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R Horn

Publications and source records attributed to R Horn.

At least 19 recordsLinked to original sources
PubMed2004

In vivo and in vitro evidence for a hepatic modulation of the leptin signal in rats.

BACKGROUND: Leptin is primarily secreted by the adipose tissue. It binds not only to hypothalamic structures involved in energy regulation but also to many peripheral tissues including the liver. Leptin circulates in free and receptor-bound forms. Both components are differentially regulated under various pathophysiological conditions and serve different physiological functions. They are released from adipose tissue but previous data suggest an additional formation outside the fat compartment. Here we tested the contribution of the liver in binding and modulating leptin in the circulation. MATERIALS AND METHODS: In vivo experiments were performed with radioactive labelled leptin with and without pretreatment with unlabelled leptin in freely moving, chronic intravenously cannulated male rats. Livers were investigated by immunohistochemistry and in situ hybridization and immunoblotting was performed, followed by ex vivo liver perfusion studies with human recombinant leptin. RESULTS: In in vivo experiments radioactively labelled leptin (at low concentrations) is avidly bound to rat liver (greater than 80% of basal serum values 90 min following i.v. infusion). Pre-treatment with excess of unlabelled leptin in vivo revealed a rapid hepatic down-regulation of leptin receptor isoforms when tested by in situ hybridization, immunoblotting or immunohistochemistry. Ex vivo perfusion of rat liver with human recombinant leptin induced a dose- and time-dependent formation of receptor-bound leptin in the perfusate. CONCLUSIONS: The present data support an active role of the liver in the modulation of the leptin signal through different regulation of the soluble leptin receptor, the bound and free forms of the hormone, which may have important implications for leptin's central efficacy and the development of 'leptin resistance'.

Adipose Tissue↗
PubMed2004

Involution of seminiferous tubules in aged hamsters: an ultrastructural, immunohistochemical and quantitative morphological study.

In this study, we examined the age-related changes on morphometric parameters and ultrastructure of seminiferous tubules, and on the expression of extracellular matrix proteins in lamina propria of Syrian hamsters. A significant decrease in the percentage of normal tubules and an increase in the percentage of hypospermatogenic and arrested maturation tubules was observed with aging. Aged animals showed a decrease in tubular diameter, tubular lumen, seminiferous epithelium volume and total tubular volume. However, the total length of seminiferous tubules was significantly increased with aging. The most important ultrastructural changes with aging were the thickening of the lamina propria, the presence of diverse abnormalities in the spermiogenesis process, degeneration of germ cells, and vacuolization and flattening of Sertoli cells showing abundant lipofucsin droplets and residual bodies. Laminin immunoreactivity was found along the lamina propria of seminiferous tubules both in young and aged animals. Fibronectin immunoreactivity was found along the lamina propria and blood vessels. Both laminin and fibronectin total volume of immunostaining per testis was increased in aged hamsters. In conclusion, the age-related changes in seminiferous tubules of hamster include: a decrease in tubular width and an increase in tubular length; widening of the lamina propria caused by a more extensive connective matrix between the peritubular cells and the basal membrane; and a strong disarrangement of the seminiferous epithelium, including germ cell degeneration and important alterations in both spermiogenesis and Sertoli cell structure.

Aging↗
PubMed2003

The pentapeptide QYNAD does not block voltage-gated sodium channels.

