Effects of Peptide Charge,Orientation, and Concentration on Melittin Transmembrane Pores

Melittin is a short cationic peptide that exerts cytolytic effects on bacterial and eukaryotic cells. Experiments suggest that in zwitterionic membranes, melittin forms transmembrane toroidal pores supported by four to eight peptides. A recently constructed melittin variant with a reduced cationic charge, MelP5, is active at 10-fold lower concentrations. In previous work, we performed molecular dynamics simulations on the microsecond timescale to examine the supramolecular pore structure of a melittin tetramer in zwitterionic and partially anionic membranes. We now extend that study to include the effects of peptide charge, initial orientation, and number of monomers on the pore formation and stabilization processes. Our results show that parallel transmembrane orientations of melittin and MelP5 are more consistent with experimental data. Whereas a MelP5 parallel hexamer forms a large stable pore during the 5-μs simulation time, a melittin hexamer and an octamer are not fully stable, with several monomers dissociating during the simulation time. Interaction-energy analysis shows that this difference in behavior between melittin and MelP5 is not due to stronger electrostatic repulsion between neighboring melittin peptides but to peptide-lipid interactions that disfavor the isolated MelP5 transmembrane monomer. The ability of melittin monomers to diffuse freely in the 1,2-dimyristoyl-SN-glycero-3-phosphocholine membrane leads to dynamic pores with varying molecularity.     

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase–encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.     

Nocturnal exposure to intermittent 60 Hz magnetic fields alters human cardiac rhythm

Heart rate variability (HRV) results from the action of neuronal and cardiovascular reflexes, including those involved in the control of temperature, blood pressure and respiration. Quantitative spectral analyses of alterations in HRV using the digital Fourier transform technique provide useful in vivo indicators of beat-to-beat variations in sympathetic and parasympathetic nerve activity. Recently, decreases in HRV have been shown to have clinical value in the prediction of cardiovascular morbidity and mortality. While previous studies have shown that exposure to power-frequency electric and magnetic fields alters mean heart rate, the studies reported here are the first to examine effects of exposure on HRV. This report describes three double-blind studies involving a total of 77 human volunteers. In the first two studies, nocturnal exposure to an intermittent, circularly polarized magnetic field at 200 mG significantly reduced HRV in the spectral band associated with temperature and blood pressure control mechanisms (P = 0.035 and P = 0.02), and increased variability in the spectral band associated with respiration (P = 0.06 and P = 0.008). In the third study the field was presented continuously rather than intermittently, and no significant effects on HRV were found. The changes seen as a function of intermittent magnetic field exposure are similar, but not identical, to those reported as predictive of cardiovascular morbidity and mortality. Furthermore, the changes resemble those reported during stage II sleep. Further research will be required to determine whether exposure to magnetic fields alters stage II sleep and to define further the anatomical structures where field-related interactions between magnetic fields and human physiology should be sought. Bioelectromagnetics 19: 98–106, 1998. © 1998 Wiley-Liss, Inc.     

Physicochemical characterisation of the ubiquitous bracken fern as useful biomaterial for preconcentration of heavy metals

Batch experiments with dry bracken fern have been done to determine cadmium and lead sequestering capacity of this biomaterial. Biomass characterisation was done by infrared spectroscopy and potentiometric analysis. The effect of pH of the metal containing solution, contact time and initial metal concentration has been studied, together with the acid-base properties of the biomaterial. Results obtained have been analysed using mathematical and modelling techniques. Effect of pH on metal sequestration has been correlated with observed acid-base properties of the natural substrate. Kinetic data analysis provided relevant information about metal sequestration rate, showing important differences between lead and cadmium. Maximum uptake was found to be the same for both metals 0.410 mmol/g. This value was also clearly correlated to the number of acidic groups determined for this material which was found to be 0.432 mmol of acidic groups per gram of fern. Results obtained indicate that acidic groups are the functional groups responsible of the sequestration of metal ions and that bracken fern is a promising material for metal preconcentration.     

