared to female controls not under diet. Under baseline conditions, log serum leptin levels were positively related to body mass index with a best fit using a non-linear regression (p < 0.001), indicating an attenuated increase in serum leptin levels with high body mass index. No subgroup with low serum leptin levels could be identified. Weight reduction induced a rapid decrease in serum leptin levels within the first 3 weeks to levels significantly lower than in body mass index-matched controls under normal diet (p < 0.001). This pattern was consistent after 6 and 12 weeks. Serum leptin levels increased again after the end of the diet but remained significantly lower than in the controls despite unrestricted calorie intake over 7 weeks. The rapid and persistent decrease in serum leptin to lower levels than expected from matched controls may explain the pertinent difficulties of obese subjects to cope with weight reduction.

Adult↗
PubMed1996

Morphological and morphometric study of early changes in the ageing golden hamster testis.

The histological and morphometric features of the aged golden hamster testis were examined and compared with those of adult animals. Three age groups (6, 12 and 18 months) were studied by light microscopy, and testosterone levels were determined. The observations showed a progressive involution of the seminiferous tubules, beginning to be perceptible at 12 months with slight hypospermatogenesis and desquamation. In 18-month-old specimens degeneration was more significant and histopathological lesions could be classified on a 6-point scale, ranging from slight hypospermatogenesis to absence of germ cells. These involutive changes were not homogeneously distributed in the testis; affected tubules close to seeming normal ones were present. The morphometric results point to a progressive diminution, in the 3 age groups, in vas deferens spermatozoa, pachytene spermatocytes, and Sertoli and Leydig cells (the latter significantly diminished only in the 18-month-old group). For morphometric purposes a 7-point scale of tubule degeneration was used, showing a significant increase, with age, in the presence of more degenerated tubule stages. Several correlations were found between the morphometric variables, outlining existing relations between age and the associated diminution of several testis cell types, and lumen diameter. No significant differences were found between groups in serum testosterone levels. In conclusion, histological changes related to age are evident in 18-month-old animals, while at 12 months a diminution in germ cell numbers and sperm production is detectable.

Aging↗
PubMed1995

The structure of U17 isolated from Streptomyces clavuligerus and its properties as an antioxidant thiol.

The predominant low-molecular-mass thiol produced by streptomycetes is a cysteine derivative previously designated as U17 [Newton, G. L., Fahey, R. C., Cohen, G. & Aharonowitz, Y. (1993) J. Bacteriol. 175, 2734-2742]. In this study we report the elucidation of the structure of the monobromobimane derivative of U17, which establishes the structure of U17 as 2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl-myo-inositol. The presence of the N-acetylcysteine moiety was indicated by formation of N-acetylcysteine-monobromobimane during acid hydrolysis of the monobromobimane derivative of U17. Complete hydrolysis released 1 mol glucosamine/mol cysteine as determined by carbohydrate and amino acid analysis. High-resolution mass spectral analysis gave a precise mass consistent with the molecular formula C27H40N4O14S. Analysis of 13C-NMR, one-dimensional 1H-NMR and two-dimensional NMR experiments identified the remaining C6H12O6 moiety as myo-inositol, confirmed the presence of N-acetylcysteine and glucosamine, and established the connectivity of the components. Two chemical properties of this novel thiol make it suitable as an intracellular storage form of cysteine and as an antioxidant thiol. First, it undergoes heavy-metal-ion catalyzed autoxidation at a rate dramatically lower than that for cysteine and markedly lower than that for glutathione or N-acetylcysteine. Secondly, the alpha-(1-->1) glycosidic link between glucosamine and myo-inositol is resistant to acid hydrolysis, hydrolysing at a rate comparable to that of the two amide bonds in the molecule.

Antioxidants↗
PubMed1995

Evidence for voltage-dependent S4 movement in sodium channels.

The mutation R1448C substitutes a cysteine for the outermost arginine in the fourth transmembrane segment (S4) of domain 4 in skeletal muscle sodium channels. We tested the accessibility of this cysteine residue to hydrophilic methanethiosulfonate reagents applied to the extracellular surface of cells expressing these mutant channels. The reagents irreversibly increase the rate of inactivation of R1448C, but not wild-type, channels. Cysteine modification is voltage dependent, as if depolarization extends this residue into the extracellular space. The rate of cysteine modification increases with depolarization and has the voltage dependence and kinetics expected for the movement of a voltage sensor controlling channel gating.

Adult↗