BACKGROUND: An endogenous pentapeptide (Gln-Tyr-Asn-Ala-Asp; QYNAD) that is present at elevated levels in human CSF from patients with demyelinating diseases has been reported to block voltage-gated sodium channels at low (10 micro M) concentrations. Objective : Because of the potential importance of sodium channel blocking activity in demyelinating disorders, this study attempted to determine the sensitivity to QYNAD of different sodium channel subtypes, including Na(v)1.6, the major sodium channel at nodes of Ranvier, and Na(v)1.2, which is expressed in axons with abnormal myelin. METHODS: Sodium channel function was assayed using patch-clamp recordings, both in heterologous expression systems and in intact neurons. RESULTS: QYNAD synthesized in 10 different batches by four different facilities failed to block sodium currents, even at concentrations as high as 500 micro M (50-fold higher than the blocking concentration originally reported). QYNAD had no effect on the currents produced by recombinant Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.7 sodium channels or on the sodium currents that are produced by native channels in adult hippocampal or dorsal root ganglion neurons. QYNAD did not interfere with conduction in the optic nerve, a myelinated fiber tract that is often affected in MS. CONCLUSIONS: These experiments do not show any sodium channel blocking effect of QYNAD. The conclusion that QYNAD contributes to the pathophysiology of inflammatory neurologic disorders by blocking voltage-gated sodium channels should therefore be viewed with caution.

Animals↗
PubMed2003

Effect of ageing on the proliferation and apoptosis of testicular germ cells in the Syrian hamster Mesocricetus auratus.

The cellular mechanisms implicated in the atrophy of seminiferous epithelium in ageing are currently under debate, although recent reports suggest that apoptosis may be the primary mechanism implicated in aged germ cell loss. Other investigators have suggested that changes in spermatogonial proliferation are also involved. In the present work, the changes in proliferation and apoptosis in the seminiferous epithelium of aged (24 months) Syrian hamsters were examined in concert and compared with those in young (6 months) animals. Proliferation of germ cells was studied by bromodeoxyuridine labelling and apoptosis was assessed by transmission electron microscopy and in situ TUNEL labelling. Aged animals showed a significant decrease in the numbers of total and proliferating spermatogonia plus preleptotene spermatocytes per unit volume and per testis and in the proliferative index (24.8 +/- 1.6%) compared with young animals (30.8 +/- 1.2%) (P < 0.05). The number of apoptotic spermatogonia plus spermatocytes per unit volume and the apoptotic index were significantly higher in aged animals (1.51 +/- 0.23% v. 0.77 +/- 0.04%; P < 0.05). Apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. In aged hamsters, tubular degeneration could be classified into several categories, showing an increase of apoptotic cells in tubular cross-sections characterized by maturation arrest in comparison with all other types. Spermatogonial proliferation was also diminished as seen in tubular cross-sections showing hypospermatogenesis, sloughing off of germ cells and maturation arrest. The results obtained in the present study suggest that the decrease in the proliferation of spermatogonia and the increase in apoptosis constitute two consecutive mechanisms correlated with the ageing of the seminiferous epithelium.

Aging↗
PubMed2002

Improved Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): assessment of macerating enzymes and sonication.

Agrobacterium -mediated transformation of shoot apices of sunflower (Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication. The frequency of explants with regenerated shoots expressing GUS (beta-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect. When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold. The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment. Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation. Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene. Regenerated plants were fertile and seeds could be harvested.

Cellulase↗
PubMed2002

Molecular mapping of the Rf1 gene restoring pollen fertility in PET1-based F1 hybrids in sunflower (Helianthus annuus L.).

Up to now a single cytoplasmic male sterility (CMS) source, PET1, is used worldwide for hybrid breeding in sunflower. Introgression of the restorer gene Rf1, responsible for fertility restoration, into new breeding material requires tightly linked markers to perform an efficient marker-assisted selection. A survey of 520 decamer primers by bulked segregant analyses identified five RAPD markers linked to the restorer gene Rf1. In a F(2) population of 183 individuals one of the RAPD markers, OPK13_454, mapped 0.8 cM from Rf1, followed by OPY10_740 with 2 cM. Bulked segregant analyses using 48 AFLP primer combinations identified 17 polymorphisms, which could be mapped in the same linkage group as Rf1. E33M61_136, and E41M48_113 were mapped 0.3 cM and 1.6 cM from the gene, respectively. Conversion of E41M48_113 into a sequence-specific marker resulted in a monomorphic pattern. However, two of the RAPD markers, OPK13_454 and OPY10_740, were successfully converted into SCAR markers, HRG01 and HRG02, which are now available for marker-assisted selection. To investigate the utility of these SCAR markers in other cross-combinations they were tested in a set of 20 lines. Comparison of the patterns of 11 restorer and nine maintainer lines of PET1 demonstrated that the markers OPK13_454/HRG01 and HRG02 were absent in all maintainer lines but present in all restorer lines, apart from the high oleic line RHA348 and the dwarf line Gio55. In addition, restorer lines developed from the interspecific hybrids Helianthus annuus x Helianthus mollis and H. annuus x Helianthus rigidus gave the same characteristic amplification products.

