Gramicidin toxicity in NG108-15 cells: protective effects of acetamidine and guanidine

Studies were conducted using a novel in vitro approach to investigate the efficacy of acetamidine hydrochloride (ACE) and guanidine hydrochloride (GUAN), previously shown to block gramicidin D (GRAM) channels in artificial membranes, in preventing the toxic effects of GRAM in NG108-15 (neuroblastoma×glioma hybrid) cells. Specifically, intracellular microelectrode techniques were employed to examine changes in membrane resting potential (V _m) and input resistance (R _in). At 1 mol/L, ACE significantly reduced loss of V _m induced by 1 or 10 g/ml GRAM, although higher concentrations of ACE did not afford enhanced antagonism. GUAN, in contrast, produced a concentration-dependent antagonism of GRAM-induced V _m and R _in loss, with high concentrations (10 or 100 mol/L) completely preventing diminutions in both V _m and R _in. In control cells superfused without GRAM, ACE produced a direct, concentration-dependent reduction in V _m and R _in, whereas GUAN hyperpolarized NG108-15 cells but did not alter R _in. These data represent the initial demonstration of the reversal of GRAM toxicity in an intact cell system.     

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.     

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori. Received: 10 May 1999 / Accepted: 7 July 1999     

Structure of the Avian Mitochondrial Cytochrome bc 1 Complex

There are now four structures of vertebrate mitochondrial bc ₁ complexes available in theprotein databases and structures from yeast and bacterial sources are expected soon. Thisreview summarizes the new information with emphasis on the avian cytochrome bc ₁ complex(PDB entries 1BCC and 3BCC). The Rieske iron–sulfur protein is mobile and this has beenproposed to be important for catalysis. The binding sites for quinone have been located basedon structures containing inhibitors and, in the case of the quinone reduction site Qi, thequinone itself.     

Molecular rearrangements of thylakoids after heavy metal poisoning, as seen by Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopy

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (F_v) and the maximal (F_m) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the -helix content (1656 cm^-1), a parallel increase in the disordered segments (1651 cm^-1), a decrease in the intramolecular -sheet (1636 cm^-1) content and the concomitant appearance of an intermolecular -structure (1621 cm^-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular -structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these lipidated proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.     

Dark reoxidation of the plastoquinone-pool is mediated by the low-potential form of cytochrome b-559 in spinach thylakoids

We have found that in isolated spinach thylakoids, plastoquinone-pool (PQ-pool), after its photoreduction, undergoes dark-reoxidation with the half-time of _1/2 = 43 ± 3 s. To explain the observed rates of PQ-pool reoxidation, a nonenzymatic plastoquinol (PQH₂) autoxidation under molecular oxygen and an enzymatic oxidation by the low-potential form of cytochrome b-559 (cyt. b-559LP), as the postulated PQ-oxidase in chlororespiration, were investigated. It was found that the autoxidation rate of PQH₂ in organic solvents and liposomes was too low to account for the observed oxidation rate of PQH₂ in thylakoids. The rate of cyt. b-559LP autoxidation in isolated Photosystem II was found to be similar (_1/2 = 26 ± 5 s) to that of the PQ-pool. This suggests that the LP form of cyt. b-559 is probably responsible for the PQ-oxidase activity observed during chlororespiration.