Reversible cell cycle inhibition and premature aging features imposed by conditional expression of p16Ink4a

The cyclin‐dependent kinase (Cdk) inhibitor p16^Ink4a (p16) is a canonical mediator of cellular senescence and accumulates in aging tissues, where it constrains proliferation of some progenitor cells. However, whether p16 induction in tissues is sufficient to inhibit cell proliferation, mediate senescence, and/or impose aging features has remained unclear. To address these issues, we generated transgenic mice that permit conditional p16 expression. Broad induction at weaning inhibited proliferation of intestinal transit‐amplifying and Lgr5+ stem cells and rapidly imposed features of aging, including hair loss, skin wrinkling, reduced body weight and subcutaneous fat, an increased myeloid fraction in peripheral blood, poor dentition, and cataracts. Aging features were observed with multiple combinations of p16 transgenes and transactivators and were largely abrogated by a germline Cdk4 R24C mutation, confirming that they reflect Cdk inhibition. Senescence markers were not found, and de‐induction of p16, even after weeks of sustained expression, allowed rapid recovery of intestinal cell proliferation and reversal of aging features in most mice. These results suggest that p16‐mediated inhibition of Cdk activity is sufficient to inhibit cell proliferation and impose aging features in somatic tissues of mammals and that at least some of these aging features are reversible.     

Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-β-boswellic acid from <Emphasis Type="Italic">Boswellia serrata</Emphasis>

Background  

Boswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-β-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE) and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 g/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 g/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.     

Spatial distribution of lead in enamel and coronal dentine of wistar rats

Lead is one of the most hazardous environmental toxins known. The assessment of lead in dental hard tissues is important in the understanding of its toxic effects on oral tissues and in estimating exposure and body burden in individuals exposed to lead from the environment. However, current information on the uptake and distribution of lead in enamel and dentine is limited. The aim of this project was to study, at high resolution, the spatial distribution of lead in enamel and coronal dentine using an experimental rat model. A dose of 40 mg/L of lead nitrate was administered to pregnant female rats during the periods of gestation and lactation through drinking water. First mandibular molar teeth were removed from their 15-d-old pups and the distribution of lead was studied using a nuclear microprobe (NMP). The distribution of lead in enamel and coronal dentine showed four distinct zones with significantly different mean lead concentrations (p<0.05). High levels of lead were observed in the superficial regions of enamel and in the dentine directly adjacent to the pulp. Additionally, the results confirmed that the NMP is capable of mapping the distribution of lead in teeth at micron resolutions with a detection limit of approx 1 microg/g.     

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat.     

Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the 'piperazinyl-phenyl-oxazolidinone' core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.     

N-[(3S)-1-benzylpyrrolidin-3-yl]-(2-thienyl)benzamides: human dopamine D4 ligands with high affinity for the 5-HT2A receptor

A series of N-[(3S)-1-benzylpyrrolidin-3-yl]-(2-thienyl)benzamides 8 has been prepared and found to bind with high affinity to the human D(4) (hD(4)) and 5-HT(2A) receptors. Several compounds displayed selectivity for these receptors versus hD(2) and alpha(1) adrenergic receptors of over 500-fold.