Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.     

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.     

In silico mutation on a mutant lipase from Acinetobacter haemolyticus towards enhancing alkaline stability

Abstract

Alkaline-stable lipases are highly valuable biocatalysts that catalyze reactions under highly basic conditions. Herein, computational predictions of lipase from Acinetobacter haemolyticus and its mutant, Mut-LipKV1 was performed to identify functionally relevant mutations that enhance pH performance under increasing basicity. Mut-LipKV1 was constructed by in silico site directed mutagenesis of several outer loop acidic residues, aspartic acid (Asp) into basic ones, lysine (Lys) at positions 51, 122 and 247, followed by simulation under extreme pH conditions (pH 8.0–pH 12.0). The energy minimized Mut-LipKV1 model exhibited good quality as shown by PROCHECK, ERRAT and Verify3D data that corresponded to 79.2, 88.82 and 89.42% in comparison to 75.2, 86.15, and 95.19% in the wild-type. Electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0???10.0). Mut-LipKV1 exhibited a mixture of neutral and positive surface charge distribution compared to the predominantly negative charge in the wild-type lipase at pH 8.0. Data of molecular dynamics simulations also supported the increased alkaline-stability of Mut-LipKV1, wherein the lipase was more stable at a higher pH 9.0 (RMSD = ～0.3?nm, RMSF = ～0.05–0.2?nm), over the optimal pH 8.0 of the wild-type lipase (RMSD = 0.3?nm, RMSF = 0.05–0.20?nm). Thus, the adaptive strategy of replacing surface aspartic acid to lysine in lipase was successful in yielding a more alkaline-stable Mut-LipKV1 under elevated basic conditions.

Communicated by Ramaswamy H. Sarma     

Non-isotopic RNA probes

Summary Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive ³⁵S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.     

Inappropriate Shock Delivered By Implantable Cardioverter Defibrillator - Cardiac Resynchronization Therapy (ICD-CRT) Due To Myopotential Oversensing

The clinical efficacy of ICD-CRT therapy depends on accurate sensing of intracardiac signals and sensing algorithms. We report the occurrence of sensing abnormality in a patient with ICD-CRT. In this patient, oversensing of myopotentials during strenuous muscular activity resulted in an inappropriate ICD-CRT discharge. Although modern ICDs are highly effective in detecting and terminating malignant tachyarrhythmias, their detection specificity must be improved. It is possible to find the mechanism of arrhythmia by EGM. Simple device reprogramming make it possible to avoid the oversensing of myopotentials.     

Some studies of the quality of irrigation water and the germination and growth of wheat at different concentrations of boron

Summary The irrigation water in use at the Punjab University New Campus is of good quality except the water of one tubewell which is relatively poor (high-salinity and low-sodium water) and has 0.45 ppm boron. Water with a boron content of upto 3.00 ppm did not affect the germination of the seeds of wheat (Triticum vulgare L. var. Mexi-Pak) and there was some stimulation of the growth of seedlings by an increase in the concentration of boron beyond 0.45 ppm. However, these seedlings, when transplanted to the soils supplied with waters containing different amounts of boron, showed a decrease in their growth at higher concentrations of boron with their ageing, and when these plants were about 100 days old and almost mature, their growth decreased significantly with an increase in the concentration of boron to 2.00 or 3.00 ppm in the water supplied to them. The growth of the plants of Mexi-Pak wheat is inversely related to the boron content of their tissues when it is more than 0.60 ppm. Mexi-Pak wheat is semi-tolerant to boron. re]19750605