Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

Phylogeography of a northeast Asian spruce, Picea jezoensis, inferred from genetic variation observed in organelle DNA markers

Range-wide genetic variation of the widespread cold-temperate spruce Picea jezoensis was studied throughout northeast Asia using maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA markers. This study assessed 33 natural populations including three varieties of the species in Japan, Russia, China, and South Korea. We depicted sharp suture zones in straits around Japan in the geographical distribution pattern of mitochondrial haplotypes (GST=0.901; NST=0.934). In contrast, we detected possible extensive pollen flow without seed flow across the straits around Japan during the past population history in the distribution pattern of chloroplast haplotypes (GST=0.233; NST=0.333). The analysis of isolation by distance of the species implied that by acting as a barrier for the movement of seeds and pollen, the sharp suture zones contributed considerably to the level of genetic differentiation between populations. Constructed networks of mitochondrial haplotypes allowed inference of the phylogeographical history of the species. We deduced that the disjunction with Kamchatka populations reflects range expansion and contraction to the north of the current distribution. Within Japan, we detected phylogeographically different types of P. jezoensis between Hokkaido and Honshu islands; P. jezoensis in Honshu Island may have colonized this region from the Asian continent via the Korean peninsula and the species in Hokkaido Island is likely to have spread from the Asian continent via Sakhalin through land bridges. Japanese endemism of mitochondrial haplotypes in Hokkaido and Honshu islands might have been promoted by separation of these islands from each other and from the Asian continent by the straits during the late Quaternary.     

Changes in sleep quality of athletes under normobaric hypoxia equivalent to 2,000-m altitude: a polysomnographic study.

This study evaluated the sleep quality of athletes in normobaric hypoxia at a simulated altitude of 2,000 m. Eight male athletes slept in normoxic condition (NC) and hypoxic conditions equivalent to those at 2,000-m altitude (HC). Polysomnographic recordings of sleep included the electroencephalogram (EEG), electrooculogram, chin surface electromyogram, and electrocardiogram. Thoracic and abdominal motion, nasal and oral airflow, and arterial blood oxygen saturation (Sa(O(2))) were also recorded. Standard visual sleep stage scoring and fast Fourier transformation analyses of the EEG were performed on 30-s epochs. Subjective sleepiness and urinary catecholamines were also monitored. Mean Sa(O(2)) decreased and respiratory disturbances increased with HC. The increase in respiratory disturbances was significant, but the increase was small and subclinical. The duration of slow-wave sleep (stage 3 and 4) and total delta power (<3 Hz) of the all-night non-rapid eye movement sleep EEG decreased for HC compared with NC. Subjective sleepiness and amounts of urinary catecholamines did not differ between the conditions. These results indicate that acute exposure to normobaric hypoxia equivalent to that at 2,000-m altitude decreased slow-wave sleep in athletes, but it did not change subjective sleepiness or amounts of urinary catecholamines.     

Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s

At least two GTP-binding proteins (G proteins) with Mr values of about 20,000 were extracted from bovine aortic smooth muscle membranes by sodium cholate. The most abundant G protein (22K G) was purified to near homogeneity by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite and Mono Q HR5/5. 22K G showed kinetic and physical properties very similar to those of smg p21, a G protein recently isolated from bovine brain and human platelet membranes, having the same effector domain as ras p21s. Moreover, 22K G was recognized specifically by the anti-smg p21 antibody. These results indicate that the major G protein in bovine aortic smooth muscle membranes is smg p21.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Confirmation of the anomeric structure of galacturonic acid in the galacturonosyl-ceramide of Sphingomonas yanoikuyae

The anomeric structure of glycosphingolipids significantly influences their activity to stimulate natural killer T cells. In this study the chemical structure of the galacturonosyl-ceramide in Sphingomonas yanoikuyae, designated GSL-1'sy, was re-examined to prove the anomeric structure of the Dgalacturonic acid (GalA) in the lipid, which was reported as beta-configuration by Naka et al., but was suggested as alpha-configuration in our preliminary study. GSL-1'sy was purified from the bacterial cells with the same procedure as Naka et al. The 1H-NMR analysis of GSL-1'sy revealed that the coupling constant of the anomeric proton of GalA was 3.0 Hz, indicating that GalA in GSL-1'sy is alpha-anomer, the configuration active for the stimulation of natural killer T cells.     

Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis.     

Characterization of a Xenopus laevis skin peptidylglycine alpha-hydroxylating monooxygenase expressed in insect-cell culture.

The C-terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two-step reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase. A Xenopus laevis PHM expressed in insect-cell culture by the baculovirus-expression-vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L-ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L-ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping-pong mechanism with respect to peptide and L-ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C-termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non-polar amino acids.     

Glutamate induces Cl− and K+ currents in the olfactory interneurons of a terrestrial slug

Glutamate-induced responses in the procerebral neurons of the terrestrial slug Limax marginatus were examined using the nystatin-perforated patch recording technique applied in the voltage-clamp mode and local application of drugs. The procerebrum contains two types of interneurons with different spontaneous activities, bursting and nonbursting neurons. In the bursting neurons, a puff of glutamate evoked a rapidly desensitizing current followed by a smaller sustained current. The reversal potential of the early component showed that the current was mediated by Cl⁻ ions, while the late component was presumed to be mediated by K⁺ ions. In the nonbursting neurons, glutamate evoked a sustained current with a strong outward rectification, and the current was mediated by K⁺ ions. Ibotenate selectively evoked the rapidly desensitizing response in the bursting neurons, whereas quisqualate evoked a non-desensitizing K⁺ current both in the bursting and nonbursting neurons. The glutamate-induced K⁺ current had similar characteristics with the spontaneous synaptic activities in the procerebrum neurons, suggesting the possibility that glutamate receptors are involved in the spontaneous oscillatory activity. Accepted: 10 February 1999     

Protocol for the production of viable bimaternal mouse embryos

A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.