Viral membrane penetration: lytic activity of a nodaviral fusion peptide

The auto-cleavage product from the C-terminal part of the capsid protein of the flock house virus, namely the ₁ peptide, was used as a model peptide to characterize the initial steps of viral membrane penetration. Monolayers at the air–water interface were used to investigate the phase behaviour of ternary lipid–peptide mixtures, whereas solid-supported membranes were used to visualize the lytic activity of the ₁ peptide. 1,2-Dipalmitoyl-sn-glycero-phospatidylcholine/1,2-dipalmitoyl-sn-glycero-phospatidylserine (4:1) membranes were used as negatively charged model membranes. By means of film balance techniques lipid/peptide discrimination was found resulting in a lipid-rich and a peptide-rich phase. Quartz crystal microbalance and scanning force microscopy experiments led to the conclusion of a detergent-like mechanism of the ₁ peptide resulting in mixed lipid–peptide micelles with a molar ratio of 2.8:1. A monolayer adsorption with an ongoing lysis of membranes was found with ₁ peptide molecules interacting at membrane defects.     

Sperm morphology: Its relevance to compensable and uncompensable traits in semen

The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure, and failure in embryogenesis, are both of seminal origin. Males also differ in the number of sperms required to reach their maximum fertilization rate. Males requiring more sperm are considered to have compensable seminal deficiencies. These include a number of known viability and morphology traits (including both abnormal heads and tails) and unknown factors (functional or molecular traits) precluding sperm access to the ovum or ability of the sperm to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These seminal deficiencies are associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis (once initiated), with most failures occurring prior to maternal recognition of pregnancy; these sperm would pre-empt fertilization by competent sperm. Evidence now exists supporting the concept that the uncompensable effect is due to chromatin aberrations in morphologically normal or near-normal fertilizing sperm present in abnormal ejaculates (elevated content of abnormal sperm). Thus, sperm morphology may be our best indication for the presence of an uncompensable deficiency, although we have yet to identify the incompetent fertilizing sperm clinically.     

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers

The genotoxic potential of bidi tobacco was evaluated by mutagenicity testing of aqueous, aqueous: ethanolic, ethanolic and chloroform extracts of processed tobacco used in the manufacture of 'bidis', indigenous forms of cigarettes smoked in India. The Salmonella/mammalian microsome test (Ames assay) was used to detect mutagenicity in tester strains TA98, TA100 and TA102. The extracts were tested in the absence and presence of metabolic activation using liver S9 from rat and hamster, and following in vitro nitrosation with sodium nitrite at acidic pH. All the extracts were non-mutagenic in the absence of nitrosation. The nitrosated aqueous extract was mutagenic in strains TA98 and TA100. While weak mutagenicity was elicited by the nitrosated aqueous: ethanolic extract in TA100, the nitrosated ethanolic extract induced a 3-fold increase in the number of revertants in the same strain. Moreover both these extracts elicited a strong mutagenic response in TA102, while the chloroform extract was non-mutagenic even after nitrite treatment. The present study indicates that workers employed in the bidi industry are exposed to potentially mutagenic and genotoxic chemicals in the course of their occupation.     

Learnt sensori-motor mappings in honeybees: interpolation and its possible relevance to navigation

We investigated the ability of bees to associate a motor parameter with a sensory one. Foragers were trained to fly along a prescribed route through a large box which was partitioned into compartments. Access from one compartment to the next was through a hole in each partition. In two of the compartments, the back wall was covered with a grating of black and white stripes. Stripe orientations and the required trajectories differed in the two compartments so giving bees the opportunity to learn that one stripe orientation signalled the need to fly leftwards and the other rightwards.We videotaped the bees' trajectories through one of these compartments in tests with the grating on the back wall in one of four possible orientations. Flight trajectories to stripes in the training orientations were appropriately to the left or to the right implying that bees had linked a given flight direction to a given stripe orientation. With gratings oriented between the training values, flight directions were, under some conditions, intermediate between the training directions. This interpolation indicates that the training regime had induced a continuous mapping between stripe orientation and trajectory direction and thus suggests that trajectory direction is a motor parameter which is encoded explicitly within the brain. We describe a simple network that interpolates much like bees and we consider how interpolation may contribute to the ability of bees to navigate flexibly within a familiar environment.     

