Human Asunder promotes dynein recruitment and centrosomal tethering to the nucleus at mitotic entry

Recruitment of dynein motors to the nuclear surface is an essential step for nucleus–centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2–Bicaudal D2 (BICD2) and Nup133– centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus–centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus–centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.     

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.     

Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide.

The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S-S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

Myeloid‐specific GPCR kinase‐2 negatively regulates NF‐κB1p105‐ERK pathway and limits endotoxemic shock in mice

G‐protein‐coupled receptor kinase 2 (GRK2) is a member of a kinase family originally discovered for its role in the phosphorylation and desensitization of G‐protein‐coupled receptors. It is expressed in high levels in myeloid cells and its levels are altered in many inflammatory disorders including sepsis. To address the physiological role of myeloid cell‐specific GRK2 in inflammation, we generated mice bearing GRK2 deletion in myeloid cells (GRK2^?mye). GRK2^?mye mice exhibited exaggerated inflammatory cytokine/chemokine production, and organ injury in response to lipopolysaccharide (LPS, a TLR4 ligand) when compared to wild‐type littermates (GRK2^fl/fl). Consistent with this, peritoneal macrophages from GRK2^?mye mice showed enhanced inflammatory cytokine levels when stimulated with LPS. Our results further identify TLR4‐induced NF‐κB1p105‐ERK pathway to be selectively regulated by GRK2. LPS‐induced activation of NF‐κB1p105‐MEK‐ERK pathway is significantly enhanced in the GRK2^?mye macrophages compared to GRK2^fl/fl cells and importantly, inhibition of the p105 and ERK pathways in the GRK2^?mye macrophages, limits the enhanced production of LPS‐induced cytokines/chemokines. Taken together, our studies reveal previously undescribed negative regulatory role for GRK2 in TLR4‐induced p105‐ERK pathway as well as in the consequent inflammatory cytokine/chemokine production and endotoxemia in mice. J. Cell. Physiol. 226: 627–637, 2011. © 2010 Wiley‐Liss, Inc.     

Self-Regulation of Anterior Insula with Real-Time fMRI and Its Behavioral Effects in Obsessive-Compulsive Disorder: A Feasibility Study

Introduction

Obsessive-compulsive disorder (OCD) is a common and chronic condition that can have disabling effects throughout the patient''s lifespan. Frequent symptoms among OCD patients include fear of contamination and washing compulsions. Several studies have shown a link between contamination fears, disgust over-reactivity, and insula activation in OCD. In concordance with the role of insula in disgust processing, new neural models based on neuroimaging studies suggest that abnormally high activations of insula could be implicated in OCD psychopathology, at least in the subgroup of patients with contamination fears and washing compulsions.

Methods

In the current study, we used a Brain Computer Interface (BCI) based on real-time functional magnetic resonance imaging (rtfMRI) to aid OCD patients to achieve down-regulation of the Blood Oxygenation Level Dependent (BOLD) signal in anterior insula. Our first aim was to investigate whether patients with contamination obsessions and washing compulsions can learn to volitionally decrease (down-regulate) activity in the insula in the presence of disgust/anxiety provoking stimuli. Our second aim was to evaluate the effect of down-regulation on clinical, behavioural and physiological changes pertaining to OCD symptoms. Hence, several pre- and post-training measures were performed, i.e., confronting the patient with a disgust/anxiety inducing real-world object (Ecological Disgust Test), and subjective rating and physiological responses (heart rate, skin conductance level) of disgust towards provoking pictures.

Results

Results of this pilot study, performed in 3 patients (2 females), show that OCD patients can gain self-control of the BOLD activity of insula, albeit to different degrees. In two patients positive changes in behaviour in the EDT were observed following the rtfMRI trainings. Behavioural changes were also confirmed by reductions in the negative valence and in the subjective perception of disgust towards symptom provoking images.

Conclusion

Although preliminary, results of this study confirmed that insula down-regulation is possible in patients suffering from OCD, and that volitional decreases of insula activation could be used for symptom alleviation in this disorder.     

