Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

Surface Ig isotypes on cells responding to lipopolysaccharide by IgM and IgG secretion.

Using the fluorescence-activated cell sorter (FACS), we have investigated the Ig isotypes present on murine B cells, which can be polyclonally activated by lipopolysaccharide (LPS) in low cell density cultures. The LPS response was partly inhibited as a result of staining with anti-IgD and anti-IgM reagents, but not with anti-IgG reagents. The IgM+, IgD+, or IgG- fractionated cell populations gave both an IgM and an IgG response comparable to controls, whereas the response of the IgM-, IgD- cells was 5- to 20-fold lower. IgG- cells separated 1 day after LPS stimulation could still mount an IgM and IgG response indistinguishable from controls at the peak of the response. It is concluded that IgM+, IgD+, IgG- cells constitute the major LPS-sensitive cell population in the low cell density culture system and that IgG is not a necessary cell surface isotype for precursors of IgG-secreting cells.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Reversal of the effects of a night interruption in lemna by inhibitors of ribonucleic Acid synthesis

The inhibition of flowering of Lemna perpusilla Torr. strain 6746 caused by a light break can be partially reversed by treatment with actinomycin D or 2-thiouracil. Actinomycin D is most active in reversing the response to a light break if the inhibitor is present in the fronds at the time the light break is administered.     

A New Insight into Structural and Functional Impact of Single-Nucleotide Polymorphisms in PTEN Gene

Phosphatase and tensin homolog (PTEN) plays essential roles in cellular processes including survival, proliferation, energy metabolism, and cellular architecture. Activating the mutations of PTEN has long been known to produce a variety of disorders, mainly diabetes and cancer in humans. Owing to the importance of PTEN gene, a functional analysis using different in silico approaches was undertaken to explore the possible associations between genetic mutations and phenotypic variation. SIFT, PolyPhen, I-Mutant 3.0, SNP&GO, and PHD-SNP were used for initial screening of functional nsSNPs. From the observed results, three mutations R47G, H61D, and V343E were selected based on their surface accessibility and total energy change. By molecular dynamics approach, H61D showed increase in flexibility, radius of gyration, solvent accessibility, and deviated more from the native structure which was supported by the decrease in the number of hydrogen bonds. Further from principal component analysis and interaction analysis, we identified significant structural changes that can reasonably explain the involvement of deviations in stability caused by mutations. Our analysis also predicts the involvement of SNPs that could potentially influence post-translational modifications in PTEN gene. These in silico predictions could provide a new insight into structural and functional impact of PTEN polymorphisms.     

In Silico Analysis of Prion Protein Mutants: A Comparative Study by Molecular Dynamics Approach

Polymorphisms in the human prion proteins lead to amino acid substitutions by the conversion of PrPC to PrPSc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt–Jakob disease, Gerstmann–Straussler–Scheinker disease and fatal familial insomnia. Cation–π interaction is a non-covalent binding force that plays a significant role in protein stability. Here, we employ a novel approach by combining various in silico tools along with molecular dynamics simulation to provide structural and functional insight into the effect of mutation on the stability and activity of mutant prion proteins. We have investigated impressions of prevalent mutations including 1E1S, 1E1P, 1E1U, 1E1P, 1FKC and 2K1D on the human prion proteins and compared them with wild type. Structural analyses of the models were performed with the aid of molecular dynamics simulation methods. According to our results, frequently occurred mutations were observed in conserved sequences of human prion proteins and the most fluctuation values appear in the 2K1D mutant model at around helix 4 with residues ranging from 190 to 194. Our observations in this study could help to further understand the structural stability of prion proteins.     

Disruption of Mitochondrial Complexes in Cancer Stem Cells Through Nano-based Drug Delivery: A Promising Mitochondrial Medicine

Mitochondria are the fulcrum for regulating cellular metabolism as well as apoptosis. The multi-lamellar vesicles (MLVs) liposome targeted against mitochondria can be formulated to disrupt mitochondrial integrity to attain programmed cell death of cancer stem cells (CSCs). The gold nanoparticles (GNPs) and a steroid nucleus (cyclopentanoperhydrophenanthrene ring) are encapsulated within MLV liposome that targets specifically to the CD44 receptor of the CSCs. Entering cytosol, it would bind distinctively to the malate–aspartate shuttle through a specifically designed ligand. Liposome fuses with the mito-membrane after associating with shuttle, thereby releasing both the components. The steroid disrupts mito-membrane’s integrity facilitating release of cytochrome c. Thus, GNPs enter into the mitosol and interact with the mitochondrial complexes to cease cellular respiration. Since the solid nano-based pharmaceutics has shown a lot of promises as a potent anticancer therapy, the role of MLV liposome can be proved to be a better weapon to terminate malignancy.     

In silico discrimination of nsSNPs in hTERT gene by means of local DNA sequence context and regularity

Understanding and predicting the significance of novel genetic variants revealed by DNA sequencing is a major challenge to integrate and interpret in medical genetics with medical practice. Recent studies have afforded significant advances in characterization and predicting the association of single nucleotide polymorphisms in human TERT with various disorders, but the results remain inconclusive. In this context, a comparative study between disease causing and novel mutations in hTERT gene was performed computationally. Out of 59 missense mutations, five variants were predicted to be less stable with the most deleterious effect on hTERT gene by in silico tools, in which two mutations (L584W and M970T) were not previously reported to be involved in any of the human disorders. To get insight into the structural and functional impact due to the mutation, docking study and interaction analysis was performed followed by 6 ns molecular dynamics simulation. These results may provide new perspectives for the targeted drug discovery in the coming future.