Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

The contribution of dynamics to macaque body and face patch responses.

Previous functional imaging studies demonstrated body-selective patches in the primate visual temporal cortex, comparing activations to static bodies and static images of other categories. However, the use of static instead of dynamic displays of moving bodies may have underestimated the extent of the body patch network. Indeed, body dynamics provide information about action and emotion and may be processed in patches not activated by static images. Thus, to map with fMRI the full extent of the macaque body patch system in the visual temporal cortex, we employed dynamic displays of natural-acting monkey bodies, dynamic monkey faces, objects, and scrambled versions of these videos, all presented during fixation. We found nine body patches in the visual temporal cortex, starting posteriorly in the superior temporal sulcus (STS) and ending anteriorly in the temporal pole. Unlike for static images, body patches were present consistently in both the lower and upper banks of the STS. Overall, body patches showed a higher activation by dynamic displays than by matched static images, which, for identical stimulus displays, was less the case for the neighboring face patches. These data provide the groundwork for future single-unit recording studies to reveal the spatiotemporal features the neurons of these body patches encode. These fMRI findings suggest that dynamics have a stronger contribution to population responses in body than face patches.

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes.

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

Molecular mechanisms of Slo2 K+ channel closure.

KEY POINTS: Intracellular Na+ -activated Slo2 potassium channels are in a closed state under normal physiological conditions, although their mechanisms of ion permeation gating are not well understood. A cryo-electron microscopy structure of Slo2.2 suggests that the ion permeation pathway of these channels is closed by a single constriction of the inner pore formed by the criss-crossing of the cytoplasmic ends of the S6 segments (the S6 bundle crossing) at a conserved Met residue. Functional characterization of mutant Slo2 channels suggests that hydrophobic interactions between Leu residues in the upper region of the S6 segments contribute to stabilizing the inner pore in a non-conducting state. Mutation of the conserved Met residues in the S6 segments to the negatively-charged Glu did not induce constitutive opening of Slo2.1 or Slo2.2, suggesting that ion permeation of Slo2 channels is not predominantly gated by the S6 bundle crossing. ABSTRACT: Large conductance K+ -selective Slo2 channels are in a closed state unless activated by elevated [Na+ ]i . Our previous studies suggested that the pore helix/selectivity filter serves as the activation gate in Slo2 channels. In the present study, we evaluated two other potential mechanisms for stabilization of Slo2 channels in a closed state: (1) dewetting and collapse of the inner pore (hydrophobic gating) and (2) constriction of the inner pore by tight criss-crossing of the cytoplasmic ends of the S6 α-helical segments. Slo2 channels contain two conserved Leu residues in each of the four S6 segments that line the inner pore region nearest the bottom of the selectivity filter. To evaluate the potential role of these residues in hydrophobic gating, Leu267 and Leu270 in human Slo2.1 were each replaced by 15 different residues. The relative conductance of mutant channels was highly dependent on hydrophilicity and volume of the amino acid substituted for Leu267 and was maximal with L267H. Consistent with their combined role in hydrophobic gating, replacement of both Leu residues with the isosteric but polar residue Asn (L267N/L270N) stabilized channels in a fully open state. In a recent cryo-electron microscopy structure of chicken Slo2.2, the ion permeation pathway of the channel is closed by a constriction of the inner pore formed by criss-crossing of the S6 segments at a conserved Met. Inconsistent with the S6 segment crossing forming the activation gate, replacement of the homologous Met residues in human Slo2.1 or Slo2.2 with the negatively-charged Glu did not induce constitutive channel opening.

Extraction of social information from gait in schizophrenia.

BACKGROUND: The human face and body are rich sources of socio-emotional cues. Accurate recognition of these cues is central to adaptive social functioning. Past studies indicate that individuals with schizophrenia (SZ) show deficits in the perception of emotion from facial cues but the contribution of bodily cues to social perception in schizophrenia is undetermined. The present study examined the detection of social cues from human gait patterns presented by computer-generated volumetric walking figures. METHOD: A total of 22 SZ and 20 age-matched healthy control participants (CO) viewed 1 s movies of a 'digital' walker's gait and subsequently made a forced-choice decision on the emotional state (angry or happy) or the gender of the walker presented at three intensity levels. Overall sensitivity to the social cues and bias were computed. For SZ, symptom severity was assessed. RESULTS: SZ were less sensitive than CO on both emotion and gender discrimination, regardless of intensity. While impaired overall, greater signal intensity did improve performance of SZ. Neither group differed in their response bias in either condition. The discrimination sensitivity of SZ was unrelated to their social functioning or symptoms but a bias toward perceiving gait as happy was associated with better social functioning. CONCLUSIONS: These results suggest that SZ are impaired in extracting social information from gait but SZ benefited from increased signal intensity of social cues. Inaccurate perception of social cues in others may hinder adequate preparation for social interactions.

