Self‐Assembly of Dendritic Macromolecules Based on the Ionic Interaction of Linear Chain Polyelectrolyte Cores with Oppositely Charged Focal Ionogenic Groups of Dendrons

Summary: A new principle for the design of dendritic macromolecules – the ionic binding of linear chain polyelectrolyte with oppositely charged focal ionogenic groups of dendrons – has been developed. The majority of the dendritic ionic complexes (DICs) are prepared with poly(styrenesulfonic acid) (PSS) as a polymeric core and L ‐aspartic acid dendrons of different generations. Two series of DICs were prepared using PSS and aspartic dendrons bearing terminal (located at the external periphery) methoxycarbonyl and hexyloxycarbonyl groups (C1‐n and C6‐n respectively where n is the generation number). Ionic binding of about 100% was found for dendrons of Generation 1–3. The solubility of the DICs was examined and the DICs prepared were studied by IR spectroscopy, ¹H NMR and viscometry.

Dendritic ionic complexes prepared using poly(styrenesulfonic acid) acid and aspartic dendrons bearing terminal methoxycarbonyl and hexyloxycarbonyl groups.     

Sleep and daytime sleepiness in retinitis pigmentosa patients

OBJECTIVE: We examined sleep, daytime sleepiness and the ability to stay awake during the day in patients affected with retinitis pigmentosa (RP), to further delineate the role of photoreceptors in the circadian cycle. METHODS: Twelve individuals diagnosed with RP (40 +/- 8 years) And 12 normally sighted healthy individuals (39 +/- 7 years) matched for age, body mass index (BMI) and sex were selected for the study. Participants had their sleep recorded on two consecutive nights and were monitored on the two following days. On the first day, their ability to stay awake and on the second, their sleep propensity were assessed using the Maintenance of Wakefulness Test (MWT) and the Multiple Sleep Latency Test (MSLT), respectively. Self-report measures were obtained using the Pittsburgh Sleep Quality Index (PSQI), the Epworth Sleepiness Scale (ESS), and the Toronto Hospital Alertness Test (THAT). RESULTS: Subjective daytime sleepiness (ESS: 9 +/- 5 vs. 6 +/- 4, P=0.053) and objectively measured sleep propensity (MSLT: 10 +/- 5 vs. 17 +/- 3 min, P < 0.000) were significantly higher in RP patients than controls, whilst their alertness (THAT: 29 +/- 9 vs. 38 +/- 7, P=0.016) and ability to stay awake (MWT: 21 +/- 9 vs. 29 +/- 2 min, P=0.006) were significantly reduced. Retinitis pigmentosa participants had more disturbed nighttime sleep, with significantly more awakenings (arousal index: 14 +/- 8 vs. 8 +/- 6 h, P=0.039), and tended to have less rapid eye movement (REM) sleep (19 +/- 5 vs. 22 +/- 3%, P=0.094). CONCLUSION: Patients with RP have increased daytime sleepiness, reduced alertness and more disturbed nighttime sleep of poorer quality than their normally sighted counterparts, suggesting an influence of photoreceptor degeneration on the circadian cycle.     

Voltage-gated potassium currents in rat vas deferens smooth muscle cells

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca(2+)-dependent K(+) channels (BK(Ca)), reduced the current amplitude significantly. When BK(Ca) channels were suppressed, fast-inactivating (I(K,f)) and delayed rectifying (I(K,dr)) components of the outward current were identified. I(K,f) was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K(d) of 0.32 mM, properties similar to those of the A-type K(+) current. In contrast, I(K,dr) activated and inactivated at more positive potentials. The time constant of activation of I(K,dr) was voltage dependent with an e-fold decrease per 21 mV depolarization. I(K,dr) was inhibited by clofilium, a blocker of voltage-gated K(+) channels, with an IC(50) of 12 micro M and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.     

