Hyperpolarization-activated cation channels: a multi-gene family.
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Amino Acid Sequence↗
Biomedical subjects
A Ludwig
Publications and source records attributed to A Ludwig.
Structure and function of cyclic nucleotide-gated channels.
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Animals↗
NH4+ conductance in Xenopus laevis oocytes.II. Effect Of hypoosmolality.
Superfusing Xenopus laevis oocytes with NH4Cl (10 mmol/l, pH 7.5) resulted in an inward current at a clamp potential of -70 mV. In paired experiments (n=22), the NH4Cl-induced peak current was -293+/-94 nA, under control conditions (osmolality: 240 mosmol/kg), and rose to -523+/-196 nA when osmolality was reduced to 144 mosmol/kg. In parallel with the rise in NH4Cl-induced inward current, membrane conductance at -70 mV doubled and the zero-current potential changed from +3.3+/-9.4 mV to -22.0+/-8.0 mV (n=22) in the presence of NH4Cl during exposure to a hypoosmolar solution. In the absence of NH4Cl, oocytes responded to hypoosmolality with a shift in zero-current potential to more negative values and an increased conductance which became partially sensitive to isosorbiddinitrate (ISDN), suggesting the activation of a volume-sensitive K+ channel. Membrane conductance in the presence of NH4Cl was decreased by ISDN to similar extents under isoosmolal and hypoosmolal conditions, indicating that NH4+ enters the oocytes through a volume-sensitive conductance separate from the ISDN-sensitive K+ channel.
Ammonium Chloride↗
Influence of the physico-chemical properties of ophthalmic viscolysers on the weight of drops dispensed from a flexible dropper bottle.
The present study investigated the influence of the rheological behaviour, the viscoelasticity and surface tension of various viscolysers on the weight of drops dispensed from a commercially available flexible dropper bottle. Furthermore, the effect of the concentration and molecular weight of some viscolysers and the angle at which the dropper bottle is held, were studied. Up to a value of 25 mPa x s, the viscosity and the rheological behaviour of the solution had no significant influence on the drop weight under conditions simulating patient manipulation. The lower the dynamic surface tension of the solution, however, the lower the weight of drops delivered. The dispensing angle (90 or 45 degrees) of the dropper bottle, on the other hand, had an impact on the weight of drops dispensed.
Chemical Phenomena↗
Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin.
Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartaterich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca2+, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C-terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.
Adenylate Cyclase Toxin↗
Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12.
Escherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N-terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA-controlled transcription started predominantly 72 bp upstream from clyA, as shown by primer extension. The corresponding putative promoter contains an unusual -10 sequence (TATGAAT) that is separated from a conventional -35 sequence by a GC-rich spacer. Site-directed deletion of the G in the -10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the G + C content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation-selective transmembrane pores that have a diameter of about 2.5-3 nm.
Bacterial Proteins↗
Bacteriophage lambda surface display of a bacterial biotin acceptor domain reveals the minimal peptide size required for biotinylation.
Phage display is a powerful technique for identifying specific ligands to a given target. In this work random peptides derived from the biotin accepting domain of the Klebsiella pneumoniae oxaloacetate decarboxylase were displayed on bacteriophage lambda heads to determine the minimal sequence length that is necessary to effect biotinylation in vivo. Phages with a functional biotinylation domain were identified after affinity purification with immobilised avidin. All biotinylated phages isolated this way were found to have a sequence of 66 amino acids from the parental protein in common. This minimal biotinylation domain is fully functional as a biotin acceptor and more resistant to proteolytic attack compared to domains of larger size derived from the same protein. The data present the first example of a posttranslational protein modification analysed in a phage display system. Moreover, a biotin domain of reduced size and improved stability was identified, that should be superior to the larger parental protein as a tag to generate biotinylated fusion proteins.
Amino Acid Sequence↗
A family of hyperpolarization-activated mammalian cation channels.
Pacemaker activity of spontaneously active neurons and heart cells is controlled by a depolarizing, mixed Na+/K+ current, named Ih (or I(f) in the sinoatrial node of the heart). This current is activated on hyperpolarization of the plasma membrane. In addition to depolarizing pacemaker cells, Ih is involved in determining the resting membrane potential of neurons and provides a mechanism to limit hyperpolarizing currents in these cells. Hormones and neurotransmitters that induce a rise in cyclic AMP levels increase Ih by a mechanism that is independent of protein phosphorylation, and which involves direct binding of the cyclic nucleotide to the channel that mediates Ih. Here we report the molecular cloning and functional expression of the gene encoding a hyperpolarization-activated cation channel (HAC1) that is present in brain and heart. This channel exhibits the general properties of Ih channels. We have also identified full-length sequences of two related channels, HAC2 and HAC3, that are specifically expressed in the brain, indicating the existence of a family of hyperpolarization-activated cation channels.
Amino Acid Sequence↗
Cyclic nucleotide-gated channels--mediators of NO:cGMP-regulated processes.
