NMR structure of C-terminally tagged gramicidin channels

A biotin group was covalently attached to the C terminus of gramicidin A (gA) through a linker arm comprising a glycine residue with either one (gAXB) or two caproyl groups (gAXXB). High-resolution two-dimensional NMR spectroscopy was used to determine the structure of these modified gA analogues and [Lys16]gramicidin A (gA-Lys) in sodium dodecyl-d25 sulphate micelles. Gated gA ion channels based on linking a receptor group to these gA analogues have been used recently as a component in a sensing device. The conformations of the gA backbones and amino acid side chains of lysinated gA and biotinylated gA in detergent micelles were found to be almost identical to that of native gA, i.e. that of an N-terminal to N-terminal (head to head) dimer formed by two right-handed, single-stranded beta 6.3 helices. The biotin tail of the gAXB and gAXXB and the lysine extremity of gA-Lys appeared to lie outside the micelle. Thus it appears that the covalent attachment of functional groups to the C terminus of gA does not disrupt the peptide's helical configuration. Further, single channel measurements of all three gA analogues showed that functioning ion channels were preserved within a membrane environment.     

Two-component blocking kinetics of open NMDA channels by organic cations

NMDA receptor channel responses were recorded from acutely isolated rat hippocampal neurons, using whole-cell patch-clamp techniques. In the continuous presence of aspartate, tetraethylammonium, tetrabutylammonium, 1-amino-3-propyl-adamantane and 9-aminoacridine caused changes in the current through NMDA channels, which were described by two-exponential functions. It was established that depending on the behavior of the amplitude of the fast component for the recovery kinetics, the blocker action can be assigned to one of five types described by the simplest models. The effects of tetraethylammonium, tetrabutylammonium and 1-amino-3-propyl-adamantane were well described by these models. Using 9-aminoacridine as an example, it was shown that the simplest models cannot describe all possible types of the blocker-channel interaction. In such cases, the method of the simplest models combination can be used. The application of the simplest kinetic models analysis allowed to make the following conclusions: at least two molecules of 1-amino-3-propyl-adamantane or 9-aminoacridine can simultaneously bind to the open channel and block it; the occupation of 9-aminoacridine blocking sites in the channel can proceed in at least two different ways; the binding of tetrabutylammonium and 9-aminoacridine prevented the closure of the activation and/or desensitization gates of the channel, while that of tetraethylammonium did not.     

Early phase wound healing by primary intention as shown by ultrasonography

Tracheooesophageal fistulae (TEF) without atresia of the oesophagus cause in children relapsing respiratory diseases. The authors present diagnostic and therapeutic procedures in seven children. The symptoms of aspiration with attacks of non-productive cough and suffocation when fed were recorded in all children since birth. In five the diagnosis was made late. In four children the diagnosis was established by oesophagoscopy, in two by cinematooesophageography and in one by Fabian's test (Koop's test). All seven children were operated by a transcervical approach. After surgery all children are in a good condition and have no respiratory complaints.     

Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides

Potential problems with the use of viral vectors for gene therapy necessitate the development of efficient nonviral vectors. The association of transferrin, or the pH-sensitive peptide GALA, with cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and its equimolar mixture with dioleoylphosphatidylethanolamine, under conditions where the liposome/DNA complex is negatively charged, drastically increased luciferase expression from pCMVluc. The percentage of cells transfected, measured by beta-galactosidase expression, was also increased by about 10-fold. The zeta potential of the ternary complexes was lower than that of the liposome/DNA complexes. Transfection activity of positively charged complexes was also enhanced by association with transferrin, GALA or the influenza hemagglutinin N terminal peptide HA-2, but to a smaller extent compared with the negatively charged complexes. The enhancement of gene delivery by transferrin or GALA was not affected significantly by the presence of serum and did not cause significant cytotoxicity. Our results indicate that negatively charged ternary complexes of cationic liposomes, DNA and transferrin, or fusigenic peptides, can facilitate efficient transfection of cultured cells, and that they may alleviate the drawbacks of the use of highly positively charged complexes for gene delivery in vivo.     

The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids

Changes in the nuclear magnetic resonance (NMR) parameters of spin-lattice relaxation (T1), spin-spin relaxation (T2), proton density (rho), and water diffusion (DNMR) were measured over time together with the histopathological status in three regions of rat brain cortex after permanent middle cerebral artery occlusion (MCA-O). Histological response ranged from severe irreversible damage (necrosis and cavitation) to relatively mild and apparently reversible damage. DNMR was the only NMR parameter which demonstrated a statistically significant change in all three regions of brain studied. Additionally, rho was significantly increased only in the region of brain studied which eventually progressed to necrosis and cavitation. Finally, data are presented which indicate that changes in T2, DNMR, and rho can occur independently of one another.     