KCNJ11 knockout morula re-engineered by stem cell diploid aggregation

KCNJ11-encoded Kir6.2 assembles with ATP-binding cassette sulphonylurea receptors to generate ATP-sensitive K+ (KATP) channel complexes. Expressed in tissues with dynamic metabolic flux, these evolutionarily conserved yet structurally and functionally unique heteromultimers serve as high-fidelity rheostats that adjust membrane potential-dependent cell functions to match energetic demand. Genetic defects in channel subunits disrupt the cellular homeostatic response to environmental stress, compromising organ tolerance in the adult. As maladaptation characterizes malignant KATP channelopathies, establishment of platforms to examine progression of KATP channel-dependent adaptive behaviour is warranted. Chimeras provide a powerful tool to assay the contribution of genetic variance to stress intolerance during prenatal or post-natal development. Here, KCNJ11 KATP channel gene knockout<-->wild-type chimeras were engineered through diploid aggregation. Integration of wild-type embryonic stem cells into zona pellucida-denuded morula derived from knockout embryos achieved varying degrees of incorporation of stress-tolerant tissue within the KATP channel-deficient background. Despite the stress-vulnerable phenotype of the knockout, ex vivo derived mosaic blastocysts tolerated intrauterine transfer and implantation, followed by full-term embryonic development in pseudopregnant surrogates to produce live chimeric offspring. The development of adult chimerism from the knockout<-->wild-type mosaic embryo offers thereby a new paradigm to probe the ecogenetic control of the KATP channel-dependent stress response.     

Effect of apolipoprotein E on the cerebral load of latent herpes simplex virus type 1 DNA

Herpes simplex virus type 1 (HSV-1) is neurotropic and enters a latent state lasting the lifetime of the host. This pathogen has recently been proposed as a risk factor for Alzheimer's disease (AD) in conjunction with apolipoprotein E4 (ApoE4). In a murine acute infection model, we showed that viral neuroinvasiveness depends directly on the overall ApoE dosage and especially on the presence of isoform ApoE4. If an interaction between ApoE and HSV-1 is involved in AD, it may occur during latency rather than during acute infection. Certainly, ApoE plays an important role in late-onset AD, i.e., at a time in life when the majority of people harbor HSV-1 in their nervous system. In the present work, wild-type, APOE knockout, APOE3, and APOE4 transgenic mice were used to analyze the influence of the ApoE profile on the levels of latent virus DNA. The knockout mice had significantly lower concentrations of the virus in the nervous system than the wild-type mice, while the APOE4 mice had very high levels in the brain compared to the APOE3 animals. ApoE4 seems to facilitate HSV-1 latency in the brain much more so than ApoE3. The APOE dosage correlated directly with the HSV-1 DNA concentration in the brain, strengthening the hypothesis that HSV-1, together with ApoE, might be involved in AD.     

Right‐handed fossil humans

Fossil hominids often processed material held between their upper and lower teeth. Pulling with one hand and cutting with the other, they occasionally left impact cut marks on the lip (labial) surface of their incisors and canines. From these actions, it possible to determine the dominant hand used. The frequency of these oblique striations in an array of fossil hominins documents the typically modern pattern of 9 right‐ to 1 left‐hander. This ratio among living Homo sapiens differs from that among chimpanzees and bonobos and more distant primate relatives. Together, all studies of living people affirm that dominant right‐handedness is a uniquely modern human trait. The same pattern extends deep into our past. Thus far, the majority of inferred right‐handed fossils come from Europe, but a single maxilla from a Homo habilis, OH‐65, shows a predominance of right oblique scratches, thus extending right‐handedness into the early Pleistocene of Africa. Other studies show right‐handedness in more recent African, Chinese, and Levantine fossils, but the sample compiled for non‐European fossil specimens remains small. Fossil specimens from Sima del los Huesos and a variety of European Neandertal sites are predominately right‐handed. We argue the 9:1 handedness ratio in Neandertals and the earlier inhabitants of Europe constitutes evidence for a modern pattern of handedness well before the appearance of modern Homo sapiens.