Physical properties of lipid bilayer membranes: relevance to membrane biological functions

Over the last 25 years one of us (WKS) has been investigating physical properties of lipid bilayer membranes. In 1991 a group led by WKS was organized into the Laboratory of Structure and Dynamics of Biological Membranes, the effective member of which is AW. Using mainly the electron paramagnetic resonance (EPR) spin-labeling method, we obtained unexpected results, which are significant for the better understanding of the functioning of biological membranes. We have developed a new pulse EPR spin-labeling method for the detection of membrane domains and evaluation of lipid exchange rates. This review will be focused on our main results which can be summarized as follows: (1) Unsaturation of alkyl chains greatly reduces the ordering and rigidifying effects of cholesterol although the unsaturation alone gives only minor fluidizing effects, as observed by order and reorientational motion, and rather significant rigidifying effects, as observed by translational motion of probe molecules; (2) Fluid-phase model membranes and cell plasma membranes are not barriers to oxygen and nitric oxide transport; (3) Polar carotenoids can regulate membrane fluidity in a way similar to cholesterol; (4) Formation of effective hydrophobic barriers to the permeation of small polar molecules across membranes requires alkyl chain unsaturation and/or the presence of cholesterol; (5) Fluid-phase micro-immiscibility takes place in cis-unsaturated phosphatidylcholine-cholesterol membranes and induces the formation of cholesterol-rich domains; (6) In membranes containing high concentrations of transmembrane proteins a new lipid domain is formed, with lipids trapped within aggregates of proteins, in which the lipid dynamics is diminished to the level of gel-phase.     

Circling mouse: possible animal model for deafness

Mutant mice with abnormalities are potentially useful as models for studying human defects. Here we report a group of mice with abnormal behavioral patterns. A new spontaneous mutant mouse exhibited hyperactive behavior at about seven days of age, followed by tight circling behavior. Breeding studies suggest that this mutation is caused by a single gene defect inherited in an autosomal recessive manner. Consequently, this mutation is referred to as a circling (cir) mouse mutation with the gene symbol cir. Auditory test results identified clearly the hearing loss of the cir, compared with wild-type mice. Pathologic studies confirmed developmental defects in cochlea and spiral ganglions that were correlated to the abnormal behavior observed in the cir mice. Thus, cir mice may be useful as a model for studying inner ear abnormalities and deafness in humans.     

An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol

The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.     

Tightly bound to DNA proteins: possible universal substrates for intranuclear processes

Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.     

Renal Fanconi syndrome: developmental basis for a new animal model with relevance to human disease

Using succinylacetone (SA), a metabolite of tyrosine excreted in excess by infants and children with hereditary tyrosinemia and the renal Fanconi syndrome (FS), we have investigated developmentally-related membrane transport events leading to emergence of the generalized renal tubular dysfunction seen in human FS. SA was found to impair sugar and amino acid uptake by both newborn renal tubules and 7-day renal brush-border membrane vesicles (BBMV). This impairment by SA was due in part to a slowing of substrate cotransport rate of 22Na+-entry into BBMV. Concentration-dependent uptake studies indicated SA inhibited the newborn high-affinity transport systems for sugars and amino acids. SA also caused an increase in membrane fluidity and a shift in the thermotropic transition temperature. The demonstrated dual nature of SA's effect on membrane fluidity and O2 consumption, together with the relative contribution of each component to SA-induced transport impairment helps to provide a basis for an understanding of the age-related increases in glucosuria, aminoaciduria and natriuria seen in infants with FS.     

Novel heme: O2 bonding of possible relevance to oxygen utilizing heme and other proteins

Solid dipyridine hemes which are unreactive toward oxygen lose both pyridine ligands upon heating under vacuum to give a solid which takes up O₂, reversibly, one O₂ per heme. Replacement of ¹⁶O₂ by ¹⁸O₂ reduces only infrared bands near 1660 and 1590 cm^?1, frequencies near the vibrational band for gaseous O₂. No FeO bands are detected. EPR spectra reveal a free radical and ferric iron; Mössbauer, NMR and infrared spectra support an iron(III) oxidation state. Limited molecular weight data indicate a dimer. Possibly two dioxygen molecules are held sandwich fashion between two porphyrins via donor-acceptor interactions, which are facilitated by electron transfer from iron(II) into the porphyrin forming a π-anion. Such O₂ bonding is not found in oxy Hb and Mb or in oxyhemerythrin but may occur with cytochrome $\text{c}$ oxidase and other oxygen utilizing (or producing) heme and other proteins.     

