Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study.

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label (3'-spiro(2'=(N-oxyl-4',4'-dimethyl-oxazolidine))5alpha-cholestane) and a fatty acid spin label (4',4'-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55 degrees C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron miccroscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.     

Inhibition of bacteriophage PBS2 replication in Bacillus subtilis by phleomycin.

Phleomycin is an effective inhibitor of the replication of Bacillus subtilis bacteriophage PBS2, whose DNA contains uracil instead of thymine. Phleomycin does not affect the induction of the known phage enzymes involved in deoxyribonucleotide metabolism. But phage DNA synthesis is severely inhibited by phleomycin, and late virion protein synthesis is eliminated. These effects appear to result from a phleomycin-induced degradation of the parental phage DNA. Similar inhibitory and degradative effects on DNA are seen in phleomyinc-treated, uninfected cells. This system is unaffected by the related antibiotic, bleomycin.     

Rhythmic oscillations in amygdala excitability during kindling

Unilateral amygdala electrodes were implanted in male Sprague-Dawley rats stimulated once daily with a 200 μamp pulse of 500 millisecond duration to produce kindling. Forty-six percent (12 of 26) of the animals that eventually developed after-discharges demonstrated rhythmic oscillations in after-discharge duration. The presence or absence of generalized bilateral clonic seizures also showed rhythmic oscillations in close association with after-discharge duration. It is suggested that during kindling some animals, independent of electrode placement, develop rhythmic oscillations in excitability of the amygdala. This model may represent a means of experimentally eliciting or uncovering neuronal substrates which show regular alterations in excitability and may be relevant to the oscillations in mood and behavior observed in the affective disorders.     

Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.     

The chromosomes of the Filariae

An understanding of the nature of the chromosomes of the filariae is expected to greatly assist the future interpretation of genome data. Filarial development is not eutelic, and there does not seem to be a fixed number of cell divisions in the way that there is in Caenorhabditis. It is not clear whether the chromosomes of the filariae have localized centromeres or whether they are holocentric. Sex determination is by a chromosomal "balance" X0 system in most filariae, but in some Onchocercidae there has been a chromosomal fusion to create a neo-XY system. It is presumed that the molecular basis of sex determination in filariae is similar to Caenorhabditis. The ancestral karyotype of the filariae is probably 5A+X0, but in some Onchocercidae this has been reduced to 4A+XY, and in O. volvulus and O. gibsoni it has been further reduced to 3A+XY. Onchocerca volvulus and O. gibsoni both have supernumary (B-) chromosomes and in O. volvulus there is a single active nucleolus organising region near the middle of the long autosome.     

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

Recurrent and founder mutations in the Netherlands—Phospholamban p.Arg14del mutation causes arrhythmogenic cardiomyopathy

Background

Recently, we showed that the c.40_42delAGA (p.Arg14del) mutation in the phospholamban (PLN) gene can be identified in 10–15 % of Dutch patients with dilated cardiomyopathy or arrhythmogenic cardiomyopathy. The arrhythmogenic burden of the p.Arg14del mutation was illustrated by the high rate of appropriate ICD discharges and a positive family history for sudden cardiac death.

Methods

Our goal was to evaluate the geographical distribution and the origin of this specific mutation in the Netherlands and to get an estimation of the prevalence in a Dutch population cohort. Therefore, we investigated the postal codes of the places of residence of PLN p.Arg14del mutation carriers and places of birth of their ancestors. In addition, a large population-based cohort (PREVEND) was screened for the presence of this mutation.

Results

By April 2012, we had identified 101 probands carrying the PLN p.Arg14del mutation. A total of 358 family members were also found to carry this mutation, resulting in a total of 459 mutation carriers. The majority of mutation carriers live in the northern part of the Netherlands and analysing their grandparents’ places of birth indicated that the mutation likely originated in the eastern part of the province of Friesland. In the PREVEND cohort we identified six heterozygous PLN p.Arg14del mutation carriers out of 8,267 subjects (0.07 %).

Conclusion

The p.Arg14del mutation in the PLN gene is the most frequently identified mutation in Dutch cardiomyopathy patients. The mutation that arose 575–825 years ago is likely to have originated from the eastern part of the province of Friesland and is highly prevalent in the general population in the northern part of the Netherlands.