Cyclic nucleotide-gated (CNG) channels represent one of the three known cellular receptor classes for cGMP. Activation of CNG channels by binding of cyclic nucleotides to a site in the C-terminus results in opening of the channel pore, entry of Ca2+ into the cell and subsequent induction of Ca2+-dependent processes. In this review we will summarize new data on the complex molecular structure and the activation mechanism of CNG channels. In addition, we will discuss the role of CNG channels as mediators of NO:cGMP-dependent cellular processes.
Animals↗
New perspectives in head and neck sonography by signal-enhanced color Doppler sonography.
Ultrasound contrast agent is used in color Doppler flow imaging for signal enhancement in perfused vessels. We present our experience with its use in the head and neck area and the results obtained. Up until now, 30 patients with carcinomas of the oral cavity or the maxillary sinus, tumors of the mandible and metastatic or inflammatory lymph nodes have been examined. Conventional B-scan sonography was supplemented by non-contrast color Doppler flow imaging. Depending on the indication, 2.5 g Levovist in bolus or 4 g fractionated was injected intravenously in a concentration of 300 or 400 mg/ml. The administration of the contrast agent was tolerated by all patients without any side effects and in all cases led to a remarkable enhancement of the Doppler signals. About 15 to 30 s after injection, an enhancement in perfused vessels was detected. The fractioned injection of 4 g led to a lengthening of signal enhancement up to more than 10 min and to the possibility of examining more parts with a single application of contrast agent. In 64% of the lymph nodes, vessels could only be identified with the administration of Levovist. The general vessel topography could be better evaluated in 83% of the cases. The lymph-node-supplying vessels were identified in 8 of 11 patients after the injection of contrast media. Additional information, which was gained in 17 patients, increased the certainty of diagnosis or of the therapeutic concept. In three cases, the examinations with the signal enhancer resulted in a change in therapy. By applying signal enhancer in color Doppler sonography the fields of examination in the head and neck areas can be expanded.
Blood Flow Velocity↗
The influence of penetration enhancers on the volume instilled of eye drops.
The influence of the physicochemical properties of various penetration enhancers on the weight of drops dispensed from flexible plastic dropper bottles was examined. Two dropper tips with a different design were compared and the dropper bottle was held in the upright position (90 degrees angle) or at a 45 degrees angle. These two angles were chosen to simulate the manipulation of an eye dropper bottle by the patient. The surface tension of the penetration enhancer solutions was determined using the dynamic drop volume method. The dynamic surface tension values ranged from 65 to 30 mN/m. The lower the surface tension of the solution, the lower the weight of the drop delivered. Both the design of the dropper tip and the manipulation technique of the dropper bottle had a moderate to important influence on the drop weight. The relationship between the surface tension of the penetration enhancer solution instilled and the comfort of the patient was evaluated by an acceptability test based on answering a questionnaire. Penetration enhancers can be used to reduce the drop size of conventional ophthalmic solutions on condition that they do not elicit local irritation.
Chemistry, Pharmaceutical↗
Submicron oil-in-water emulsion formulations for mefloquine and halofantrine: effect of electric-charge inducers on antimalarial activity in mice.
Stearylamine, oleic acid, phosphatidylserine and dicetylphosphate have been studied to determine their capacity to induce electric charge on non-ionic submicron emulsions containing halofantrine and mefloquine. The in-vivo antimalarial activity of drug-loaded emulsions, evaluated in mice, was affected by the nature of the additives used. In particular, the electric-charge inducers markedly affected the pharmacological activity of mefloquine, but not of halofantrine. After subcutaneous administration ED50 values (the doses affording 50% protection) were 3 and 15 mg kg(-1), respectively, for halofantrine and mefloquine emulsions without charge inducers. The mefloquine-loaded emulsions with charge inducers were active at 10 mg kg(-1) for dicetylphosphate, 17 mg kg(-1) for phosphatidylserine, 23 mg kg(-1) for oleic acid and 27 mg kg(-1) for stearylamine, again after subcutaneous administration. This work has enabled the formulation of stable emulsions, incorporating drugs with high antimalarial activity, which are proposed for parenteral delivery of these fairly soluble drugs.
Animals↗
The structure of the murine calcium channel gamma-subunit gene and protein.
The gamma-subunit of voltage-gated calcium channels is a membrane protein that is associated with the skeletal muscle type of voltage gated calcium channels. Using a subunit-specific polyclonal antibody, the gamma-protein was detected in mouse skeletal muscle but not in brain, where at least five additional types of voltage-gated calcium channels are expressed. Murine genomic clones containing the full coding sequence of the gamma-subunit were isolated, the exons were mapped and sequenced. The murine gamma-subunit is encoded by a single copy gene containing 4 translated exons which are distributed over approximately 14 kilobases of DNA. The intron placement within the mouse gene correlates with the previously revealed organization of the human gamma-subunit gene, although the primary structures of the gamma-subunits are only moderately conserved between the murine, human, rat and rabbit proteins (75% identity).