Identification of the region that plays an important role in determining antibacterial activity of bovine seminalplasmin

Seminalplasmin (SPLN) is a 47-residue protein isolated from bovine seminal plasma having potent antimicrobial activity against a broad spectrum of microorganisms. SPLN, also known as caltrin, acts as a calcium transport regulator in bovine sperms. Analysis of the sequence of SPLN reveals a 27-residue stretch with the sequence SLSRYAKLANRLANPKLLETFLSKWIG more hydrophobic than the rest of the protein. It is demonstrated that a synthetic peptide corresponding to this 27-residue segment has antimicrobial activity comparable to that of SPLN. It does not exhibit hemolytic activity at concentrations where antibacterial activity is observed. Since P27 can be conveniently obtained in large amounts by chemical synthesis, it could serve not only as a starting compound to obtain peptides with improved antibacterial activity but also to understand the role of SPLN in reproductive physiology.     

The Effect of Inkjet Printing over Polymeric Films as Potential Buccal Biologics Delivery Systems

The buccal mucosa appears as a promissory route for biologic drug administration, and pharmaceutical films are flexible dosage forms that can be used in the buccal mucosa as drug delivery systems for either a local or systemic effect. Recently, thin films have been used as printing substrates to manufacture these dosage forms by inkjet printing. As such, it is necessary to investigate the effects of printing biologics on films as substrates in terms of their physical and mucoadhesive properties. Here, we explored solvent casting as a conventional method with two biocompatible polymers, hydroxypropyl methylcellulose, and chitosan, and we used electrospinning process as an electrospun film fabrication of polycaprolactone fibers due to its potential to elicit mucoadhesion. Lysozyme was used as biologic drug model and was formulated as a solution for printing by thermal inkjet printing. Films were characterized before and after printing by mechanical and mucoadhesive properties, surface, and ultrastructure morphology through scanning electron microscopy and solid state properties by thermal analysis. Although minor differences were detected in micrographs and thermograms in all polymeric films tested, neither mechanical nor mucoadhesive properties were affected by these differences. Thus, biologic drug printing on films was successful without affecting their mechanical or mucoadhesive properties. These results open way to explore biologics loading on buccal films by inkjet printing, and future efforts will include further in vitro and in vivo evaluations.     

Surface Thermodynamics of Mucoadhesive Dry Powder Formulation of Zolmitriptan

Microparticle powders for nasal delivery were formulated to contain the model drug, zolmitriptan, and varying proportions of different polymers. The objective of the study was to investigate the effects of these formulative parameters on the surface chemistry of the spray-dried microparticles and their potential for adhesion to the tested substrates, porcine mucin, and nasal tissue. The polymers used were chitosans of varying ionization states and molecular weights and hydroxypropyl methyl cellulose. The surface energies of the surfaces of the microparticles were determined using contact angle measurements and the van Oss model. The theory of surface thermodynamics was applied to determine the theoretical potential for the different materials to adhere to the substrates. It was found that the drug or polymers alone, as well as the various formulations, were more likely to adhere to mucin than to nasal tissue. Further, there was a trend for higher molecular weight chitosans to adhere better to the substrates than lower molecular weight chitosans. Similarly, adhesion was improved for formulations with a higher content of polymers. These theoretical predictions may be compared with further experimental results and be of use in making informed decisions on the choice of formulations for future expensive bio-studies.     

Choline: Selective enhancement of serial learning and encoding of low imagery words in man

The effect of a single oral dose of choline chloride (10g) on two kinds of memory measures (serial learning and a selective reminding task) was studied in 10 normal volunteers. Choline decreased significantly the number of trials required to master a serial learning word test. On the selective reminding task only the recall of poorly imageable words (which are ordinarily difficult to remember) was enhanced by choline. Words which were highly imageable (and ordinarily easy to remember) were unaffected. The degree of enhancement with choline correlated inversely with performance of subjects on placebo. Our data indicate that brain acetylcholine may play a facilitatory role in encoding and storage of specific information items in man.