The influence of focal cerebellar lesions on the control and adaptation of gait.

Cerebellar ataxic gait is influenced greatly by balance disorders, most likely caused by lesions of the medial zone of the cerebellum. The contributions of the intermediate and lateral zone to the control of limb dynamics for gait and the adaptation of locomotor patterns are less well understood. In this study, we analysed locomotion and goal-directed leg movements in 12 patients with chronic focal lesions after resection of benign cerebellar tumours. The extent of the cortical lesion and possible involvement of the cerebellar nuclei was determined by 3D-MR imaging. The subjects (age range 13-39 years, mean 20.3; seven female; ICARS score: mean 5.7, SD 6.3) performed three tasks: goal-directed leg placement, walking and walking with additional weights on the shanks. Based on the performance on the first two tasks, patients were categorized as impaired or unimpaired for leg placement and for dynamic balance control in gait. The subgroup with impaired leg placement but not the subgroup with impaired balance showed abnormalities in the adaptation of locomotion to additional loads. A detailed analysis revealed specific abnormalities in the temporal aspects of intra-limb coordination for leg placement and adaptive locomotion. These findings indicate that common neural substrates could be responsible for intra-limb coordination in both tasks. Lesion-based MRI subtraction analysis revealed that the interposed and the adjacent dentate nuclei were more frequently affected in patients with impaired compared to unimpaired leg placement, whereas the fastigial nuclei (and to a lesser degree the interposed nuclei) were more frequently affected in patients with impaired compared with unimpaired dynamic balance control. The intermediate zone appears thus to be of particular importance for multi-joint limb control in both goal-directed leg movements and in locomotion. For locomotion, our results indicate an influence of the intermediate zone on dynamic balance control as well as on the adaptation to changes in limb dynamics.

Qualitative in vitro NMR analysis of creatine ethyl ester pronutrient in human plasma.

There are a number of forms of creatine available that attempt to improve the solubility and permeability, with the anticipation this will result in an improved pharmacokinetic profile and ultimately an enhanced ergogenic response. Previous research has shown that the different salt forms can improve solubility resulting in slightly altered pharmacokinetic profiles, however specific data exploring the conversion of esterified derivatives to creatine is lacking. The purpose of this study was to examine the assertion that creatine ethyl ester undergoes enzymatic conversion to creatine in human tissues. The IN VITRO response of creatine ethyl ester to incubation in human plasma was examined by H-NMR analysis. Lyophilized human plasma was reconstituted in D2O and phosphate-buffered saline and 1.5 mg of the analyte was added. Following incubation at 37 degrees C for 4 h and subsequent protein precipitation, the supernatant was analyzed by NMR, utilizing the diagnostic chemical shift of the methylene signal to determine the species present in solution, I.E. creatine ethyl ester, creatine, or creatinine. Both creatine and creatinine were run in parallel as control experiments and each assay was run in triplicate. As expected both creatine and creatinine remained unchanged. However, conversion of creatine ethyl ester to creatine by the esterases in human plasma was not observed to any detectable extent and the only species detected after the incubation period was creatinine. While not a definitive characterization of the IN VIVO behavior, these results strongly warrant a complete IN VIVO pharmacokinetic analysis of creatine ethyl ester since it appears these "pronutrients" may actually provide large exogenous sources of pharmacologically inactive creatinine rather than ergogenic creatine.

Structure-based analysis of CysZ-mediated cellular uptake of sulfate.

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

[West Nile virus--causative agent of a zoonosis with increasing significance?]. / Ubersichtsartikel: West-Nil-Virus - Ursache einer Zoonose mit zunehmender Bedeutung?