Chromosome Mapping↗
PubMed2001

Slow photo-cross-linking kinetics of benzophenone-labeled voltage sensors of ion channels.

Voltage-gated ion channels have voltage sensors that move in response to changes in membrane potential. This movement regulates the gates that control access of ions to the permeation pathway. To study the coupling between voltage sensors and gates, we immobilize the voltage sensors, using a bifunctional photo-cross-linking reagent that can be attached to an introduced cysteine, and observe the consequences for gate movement [Horn, R., Ding, S., and Gruber, H. J. (2000) J. Gen. Physiol. 116, 461-475]. UV irradiation of the benzophenone adduct attached to the cysteine residue immobilizes the voltage sensors, S4 segments, of both Na(+) and Shaker K(+) channels. Here we examine the kinetics of S4 immobilization after a brief UV flash. Immobilization has an exponential time course with time constants of >200 ms for Shaker and 17 ms for Na(+) channels, whereas the triplet excited state lifetime of the benzophenone adduct is <1 ms. This result suggests that H-atom abstraction by benzophenone is rapid and that the rate-limiting step in immobilization is the recombination of alkyl and ketyl free radicals generated by H-abstraction. H-Abstraction is also 2.7-fold more efficient at a hyperpolarized voltage than at a depolarized membrane potential in Shaker S4 segments. S4 immobilization after a UV flash can be prevented by depolarization of Shaker channels, suggesting that movement in the activation pathway is capable of separating the ketyl and alkyl free radicals. Exploiting the unique charge movement and gating properties of the L382V mutant of Shaker, we show that free radical separation follows S4 movement itself and is relatively independent of the movement of activation gates.

Benzophenones↗
PubMed2001

Randomized, double-blind, multicenter trial comparing clinafloxacin with imipenem as empirical monotherapy for febrile granulocytopenic patients.

In a double-blind, multicenter trial, 541 febrile granulocytopenic patients were randomized to receive either intravenous (iv) clinafloxacin (200 mg every 12 h) or i.v. imipenem (500 mg every 6 h) as empirical monotherapy. More baseline pathogens were susceptible to clinafloxacin (259 [99%] of 262 organisms) than to imipenem (253 [95%] of 265; P=.03). Initial favorable clinical response rates for clinafloxacin (88 [32%] of 272 patients) and imipenem (89 [33%] of 269) were similar. After addition of other antimicrobial agents, overall response rates were 259 (95%) of 272 for clinafloxacin and 251 (93%) of 269 for imipenem. During the study, only 13 clinafloxacin (5%) and 18 imipenem (7%) recipients died. Both drugs were generally well tolerated. Drug-related skin rash occurred more often with clinafloxacin (11% vs. 6%; P=.07), whereas nausea (2% vs. 5%; P=.16), Clostridium-difficile-associated diarrhea (3% vs. 8%; P=.02), and seizures (0% vs. 2%; P=.06) occurred more often with imipenem. These results suggest that clinafloxacin and imipenem have similar efficacy as empirical monotherapy in febrile granulocytopenic patients.

Adolescent↗
PubMed2001

Effects of insulin and glucocorticoids on the leptin system are mediated through free leptin.