Alternative Splicing↗
[WinDiet. Support decision system for the assessment of nutritional status and diet prescription].
A "Sistema de Apoio à Decisão para Avaliação do Estado Nutricional e Prescrição de Dietas" called "WinDiet" was developed. The system was developed on a computer compatible to IBM-PC standard. For system processing procedures through the computer, that is, interpretation, compilation, table generation en others, it was selected the Delphi version 1.0 software, from Borland International, for its using facilities, data basis accomplishment, communication environment and mainly an ample availability of mathematic, impression and programming resources besides allowing for all the programming operations to be accomplished on Windows environment. Knowing the procedures for nutritional evaluation and diet prescription was possible by consulting to text books, periodicals and the area experts. This system allows for individual nutritional assessment applying the anthropometric methods such as weight, height, skinfold thickness measurings, the lab methods such as albumin, transferrin, height/creatinin index and others, the dietary methods by food consumption recall, and the clinical methods that give emphasis to pathological antecedents and indicative sings of nutritional status. The software also turns possible to prescribe and calculate diets from recommendation besides having an actual data basis which may be changed or modified. Yet it maintains the individual description from all evaluation and menus, emitting its complete reports. It was verified that it reduce significantly the working time and increase the precision of estimate and nutritional diagnosis. Thus, the WinDiet may be used in nutritionist's offices, ambulatories, hospitals as well as on nutritional researches and education.
Diet Therapy↗
The CXC-chemokine neutrophil-activating peptide-2 induces two distinct optima of neutrophil chemotaxis by differential interaction with interleukin-8 receptors CXCR-1 and CXCR-2.
The CXC-chemokines interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and melanoma growth-stimulatory activity (MGSA) are chemoattractants with high selectivity for neutrophils. Although IL-8 has been shown to act as an extremely potent mediator, reports on NAP-2 and MGSA are still contradictory. Here we show for the first time that NAP-2 and MGSA induce two distinct optima of neutrophil chemotaxis. A first optimum is elicited within a concentration range as low as it is characteristic for IL-8. However, a second optimum appears at more than 200-fold higher stimulus concentrations, at which IL-8 is inactive. Investigating the involvement of the two chemokine receptors CXCR-1 and CXCR-2 in NAP-2-mediated chemotaxis, we observe that the cells become desensitized to the first optimum of the chemokine after selective downregulation of CXCR-2, while both optima disappear upon simultaneous downregulation of both receptors. Blocking monoclonal antibodies (MoAbs) specific for CXCR-2 or CXCR-1 either suppress the first optimum of NAP-2-induced chemotaxis or drastically reduce the second one, respectively. These results provide evidence that both receptors are involved in NAP-2-induced neutrophil chemotaxis, with CXCR-2 rendering the cells responsive to low dosages of the chemokine, and with CXCR-1 extending their responsiveness to NAP-2 dosages higher by several orders of magnitude.
Antibodies, Monoclonal↗
Regional expression and cellular localization of the alpha1 and beta subunit of high voltage-activated calcium channels in rat brain.
The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different alpha1 and four different beta subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The beta subunit-specific antibodies identified the presence of beta1a in skeletal muscle; beta2 in heart; and beta2, beta3, and beta4 in brain. The beta3 protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The beta4 protein was also widely expressed, most prominently in the cerebellum. beta2 protein was expressed at only low levels. In situ hybridization with beta subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the alpha1A, alpha1B, alpha1C, and alpha1D subunits showed a broad distribution of alpha1A and alpha1B transcripts, whereas the expression level of alpha1C and alpha1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that beta4 may associate predominantly, but probably not exclusively, with the alpha1A subunit, and beta3 with the alpha1B subunit. In certain brain areas such as the habenula, the beta3 subunit may associate with other alpha1 subunits too. Furthermore, the beta2 subunit may form complexes with different alpha1 subunits in brain and cardiac muscle. These results demonstrate that a given beta subunit may associate with different alpha1 subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.
Animals↗
Regulation of neutrophil activation by proteolytic processing of platelet-derived alpha-chemokines.
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Animals↗
[Lung edema and erosive gastroduodenitis as a sequela of inappropriate use of an adrenaline dose aerosol after wasp sting].
Epinephrine (adrenaline) is an important drug in the treatment of severe anaphylactic reactions. Along with other drugs such as H1-antihistamines and glucocorticosteroids, it is found in every first aid kit for at-risk individuals, such as those who are allergic to insect stings. Subcutaneous or intramuscular injections if carried out by an untrained individual or the patient himself might give rise to potential problems. Therefore, it is common to prescribe epinephrine pressure aerosol as a safer alternative. If epinephrine aerosol is overused, it can cause serious problems. A patient developed by self-medication following a wasp sting lung edema as well as an erosive gastroduodenitis. She consumed two aerosol vials each of which contained about 73 mg of adrenaline. In order to avoid such incidents it is crucial that every doctor provides his or her patient with sufficient oral and written information regarding the correct use fo epinephrine inhalers.
Administration, Inhalation↗