The epidemic West Nile Virus (WNV) infections observed in the last years, particularly those in the USA in 1999 and the following years, have led to an increasing interest in this zoonotic infection. Here, the most prominent aspects of WNV biology and epidemiology are presented. Clinical signs observed in men and horses are described, as well as the current state of diagnostics and immunoprophylaxis. Preliminary results of investigations on the prevalence of WNV in Germany show that migrating birds have been in contact with WNV; there is however no indication for the presence of this virus. While WNV is endemic in many parts of the "Old World", thus inducing "natural immunity" in (migrating) birds and vertebrates, a susceptible bird population with no existing immunity against this virus was exposed in the "New World".

Experimental investigation of anion-π interactions--applications and biochemical relevance.

Anion-π interactions, intuitively repulsive forces, turned from controversial to a well-established non-covalent interaction over the past quarter of a century. Within this time frame the question "Anion-π interactions. Do they exist?" could be answered and even more importantly its functional relevance was proven. The present feature article summarizes the experimental findings of anion-π studies in the gas phase, solution and in the solid state and highlights the application of anion-π interactions in anion recognition, sensing and transport as well as in catalysis. Moreover, the biochemical relevance of this weak intermolecular force is comprehensively reviewed.

The effect of surgery and intracerebral injections on motor skill learning in rats: results from a database analysis.

Male Long-Evans rats are often used to investigate neural mechanisms of learning in the motor system. Successful acquisition of a skilled motor task is influenced by various variables such as animal supplier and batch membership. In this retrospective analysis of our laboratory database, we investigate how head and brain surgery as well as intracerebral injections that were performed to address particular scientific questions affect motor learning. Overall, invasive interventions (n=90) slow the acquisition of a skilled-reaching task when compared to naïve animals (n=184; P=0.01). With respect to subgroups, this detrimental effect widely differs between particular procedures: whereas epidural implantations of thin-film electrode arrays and punctual injection through pre-implanted cannulas into primary motor cortex (M1) do not interfere with learning, skill acquisition is slowed after chronic infusion using osmotic minipumps into M1 and skill acquisition is lastingly impaired after bilateral cannula implantation within the dorsal striatum. In line with previous reports, breeder-specific differences could be observed in the analysis of the overall population. In summary, interventions may impair learning-behavior in an unpredictable fashion. Thus, a comparison of behavioral data to a naïve population is recommended to be aware of these drawbacks.

Parametric population representation of retinal location: neuronal interaction dynamics in cat primary visual cortex.

Neuronal interactions are an intricate part of cortical information processing generating internal representations of the environment beyond simple one-to-one mappings of the input parameter space. Here we examined functional ranges of interaction processes within ensembles of neurons in cat primary visual cortex. Seven "elementary" stimuli consisting of small squares of light were presented at contiguous horizontal positions. The population representation of these stimuli was compared to the representation of "composite" stimuli, consisting of two squares of light at varied separations. Based on receptive field measurements and by application of an Optimal Linear Estimator, the representation of retinal location was constructed as a distribution of population activation (DPA) in visual space. The spatiotemporal pattern of the DPA was investigated by obtaining the activity of each neuron for a sequence of time intervals. We found that the DPA of composite stimuli deviates from the superposition of its components because of distance-dependent (1) early excitation and (2) late inhibition. (3) The shape of the DPA of composite stimuli revealed a distance-dependent repulsion effect. We simulated these findings within the framework of dynamic neural fields. In the model, the feedforward response of neurons is modulated by spatial ranges of excitatory and inhibitory interactions within the population. A single set of model parameters was sufficient to describe the main experimental effects. Combined, our results indicate that the spatiotemporal processing of visual stimuli is characterized by a delicate, mutual interplay between stimulus-dependent and interaction-based strategies contributing to the formation of widespread cortical activation patterns.

Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures.

The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L. cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies. Spider plant shoots were exposed to 7.1 [mu]L L-1 (8.5 mg m-3) gaseous [14C]-formaldehyde over 24 h. Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components. Similar results were obtained upon feeding [14C]formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures. Serine and phosphatidylcholine were identified as major metabolic products. Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD. Only the latter enzyme activity required glutathione as an obligatory second cofactor. It had an apparent Km value of 30 [mu]M for formaldehyde and an isoelectric point at pH 5.4. Total cell-free dehydrogenase activity corresponded to 13 [mu]g formaldehyde oxidized h-1 g-1 leaf fresh weight. Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.). The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant. Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.

