Recovery of Aging-Related Size Increase of Skin Epithelial Cells: In vivo Mouse and In vitro Human Study

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20–40% for cells of older passage (6–8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.     

Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.     

Proton Pump Inhibitor Usage and the Risk of Myocardial Infarction in the General Population

Background and Aims

Proton pump inhibitors (PPIs) have been associated with adverse clinical outcomes amongst clopidogrel users after an acute coronary syndrome. Recent pre-clinical results suggest that this risk might extend to subjects without any prior history of cardiovascular disease. We explore this potential risk in the general population via data-mining approaches.

Methods

Using a novel approach for mining clinical data for pharmacovigilance, we queried over 16 million clinical documents on 2.9 million individuals to examine whether PPI usage was associated with cardiovascular risk in the general population.

Results

In multiple data sources, we found gastroesophageal reflux disease (GERD) patients exposed to PPIs to have a 1.16 fold increased association (95% CI 1.09–1.24) with myocardial infarction (MI). Survival analysis in a prospective cohort found a two-fold (HR = 2.00; 95% CI 1.07–3.78; P = 0.031) increase in association with cardiovascular mortality. We found that this association exists regardless of clopidogrel use. We also found that H₂ blockers, an alternate treatment for GERD, were not associated with increased cardiovascular risk; had they been in place, such pharmacovigilance algorithms could have flagged this risk as early as the year 2000.

Conclusions

Consistent with our pre-clinical findings that PPIs may adversely impact vascular function, our data-mining study supports the association of PPI exposure with risk for MI in the general population. These data provide an example of how a combination of experimental studies and data-mining approaches can be applied to prioritize drug safety signals for further investigation.     

A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1

Background

Pandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. However, some subtype A and C viruses exhibit an unusual gene arrangement in which the first exon of rev (rev1) and the vpu gene are placed in the same open reading frame. Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance is unknown.

Results

Examining infectious molecular clones (IMCs) of HIV-1 that encode the rev1-vpu polymorphism, we show that a fusion protein is expressed in infected cells. Due to the splicing pattern of viral mRNA, however, these same IMCs also express a regular Vpu protein, which is produced at much higher levels. To investigate the function of the fusion gene, we characterized isogenic IMC pairs differing only in their ability to express a Rev1-Vpu protein. Analysis in transfected HEK293T and infected CD4+ T cells showed that all of these viruses were equally active in known Vpu functions, such as down-modulation of CD4 or counteraction of tetherin. Furthermore, the polymorphism did not affect Vpu-mediated inhibition of NF-кB activation or Rev-dependent nuclear export of incompletely spliced viral mRNAs. There was also no evidence for enhanced replication of Rev1-Vpu expressing viruses in primary PBMCs or ex vivo infected human lymphoid tissues. Finally, the frequency of HIV-1 quasispecies members that encoded a rev1-vpu fusion gene did not change in HIV-1 infected individuals over time.

Conclusions

Expression of a rev1-vpu fusion gene does not affect regular Rev and Vpu functions or alter HIV-1 replication in primary target cells. Since there is no evidence for increased replication fitness of rev1-vpu encoding viruses, this polymorphism likely emerged in the context of other mutations within and/or outside the rev1-vpu intergenic region, and may have a neutral phenotype.     

Nocardia bhagyanesis sp. nov., a novel actinomycete isolated from the rhizosphere of Callistemon citrinus (Curtis), India

A novel actinomycete strain, designated VRC07^T, was isolated from a Callistemon citrinus rhizosphere sample collected from Hyderabad, India. Its taxonomic status was determined by using polyphasic approach. It is a Gram-positive, aerobic, non-motile, weakly acid-fast strain. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain VRC07^T is a member of the genus Nocardia. The highest levels of 16S rRNA gene sequence similarity was found between the strains Nocardia niwae W9241^T (99.6 %), Nocardia amikacinitolerans W9988^T (99.3 %) and Nocardia arthritidis IFM 10035^T (98.9 %); similarity to other type strains of the genus Nocardia was below 98.7 %. The organism had chemical and morphological features consistent with its classification in the genus Nocardia such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan. Arabinose and galactose as the diagnostic sugars. Diagnostic polar lipids were phosphatidylinositol, diphosphatidylglycerol, and phosphatidylglycerol. The predominant menaquinone was MK-8(H_{4, ω-cycl}). The major fatty acids were C_16:0, C_18:0, C_{18:1 w9c}, C_{18:0 10-methyl TBSA} and sum in feature 3 (16:1 w7c/16:1 w6c). The G+C content of the genomic DNA was 68.5 mol%. The DNA–DNA relatedness data, together with phenotypic differences clearly distinguished the isolate from its closest relatives. On the basis of these phenotypic and genotypic data, the isolate represents a novel species, for which the name Nocardia bhagyanesis sp. nov., is proposed. The type strain is VRC07^T (=KCTC 29209^T = MTCC 11725^T = ATCC BAA-2548).     

Pathogenesis-related genes and proteins in forest tree species

Trees occupy more than 30% of the land biosphere. They are important from both ecological and environmental standpoints and provide some of the most valuable commodities in the world economy. The perennial nature and size of trees are the critical determinants of their survival in response to biotic and abiotic stresses. The identification of the defense pathways at biochemical and genetic levels in tree pathosystems are beginning to be addressed. The basic physiological and biochemical mechanisms in woody perennials in response to pathogen is homologous to the model annual crop like Arabidopsis, but their secondary metabolic processes and ecological survival strategies are likely to be divergent from their annual counterparts. The limited domestication in tree species makes its molecular mechanisms less comparable to the highly pedigreed crop species. Recent reports have highlighted that the possible difference in genetic programs responding to invasive pathogens between annuals and perennials could be the spatial and temporal pattern of gene regulation. Several reviews on pathogen defense with reference to crop species are available, while similar reports from the tree species are limited to few commercially important species like Populus, Pinus, Picea, Eucalyptus, Castanea, and Pseudotsuga. This paper reviews the present status of pathogenesis-related genes and proteins from tree species with emphasis on the resistant genes and the proteins induced during systemic acquired resistance and highlights the ecological and evolutionary significance of defense-related genes from tree species.     

2,6-Dithienyl-4-furyl pyridines: Synthesis,topoisomerase I and II inhibition,cytotoxicity, structure–activity relationship,and docking study

For the development of novel antitumor agents, 2,6-dithienyl-4-furyl pyridine derivatives were prepared and evaluated for their topoisomerase I and II inhibitory activity as well as cytotoxicity against several human cancer cell lines. Among the 21 prepared compounds, compound 24 exhibited strong topoisomerase I inhibitory activity. In addition, a docking study with topoisomerase I and compound 24 was performed.