OBJECTIVE: Insulin and glucocorticoids are known to increase total leptin levels. However, the effects of insulin and glucocorticoids on the components of the leptin system - free leptin (FL), bound leptin (BL) and soluble leptin receptor (SR) - have not been elucidated. The aim of this study was to determine if there is a differential effect of insulin and glucocorticoids on the leptin system. MATERIAL AND METHODS: In the first of two studies (study 1), we measured free leptin (FL), bound leptin (BL), a soluble leptin receptor (SR) and insulin, by specific RIA methods, in six healthy subjects on a control day, and subsequently during a hyperinsulinaemic euglycaemic clamp study. In the second study (study 2) we measured the same parameters in six healthy subjects, before (day 1) and during administration of dexamethasone over 3 consecutive days. RESULTS: In study 1, on the control day FL levels rose over the 16 h monitoring period (P = 0.057) and SR levels declined (P < 0.001), but there was no change of BL levels. Even after accounting for diurnal variation, FL levels increased even more substantially over 12 h of insulin infusion than observed on the control day (P < 0.001). In study 2, mean FL concentration doubled from day 1 to day 2 (P = 0.01) and remained elevated subsequently. In contrast to FL, BL and SR levels remained unchanged during the study. Fasting insulin levels (pmol/l) increased from day 1 to day 2, but this rise only approached significance on day 4 (P = 0.05). CONCLUSION: We conclude that insulin and dexamethasone increase free leptin levels, but do not change the concentrations of bound leptin and soluble leptin receptor. Furthermore, the dexamethasone-induced rise in leptin levels is (at least partially) independent of the effects of glucocorticoid-induced hyperinsulinaemia.

Adult↗
PubMed2001

Hepatitis C and the leptin system: bound leptin levels are elevated in patients with hepatitis C and decrease during antiviral therapy.

BACKGROUND: Serum leptin levels are elevated in alcoholic liver cirrhosis and thus might be involved in the anorexia and hypermetabolism often seen in those patients. We hypothesized that the leptin system is modulated in patients with hepatitis C and might be affected by antiviral therapy. The aim of this study was to investigate the different leptin components in serum of patients with hepatitis C before, during and after interferon alpha and ribavirin therapy and in controls. METHODS: 25 patients (11 female, 14 male) with chronic hepatitis C were compared with body mass index, gender and age-matched controls (n = 25). Patients were treated with interferon alpha alone (3 MU tiw) or in combination with ribavirin for 6-12 months. Free leptin and bound leptin levels were measured using specific radioimmunoassays before interferon therapy, at 12 weeks of therapy and after 3 months of follow-up. RESULTS: Free leptin levels were higher in female than in male subjects, both for patients (P < 0.01) and controls (P < 0.05). Bound leptin levels were elevated in both female (P < 0.05) and male (P < 0.001) patients compared to controls. No alteration of free leptin levels was found during therapy, whereas bound leptin levels decreased after 3 months of therapy (P < 0.005) and re-increased to the baseline levels 3 months after therapy was stopped. Responder but not non-responder had decreased bound leptin levels (P < 0.01) comparing pre- and posttreatment levels. However, no significant correlations were determined between any of the leptin components to virus load, ALT, TNF alpha receptor levels (sTNFR-75, sTNFR-55) and histopathology at any time point. CONCLUSION: Since no correlation was found between the different leptin components and any of the inflammatory markers, the decrease in bound leptin levels during antiviral therapy suggests either a direct interferon-dependent effect on the leptin system or an alteration of other leptin secretagogues.

Adult↗
PubMed2001

Susceptibility of the Bacteroides fragilis group to newer quinolones and other standard anti-anaerobic agents.

The susceptibilities of 200 clinical isolates of the Bacteroides fragilis group to four quinolones (moxifloxacin, clinafloxacin, trovafloxacin and ciprofloxacin) were determined, as well as to cefoxitin, clindamycin, metronidazole, imipenem and ticarcillin-clavulanic acid. The results for the latter five agents were compared with those of a study on 200 isolates done 6 years previously. Clinafloxacin and trovafloxacin were the most active agents tested with MIC90s lower than all other antimicrobials except imipenem. Susceptibility rates for imipenem, ticarcillin- clavulanic and metronidazole continue to be high, although resistant strains are emerging. For ticarcillin-clavulanic acid and metronidazole, MIC90s increased four- to eight-fold for the B. fragilis species between the two study periods.