Diagnosis, treatment and follow-up control in 124 patients with basal cell carcinoma of the maxillofacial region treated from 1992 to 1997.

AIM: The aim of this study was to analyze diagnostic and therapeutic procedures and the outcome of patients treated for the most common malignant tumor of the facial skin, basal cell carcinoma. PATIENTS AND METHODS: The files of patients with basal cell carcinoma (BCC) treated over a period of 6 years were evaluated. Emphasis was placed on the frequency of second interventions, local recurrences and histological subtyping of tumors. RESULTS: One-hundred and twenty-four patients were treated for 216 basal cell carcinomas (solitary: 67%, multiple: 33%). The tumors were predominantly located in the skin covering the middle third of the face. The tumors were 30 mm or less in diameter in 86%. Treatment was exclusively surgical. Histopathological subtyping revealed solid (83%), sclerodermiform (10%), metatypical (4%) and multicentric (3%) tumors. Resection of adjacent bone was mandatory in 12 patients and orbital exenteration in 2. Further local resections were necessary after thorough histological investigation in 71% of patients. Local recurrences occurred in 14 patients, predominantly within the first year after ablative surgery. Relative to the small number of sclerodermiform BCC, this subtype was the most frequent tumor that developed local recurrences. CONCLUSION: Basal cell carcinoma is a malignant tumor, slowly growing and often showing wide extension to macroscopically non-affected sites. Resection of tumors is delicate in the maxillofacial region due to the predilection for sites of origin adjacent to structures of eminent importance for facial appearance. The sclerodermiform subtype is prone to local recurrence and these patients should be followed up carefully.

Sub-processes of motor learning revealed by a robotic manipulandum for rodents.

Rodent models are widely used to investigate neural changes in response to motor learning. Usually, the behavioral readout of motor learning tasks used for this purpose is restricted to a binary measure of performance (i.e. "successful" movement vs. "failure"). Thus, the assignability of research in rodents to concepts gained in human research - implying diverse internal models that constitute motor learning - is still limited. To solve this problem, we recently introduced a three-degree-of-freedom robotic platform designed for rats (the ETH-Pattus) that combines an accurate behavioral readout (in the form of kinematics) with the possibility to invasively assess learning related changes within the brain (e.g. by performing immunohistochemistry or electrophysiology in acute slice preparations). Here, we validate this platform as a tool to study motor learning by establishing two forelimb-reaching paradigms that differ in degree of skill. Both conditions can be precisely differentiated in terms of their temporal pattern and performance levels. Based on behavioral data, we hypothesize the presence of several sub-processes contributing to motor learning. These share close similarities with concepts gained in humans or primates.

Cytokine expression in a rat model of Staphylococcus aureus endophthalmitis.

PURPOSE: To examine the ability of viable Staphylococcus aureus to induce the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, cytokine-induced neutrophil chemoattractant (CINC), and interferon (IFN)-gamma after intravitreal injection. METHODS: Experimental rat eyes were injected with a 25-microl volume of approximately 80 colony-forming units of viable S. aureus; control eyes received sterile saline. Eyes were graded daily for signs of clinical inflammation and were removed 6, 24, 48, and 72 hours after injection. One group was prepared for histologic analysis, and vitreous was removed from the other group for cytokine analysis, using standard enzyme-linked immunosorbent assay procedures. RESULTS: TNF-alpha, IL-1beta, CINC, and IFN-gamma were detected in experimental vitreous samples at increased levels that peaked at 24 hours. TNF-alpha, IL-1beta, and CINC declined at 48 hours, but IFN-gamma remained elevated. At 72 hours, levels returned to baseline. Statistically significant elevations of TNF-alpha, IL-1beta, and CINC were detected in experimental samples at 24, but not at 6 and 48 hours compared with levels in saline control samples (P < 0.03). A statistically significant increase in IFN-gamma was detected at 24 and 48 hours compared with control levels (P < 0.03). In experimental animals, clinical inflammation and inflammatory cells peaked at 24 hours, persisted at 48 hours, and began to decline thereafter. Neutrophils were the predominant inflammatory cell detected at 24 (72.3% of cells) and 48 (60.1%) hours. By 72 hours, the total number of inflammatory cells had decreased by 75.0%, and the cellular infiltrate had changed so that neutrophils equaled monocytes-macrophages. CONCLUSIONS: S. aureus induced the expression of TNF-alpha, IL-1beta, CINC, and IFN-gamma. The time course of these cytokine levels could account for the clinical inflammatory responses and the entry and decline of vitreous cells in this model of bacterial endophthalmitis.