Ionic basis of the resting membrane potential and action potential in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of C. elegans is a rhythmically active muscle that pumps bacteria into the gut of the nematode. This activity is maintained by action potentials, which qualitatively bear a resemblance to vertebrate cardiac action potentials. Here, the ionic basis of the resting membrane potential and pharyngeal action potential has been characterized using intracellular recording techniques. The resting membrane potential is largely determined by a K(+) permeability, and a ouabain-sensitive, electrogenic pump. As previously suggested, the action potential is at least partly dependent on voltage-gated Ca(2+) channels, as the amplitude was increased as extracellular Ca(2+) was increased, and decreased by L-type Ca(2+) channel blockers verapamil and nifedipine. Barium caused a marked prolongation of action potential duration, suggesting that a calcium-activated K(+) current may contribute to repolarization. Most notably, however, we found that action potentials were abolished in the absence of external Na(+). This may be due, at least in part, to a Na(+)-dependent pacemaker potential. In addition, the persistence of action potentials in nominally free Ca(2+), the inhibition by Na(+) channel blockers procaine and quinidine, and the increase in action potential frequency caused by veratridine, a toxin that alters activation of voltage-gated Na(+) channels, point to the involvement of a voltage-gated Na(+) current. Voltage-clamp analysis is required for detailed characterization of this current, and this is in progress. Nonetheless, these observations are quite surprising in view of the lack of any obvious candidate genes for voltage-gated Na(+) channels in the C. elegans genome. It would therefore be informative to re-evaluate the data from these homology searches, with the aim of identifying the gene(s) conferring this Na(+), quinidine, and veratridine sensitivity to the pharynx.     

Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.     

Processing of influenza HA protein in MDCK cells: Components with different mobilities in polyacrylamide gel electrophoresis and their precursor-product relationships

Summary In influenza virus-infected MDCK cells labelled with¹⁴C-chlorella hydrolysate or³⁵S-methionine a virus-specific protein component is revealed migrating slightly faster than HA protein in polyacrylamide gel electrophoresis. Under chase conditions the component disappears either completely or partially, with a concomitant intensification of the HA band. The rate and extent of this transition are strain-dependent. Both the HA band and the faster moving component are not revealed if the cells are labelled in the presence of 20mM of D-glucosamine. In primary cell cultures of chick embryos a single HA band with a mobility similar to that of the faster moving component in MDCK cells has been observed. It is suggested that the transition of the label from the faster moving component to the HA band reflects the final step of HA processing specific for MDCK cells.With 7 Figures     

Muscle-epidermis interactions affect exoskeleton patterning in Caenorhabditis elegans.

The C. elegans hypodermis is a single epithelial cell layer separated from the musculature by a thin basement membrane on its basal surface. The hypodermis secretes the extracellular material of the cuticle from its apical surface. The regulation of cuticle synthesis and apical secretion is not well understood. UNC-95 is a component of the muscle dense bodies and M-lines, which are integrin-based adhesion complexes required for force transduction to the cuticle. Using gene expression profiling and in vivo assays, we show that, in unc-95 mutant worms, there is an increase in expression levels of a group of hypodermal and pharyngeal genes related to cuticle structure and molting. Moreover, the cuticle structure of unc-95 mutant adult is impaired. Our findings suggest that aberrant force transduction from the structurally impaired muscle attachments across the basement membrane to the underlying hypodermis elicits intercellular signaling that plays a role in regulating cuticle synthesis and patterning.     

Tetrasomy 12pter-12p13.31 in a girl with partial Pallister-Killian syndrome phenotype

A dysmorphic patient was shown to carry a small supernumerary marker chromosome. Multicolor, centromere-multicolor and regular FISH experiments proved the marker to be an analphoid 12pter derived isochromosome. Microdissection of the marker followed by reverse painting and array CGH analysis showed that the isochromosome contains approximately 6 Mb of 12pter-12p13.31 derived sequence. This is only the second report of a marker with a neocentromere 12pter and the molecular fine mapping of the duplicated region further refines the 12p region defining the Pallister-Killian syndrome phenotype. In addition, we show the feasibility of using microdissected chromosomes or chromosomal fragments to molecularly map the chromosomal breakpoints on array CGH. This technology may aid in the identification of chromosomal translocation breakpoints.     

Species-Specific Differences in Organization of Orthopoxvirus Kelch-Like Proteins

Organization of orthopoxvirus proteins of the kelch superfamily and their genes were analyzed and compared. Complete genomic sequences of variola (VAR), monkeypox (MPV), vaccinia (VAC), and species-specific regions of cowpox (CPV) viruses were used in the work. Despite the multiplicity of kelch-like proteins in orthopoxviruses, their function is still vague. It has been discovered that the genes of orthopoxvirus kelch-like proteins are localized only to the terminal variable regions of the genome and display species-specific differences in the lengths of the proteins they potentially encode. All the genes belonging to kelch superfamily in the genome of VAR, which has the only host–the man, are mutationally destroyed. However, CPV, displaying the widest host range among orthopoxviruses, encode the most numerous set of kelch-like proteins. Weak homologies between kelch-like proteins of one virus were demonstrated as well as high homologies between isologues of different orthopoxvirus species. The comparison performed suggest that CPV virus is most ancient and may be considered as the ancestor of other orthopoxviruses pathogenic for humans.