Anti-Infective Agents↗
PubMed2000

Free and protein bound leptin are distinct and independently controlled factors in energy regulation.

AIMS/HYPOTHESIS: Leptin exerts important regulating effects on energy homeostasis and could have a central role in our understanding of obesity, diabetes mellitus and the metabolic syndrome. Leptin circulates in a free and protein bound form. The aim of the present study was to test whether both fractions of the leptin system can be selectively regulated and thus serve independent physiological roles. METHODS: Using specific radioimmunoassays we measured both leptin components in relation to BMI in healthy subjects before and after weight reduction and in hyperthyroid patients during correction of thyrotoxicosis. In the latter group body composition and resting energy expenditure was monitored. In addition, we measured serum and cerebrospinal fluid concentrations of free and bound leptin in patients with neurological disorders. RESULTS: Under all conditions free leptin concentrations reflected body fat mass. Bound leptin concentrations decreased during weight reduction but also after treatment of hyperthyroidism despite an increase in fat mass. Direct measurement of resting energy expenditure and bound leptin in hyperthyroid patients and under thyrostatic treatment showed a significant positive correlation of both variables. In contrast to free leptin whose transport into the cerebrospinal fluid appears to be saturated at low physiological concentrations of serum free leptin, bound leptin concentrations in the cerebrospinal fluid increased in parallel to serum concentrations over the whole physiologically relevant range. CONCLUSION/INTERPRETATION: Our data indicate a distinct role of free and bound leptin in the feedback regulating energy intake and expenditure and could have important implications for our understanding of the physiology and pathophysiology of leptin-dependent signalling.

Adolescent↗
PubMed2000

Determinants of within-subject variation of fasting serum leptin concentrations in healthy subjects.

The hormone leptin is considered to contribute to body weight regulation through modulation of feeding behavior and energy expenditure. The aim of the present study was 1) to assess the day-to-day within-subject variation (biovariability) of serum leptin concentrations in healthy subjects and 2) to investigate whether this variation is associated with food intake, exercise, anthropometric measurements or various metabolic covariates (insulin, C-peptide and glucagon, glucose, lactate, 3-hydroxybutyrate (3-OHB), triglycerides, non-esterified-fatty acids and glycerol). Serum leptin levels were taken daily on 12 consecutive days after an overnight fast in 12 healthy subjects with a mean (SD) age of 22.7 (1.5) yr. and a BMI of 22.8 (1.6) kg/m2. Food intake, exercise, anthropometric measurements and various metabolic covariates were also determined during this period. The overall mean of serum leptin concentration was 33.3 pmol/L with a within-subject SD range of 27-41 pmol/L and a between-subject SD range of 18-61 pmol/L. The within-subject variance of serum leptin as a proportion of total variance was 9.5%. Within-subject variation of serum leptin concentrations is small in relation to between-subject variation in healthy, normal weight subjects. This has implications for the power of interventional or prospective studies. In men, 6.7% of the variation in serum leptin concentration was associated with body weight measured on the same day (p= 0.037). In women, however, 66% of the variation was negatively associated with 3-OHB measured on both the same and the previous day (p=0.0003 and 0.002), and positively associated with triglyceride concentration measured on the previous day (p=0.0017) and insulin measured on the same day (p=0.0002). Within-subject associations in women could be due to phasic changes in unmeasured variables, possibly related to the menstrual cycle or might suggest that energy balance may exert a delayed influence on serum leptin levels, with plasma 3-OHB and triglycerides acting as markers for the state of the fat stores that regulate leptin secretion. The differences between the genders remain unexplained, however.

3-Hydroxybutyric Acid↗
PubMed2000

Role of domain 4 in sodium channel slow inactivation.

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.

Cell Line, Transformed↗
PubMed2000

Immobilizing the moving parts of voltage-gated ion channels.

Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

Base Sequence↗