Adhesion molecule expression in a rat model of Staphylococcus aureus endophthalmitis.

PURPOSE: To determine whether Staphylococcus aureus and its components induce expression of E-selectin and intercellular adhesion molecule (ICAM)-1 in rat ocular tissues and on human endothelial cells in culture. METHODS: Experimental and control rat eyes were injected with 80 colony-forming units of viable S. aureus and lipopolysaccharide-free sterile saline (NS), respectively. Eyes were enucleated and immediately frozen. E-selectin and ICAM-1 expression were evaluated on frozen sections by using standard immunohistochemical techniques. Using an enzyme-linked immunoassay, in vitro expression of E-selectin and ICAM-1 was evaluated on macrovascular endothelial cells after stimulation with S. aureus and selected purified components. RESULTS: In S. aureus-injected eyes, E-selectin and ICAM-1 expression peaked at six to 24 hours, decreased slightly at 24 and 48 hours, and further declined by 72 hours. However, in NS-injected eyes, peak levels of E-selectin and ICAM-1 were seen at 6 hours, after which expression declined in the areas in which an increase was previously observed. In in vitro assays, peptidoglycan (0.01 microg/ml) induced a fourfold increase in E-selectin (P < 0.0001) and a twofold increase in ICAM-1 (P < 0.002) expression. Ribitol teichoic acid (RTA) (1 microg/ml) induced a twofold increase in E-selectin (P < 0.0001) and a threefold increase in ICAM-1 (P < 0.0001) expression. CONCLUSIONS: Eyes injected with S. aureus demonstrated a more intense and prolonged expression of both E-selectin and ICAM-1 than did eyes injected with NS. In addition, S. aureus components induced the in vitro expression of these adhesion molecules on macrovascular endothelial cells. The relevance of these findings to microvascular endothelial cells is yet to be determined.

Complement system and host defense against staphylococcal endophthalmitis.

PURPOSE: The authors studied the role of the complement system in host defense against Staphylococcus epidermidis and S. aureus endophthalmitis. METHODS: Guinea pigs in the S. epidermidis model received an intravitreal injection of 7000 viable organisms, and guinea pigs in the S. aureus model received 50 viable organisms. The experimental animals in each model were decomplemented with intraperitoneal (IP) injections of cobra venom factor, whereas the control animals received IP injections of normal saline. Mean log bacterial counts in the vitreous and mean serum complement titers were compared in the experimental and control animals in each model on days 1, 2, 3, and 7. RESULTS: In the S. epidermidis model, mean log bacterial counts in the vitreous were significantly higher in the experimental group than the control group on days 1 and 2 (P < 0.01) and on day 3 (P < 0.05). Mean serum complement titers were significantly lower in the experimental group at all days (P < 0.01). In the S. aureus model, mean log bacterial counts in the vitreous were significantly higher in the experimental group than the control group on day 2 (P < 0.05) and day 3 (P < 0.01). Mean serum complement titers were significantly lower in the experimental group on days 1, 2, and 3 (P < 0.01), but not on day 7. CONCLUSION: These results suggest that decomplemented guinea pigs show impaired host defense to S. epidermidis and S. aureus endophthalmitis and that this defense is restored as complement levels approach normal.

Blocking of interferon synthesis in murine macrophages by pretreatment with interferon.

The influence of pretreatment with interferon (IFN) on subsequent IFN synthesis was investigated in macrophage cultures of DBA/2 and C57BL/6 mice. The doses of IFN alpha/beta for pretreatment ranged from 10,000 U/ml to 100 U/ml and the incubation time was between 18 and 2 h. No blocking effect was observed for chemical induction with poly I:poly C or CMA. However, for viral infection with NDV, blocking was observed. This inhibition of IFN synthesis was dependent on the dose and time of IFN pretreatment and of the titer of the inducing virus. Similarly in mouse fibroblast cultures no blocking activity was observed for induction with poly I:poly C/DEAE-dextran. Again, with NDV as inducer, pretreatment with IFN resulted in inhibition of interferon synthesis. Thus, our data show that blocking occurs only with a viral inducer and suggest that it is caused by an antiviral effect.