CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes

Background

CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes.

Results

We now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [³H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization.

Conclusions

These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease.     

Genetics of Antimicrobial Resistance

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.     

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.     

Age and Sex Dependent Alteration in Presenilin Expression in Mouse Cerebral Cortex

(1) Presenilin (PS) expression is regulated by several cellular and extracellular factors which change with age and sex. Both age and sex are key risk factors for Alzheimer’s disease (AD), which is linked to mutations in PS genes. (2) We have analyzed the effect of age and sex on PS expression by northern hybridization and western blot analysis using the cerebral cortex of adult (24 ± 2 weeks) and old (65 ± 5 weeks) mice. (3) Our results demonstrate that PS1 was downregulated and PS 2 was upregulated in old mice of both sexes. The level of PS 1 was relatively higher and that of PS 2 was lower in female than male mice of same age group. Taken together, these findings show age and sex dependent alteration in PS expression, which in turn may influence the signal transduction pathways and consequently brain functions.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: Lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Micropropagation and germplasm conservation of Central Park Splendor Chinese elm (<Emphasis Type="Italic">Ulmus parvifolia</Emphasis> Jacq. ‘A/Ross Central Park’) trees

Micropropagation offers opportunities to propagate, preserve and ship tree germplasm. It also reduces the risk of moving pathogens and insects with the germplasm due to built-in pathogen detection capabilities of aseptic cultures. For the past few decades, our laboratory has been involved in a project to preserve and restore a large, cold hardy, and historically important Chinese elm (Ulmus parvifolia Jacq. ‘A/Ross Central Park’) tree. Here we present three simple and efficient systems for its micropropagation, germplasm conservation and distribution: (1) in vitro plant formation from meristematic nodules (MNs), (2) plantlet generation from axillary buds, and (3) in vitro rooting of micro-cuttings from 20-years-old hedged stock plants. Newly flushed nodal segments were used as explants. WPM with 0.5 mg/l BA was found to be the best medium for meristematic shoot development and WPM supplemented with 2.0 mg/l 4-CPPU and 0.5 mg/l TDZ was best for meristematic nodule formation. Rhizogenesis of regenerants and micro-cuttings was best achieved on WPM with 1.0 mg/l NAA and 2% sucrose. Rooted plants were readily acclimatized to the greenhouse ambient environment and continued to grow well under greenhouse conditions. The survival rate of acclimatized plantlets under ex vitro conditions was 100% after 4 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 1,000 plants. Cycling of shoot explants and MNs through repetitive cultures was effective in scaling-up propagules.