Signal peptide mimics conjugated to peptide nucleic acid: a promising solution for improving cell membrane permeability

The specific binding ability and biostability of PNA (peptide nucleic acid) with DNA or RNA make PNA not only a good tool for the studies of molecular biology but also the candidate for gene-targeting drugs. However, the main obstacle for its potential usage as a therapeutic is the low cell uptake caused by the poor cell membrane permeability. In this paper the hydrophobic pentadecapeptide and two signal peptide mimics, hexa- and decapeptides ending with a positively charged amino acid, were proposed as the linked carrier for the transportation of PNA T10 through the cell membrane; stable spin label was coupled to the peptide-PNA conjugate so that the ESR measurements can be used for the assessment of their transmembrane movements. The syntheses of spin-labeled peptide-PNA conjugates were carried out on MBHA resin with Boc strategy. The cell membrane permeability of the spin-labeled conjugates of peptides and PNA can be determined with ESR, during the incubation of erythrocyte with the samples. According to ESR measurements, the three conjugates exhibit enhanced uptake into erythrocytes. The hexa- and decapeptide-modified PNA showed suitable water solubility. The peptide-PNA conjugates retained their binding ability to complementary DNA. The results suggest that peptide modification of PNA might be a promising solution for improving cell membrane permeability toward PNA.     

Light affects mitochondria to cause apoptosis to cultured cells: possible relevance to ganglion cell death in certain optic neuropathies

Retinal ganglion cell axons within the globe are laden with mitochondria that are unprotected from light (400–760 nm) impinging onto the retina. Light can be absorbed by mitochondrial enzymes such as cytochrome and flavin oxidases causing the generation of reactive oxygen species, and we have suggested this may pose a risk to ganglion cell survival if their energy state is compromised, as may be so in glaucoma or in Leber's Hereditary Optic Neuropathy. Here, we demonstrate that light (400–760 nm) provokes apoptosis in cultured retinal ganglion-5 cells, and that this effect is enhanced in low serum, and attenuated by various antioxidants. Apoptosis is shown to be caspase independent, involving reactive oxygen species generation and the activation of poly(ADP-ribose) polymerase-1 and apoptosis-inducing factor. We further show that light-induced apoptosis requires the participation of the mitochondrial respiratory chain. This was demonstrated by culturing fibroblasts (BJhTERT cells) in ethidium bromide for 40 days to deplete their mitochondrial DNA and perturb their mitochondrial respiratory chain function (BJhTERT rh0 cells). Only BJhTERT cells, with intact mitochondrial respiratory chain function were affected by light insult. Finally, we show that exposure of anaesthetized pigmented rat eye to white, but not red light, causes changes in the expression of certain retinal mRNAs (neurofilament light, Thy-1 and melanopsin) and optic nerve proteins (neurofilament light and tubulin), suggesting that ganglion cell survival is affected. Our findings support the proposal that the interaction of light, particularly the blue component, with intra-axonal ganglion cell mitochondria may be deleterious under certain circumstances, and suggest that reducing the light energy impinging upon the retina might benefit patients with certain optic neuropathies.     

An introduction to peptide nucleic acid

Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.     

Resistance of veillonella to hydrosoluble pristinamycins: a possible outer membrane barrier effect

An in vitro study on 25 Veillonella strains showed a consistent clindamycin susceptibility with resistance to erythromycin and to the hydrosoluble pristinamycins, quinupristin and dalfopristin and its 30:70 combination, the synergistin RP59500. Double erythromycin-clindamycin disk tests did not show any inducible resistance pattern. The addition of 10 or 50 mug/mL of polymyxin B nonapeptide, an outer membrane permeabilizing agent, consistently reduced quinupristin and dalfopristin MICs in most strains. This result suggests that the Veillonella outer membrane may act as a permeability barrier to these antibiotics, as in the case of other Gram-negative bacteria.