Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.     

Antibacterial Activity of Long-Chain Primary Alcohols from <Emphasis Type="Italic">Solena amplexicaulis</Emphasis> Leaves

Extraction, thin layer chromatography and gas chromatography–mass spectrometry of Solena amplexicaulis (Lam.) Gandhi, commonly known as creeping cucumber, (Cucurbitaceae) leaves revealed 21 long-chain primary alcohols, and 100 g leaves indicated presence of 3651.59 ± 327.18 SE µg long-chain primary alcohols. 1-Heptadecanol and 1-triacontanol were the predominant and least abundant primary alcohols, representing for 780.44 ± 42.59 and 3.28 ± 0.55 SE μg, respectively. Antibacterial property of the complete synthetic blend (0.1%), comparable to long-chain alcohols as detected by GC-FID of 100 g S. amplexicaulis leaf extracts was evaluated on the pathogenic bacteria Salmonella gallinarum by agar well diffusion method, and exhibited 20.4, 26.7 and 38.2 mm zone of inhibition at 25, 50 and 100 μl doses, respectively. One hundred µl dose of 6 individual pure synthetic compounds, 1-tridecanol, 1-pentadecanol, 1-heptadecanol, 1-nonadecanol, 1-eicosanol and 1-tricosanol comparable to the amounts present in 0.1% solution of pure isolated alcohols from S. amplexicaulis leaves displayed 16.2, 17.7, 18.6, 22.8, 15.8 and 14.5 mm zone of inhibition against this bacterium, respectively. Hundred µl dose from a synthetic blend of above 6 compounds (comparable to the proportions as present in 0.1% solution of pure isolated alcohols from 100 g S. amplexicaulis leaves) exhibited 38.1 mm zone of inhibition against this bacterium. Furthermore, 100 μl dose from a mixture (1:1) comprising of chloramphenicol (1 µg/ml) and a synthetic blend of above 6 compounds displayed 38.8 mm inhibition zone against S. gallinarum, and hence, this combination might be used against this pathogenic bacteria.     

Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle.

Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBS(Thr695)) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBS(Thr695) of MBP and caused MBP activation. Preexposure to N(G)-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBS(Thr695) phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBS(Thr695). Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBS(Thr695) phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.     

Diploid/polyploid syntenic shuttle mapping and haplotype-specific chromosome walking toward a rust resistance gene (Bru1) in highly polyploid sugarcane (2n approximately 12x approximately 115)

The genome of modern sugarcane cultivars is highly polyploid ( approximately 12x), aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. Here we report on the first attempt to isolate a gene from sugarcane by map-based cloning, targeting a durable major rust resistance gene (Bru1). We describe the genomic strategies that we have developed to overcome constraints associated with high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13 markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. These approaches will pave the way for the development of future map-based cloning approaches for sugarcane and other complex polyploid species.     

Zinc,Manganese, Calcium,Copper, and Cadmium Level in Scalp Hair Samples of Schizophrenic Patients

The purpose of the study was to determine the concentration of trace elements present in scalp hair sample of schizophrenic patients and to find out the relationship between trace elements level and nutritional status or socioeconomic factors. The study was conducted among 30 schizophrenic male patients and 30 healthy male volunteers. Patients were recruited from Bangabandhu Sheikh Mujib Medical University by random sampling. Hair trace element concentrations were determined by flame atomic absorption spectroscopy and analyzed by independent t test, Pearson’s correlation analysis, regression analysis, and analysis of variance (ANOVA). Mn, Zn, Ca, Cu, and Cd concentrations of schizophrenic patients were 3.8 ± 2.31 μg/gm, 171.6 ± 59.04 μg/gm, 396.23 ± 157.83 μg/gm, 15.40 ± 5.68 μg/gm, and 1.14 ± 0.89 μg/gm of hair sample, while those of control subjects were 4.4 ± 2.32 μg/gm, 199.16 ± 27.85 μg/gm, 620.9 ± 181.55 μg/gm, 12.23 ± 4.56 μg/gm, and 0.47 ± 0.32 μg/gm of hair sample, respectively. The hair concentration of Zn and Ca decreased significantly (p = 0.024; p = 0.000, respectively) and the concentration of Cu and Cd increased significantly (p = 0.021; p = 0.000, respectively) in schizophrenic patients while the concentration of Mn (p = 0.321) remain unchanged. Socioeconomic data reveals that most of the patients were poor, middle-aged and divorced. Mean body mass indices (BMIs) of the control group (22.26 ± 1.91 kg/m²) and the patient group (20.42 ± 3.16 kg/m²) were within the normal range (18.5−25.0 kg/m²). Pearson’s correlation analysis suggested that only Ca concentration of patients had a significant positive correlation with the BMI (r = 0.597; p = 0.000) which was further justified from the regression analysis (R ² = 44%; t = 3.59; p = 0.002) and one-way ANOVA test (F = 3.62; p = 0.015). A significant decrease in the hair concentration of Zn and Ca as well as a significant increase in the hair concentration of Cu and Cd in schizophrenic patients than that of its control group was observed which may provide prognostic tool for the diagnosis and treatment of this disease. However, further work with larger population is suggested to examine the exact correlation between trace element level and the degree of disorder.     

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

Background

Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number {"type":"entrez-protein","attrs":{"text":"Q8BGW0","term_id":"81896053","term_text":"Q8BGW0"}}Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 ({"type":"entrez-protein","attrs":{"text":"Q91YX0","term_id":"73920022","term_text":"Q91YX0"}}Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.

Methodology/Principal Findings

Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase ({"type":"entrez-protein","attrs":{"text":"P25911","term_id":"2507208","term_text":"P25911"}}P25911), the Rho guanine nucleotide exchange factor, Vav ({"type":"entrez-protein","attrs":{"text":"P27870","term_id":"137483","term_text":"P27870"}}P27870), and the adaptor protein Grb2 ({"type":"entrez-protein","attrs":{"text":"Q60631","term_id":"2498425","term_text":"Q60631"}}Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.

Conclusions/Significance

We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.     

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (−)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.     

An evolutionarily conserved TNF-alpha-responsive enhancer in the far upstream region of human CCL2 locus influences its gene expression

Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.