Lipid bilayer electrostatic energy, curvature stress, and assembly of gramicidin channels

Hydrophobic interactions between lipid bilayers and imbedded membrane proteins couple protein conformation to the mechanical properties of the bilayer. This coupling is widely assumed to account for the regulation of membrane protein function by the membrane lipids' propensity to form nonbilayer phases, which will produce a curvature stress in the bilayer. Nevertheless, there is only limited experimental evidence for an effect of bilayer curvature stress on membrane protein structure. We show that alterations in curvature stress, due to alterations in the electrostatic energy of dioleoylphosphatidylserine bilayers, modulate the structurally well-defined gramicidin A monomer <--> dimer reaction. Maneuvers that decrease the electrostatic energy of the unperturbed bilayer promote channel dissociation; we measure the change in interaction energy. The bilayer electrostatic energy thus can affect membrane protein structure by a mechanism that does not involve the electrostatic field across the bilayer, but rather electrostatic interactions among the phospholipid head groups in each monolayer which affect the bilayer curvature stress. These results provide further evidence for the importance of mechanical interactions between a bilayer and its imbedded proteins for protein structure and function.     

Pharmacokinetics and anti-HIV-1 efficacy of negatively charged human serum albumins in mice

Changes of blood metabolites and hormones were studied in female breeding calves before, during and after weaning from 4 to 18 weeks of age. Calves were initially fed increasing amounts of whole milk (up to 7 kg/day in week 8 of life). Milk intake was then gradually decreased up to the age of 16 weeks, when calves were completely weaned and only fed hay and concentrates. Average daily gain was 0.85 kg. Postprandial concentrations of glucose, insulin, insulin-like growth factor-I and 3.5.3'-triiodothyronine concentrations gradually decreased (P < 0.05) with age, while those of beta-hydroxybutyrate, protein, albumin, haemoglobin and iron increased (P < 0.05). Concentrations of cholesterol transiently increased, whereas those of urea reversibly decreased. Non-esterified fatty acids, triglycerides and growth hormone did not consistently change during the duration of the study. In conclusion, changes of glucose, beta-hydroxybutyrate, haemoglobin, iron, insulin, insulin-like growth factor-I and 3.5.3'-triiodothyronine were markedly different from those usually seen in veal calves of the same age.     

Thermally denatured ribonuclease A retains secondary structure as shown by FTIR

Fourier transform-infrared (FTIR) spectroscopy has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A) at pH* 2.0 (uncorrected pH measured in D2O). The amide I spectral region of the native and thermally denatured protein was compared. A substantial decrease in the amount of beta-sheet and alpha-helix and a corresponding increase in the amount of turn and unordered structure was observed on thermal denaturation. The results indicate that thermally denatured RNase A contains significant amounts of secondary structure (11% helix and 17% beta-sheet), consistent with previous results reported for circular dichroism, and with a relatively compact structure, as revealed by dynamic light scattering. These results are in contrast to those of amide protection experiments reported recently [Robertson, A.D., & Baldwin, R.L. (1991) Biochemistry 30, 9907-9914] which indicated no stable hydrogen-bonded structure under these experimental conditions. Possible explanations for this apparent discrepancy are given.     

Various advantages of rigid bronchoscopy as shown by a case report]

The clinical effect of a large biopsy of a bronchial adenoma via the bronchoscope is described. The author explains why, for diagnosis and therapy of bronchial adenoma, the use of the bronchoscope is to be prefered to the use of the fiberscope.     

Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

Interaction of hemoglobin with red blood cell membranes as shown by a fluorescent chromophore

Hemoglobin quenching of the fluorescence intensity of 12-(9-anthroyl)stearic acid (AS) embedded in the red blood cell membrane occurs through an energy transfer mechanism and can be used to measure the binding of hemoglobin to the membrane. The binding of hemoglobin to red cell membranes was found to be reversible and electrostatic in nature. Using a theory of energy transfer based on F?rster formulation, the quantitative data for the binding were derived. The number of binding sites was found to be 1.4 +/- 0.2 X 10(6) molecules per cell and the binding constant was 0.85 X 10(8) M-1.     

Extra-hypothalamic vasopressin neurons coexpress galanin messenger RNA as shown by double in situ hybridization histochemistry

Vasopressin (VP) neurons in the bed nucleus of the stria terminalis (BNST) and medial amygdala (AMe) exhibit sexual dimorphism and steroid dependency. VP neurons in the supraoptic nucleus and paraventricular nucleus have been shown to coexpress other transmitters including galanin (GAL). However, little is known about what other neurotransmitters may be colocalized with VP in the BNST and AMe. Here, we have used radio-labeled and digoxigenin-labeled cRNA probes to perform double in situ hybridization histochemistry for VP and GAL in the BNST and AMe of intact, adult male rats. We provide evidence that in the basal state, the majority of VP-synthesizing cells in the BNST and AMe of the adult male rat also express galanin mRNA. Likewise, the majority of GAL-expressing neurons in these regions also contain VP mRNA. These findings give further evidence for the similarity of the BNST and AMe and provide a rationale for studies investigating the role of GAL in functions involving extrahypothalamic VP pathways.