Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets.

Changes in cytosolic free calcium ([Ca2+]i) have been continuously imaged during the interaction of the H-2Kb specific cytotoxic T cell lymphocyte (CTL) BM 3.3, with either the H-2Kb EL4.BU or the H-2Kk RDM4 cell lines. Activation of the CTLs by EL4.BU raises [Ca2+]i to several hundred nanomolar in the CTL. Frequently [Ca2+]i is preferentially elevated in the region of the CTL furthest from the site of target contact. These responses require external Ca2+ suggesting that they are generated by the plasma membrane and not internal stores. Inappropriate targets such as RDM4 evoke no changes in [Ca2+]i. Activation of the BM 3.3 CTL is followed by increases of [Ca2+]i to several micromolar or higher in the EL4.BU targets. This massive increase can be mimicked by direct application of cytolytic granules isolated from rat natural killer cells. The increase in plasma membrane permeability is ion-specific since external Mn2+ can also readily enter target cells that have been 'hit', as evidenced by the rapid selective quenching of fura-2 in those targets. The flood of Ca2+ into the target cell is followed by a leakage of the trapped fura-2. Since both processes continue after the CTL has disengaged, they provide a useful assay for the lethal hit. Furthermore, this technique can be used to follow complete cycles of CTL activation and lethal hit delivery, which under some circumstances can be as rapid as 6 min per cycle.     

The molecular basis of potassium nutrition in plants

Over the last five years, the cloning and characterization of K⁺ transport genes corresponding to K⁺ channels (KAT1, AKT1, KST1, AKT2), associated subunits (KAB1) and a high-affinity transporter (HKT1) has opened up important new avenues for research on plant K⁺ nutrition. With the abundance of molecular data now available it seems timely to link this information with the wealth of data previously accumulated on the physiology of plant K⁺ acquisition. The ultimate goal of all this research is to gain a better understanding of K⁺ transport and nutrition in the intact plant. Thus it is important to begin to integrate the molecular research with results from biochemical and physiological research conducted at the cellular, root and whole plant levels. This article will focus on describing the features of the cloned K⁺ transporters and their possible roles in mediating high- and low-affinity K⁺ uptake from the soil, as well as how K⁺ acquisition may be regulated.Abbreviations NEM N-ethyl maleimide - PCMBS p-chloromercuribenzene sulphonic acid     

Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides

4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl-alpha-L-arabinofuranosides and of 2-, 3-, and 4-O-(E)-feruloyl- and 2-, 3- and 4-O-acetyl-beta-D-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl-alpha-L-arabinofuranoside. On the contrary, type-B and -C feruloyl esterases, represented by FoFaeI from F. oxysporum and TsFaeC from Talaromyces stipitatus, acted on almost all ferulates with exception of 4- and 3-O-feruloyl-beta-D-xylopyranoside. 5-O-Feruloyl-alpha-L-arabinofuranoside was the best substrate for both TsFaeC and FoFaeI, although catalytic efficiency of the latter enzyme toward 2-O-feruloyl-alpha-L-arabinofuranoside was comparable. In comparison with acetates, the corresponding ferulates served as poor substrates for the carbohydrate esterase family 1 feruloyl esterase from Aspergillus oryzae. The enzyme hydrolyzed all alpha-L-arabinofuranoside and beta-D-xylopyranoside acetates. It behaved as a non-specific acetyl esterase rather than a feruloyl esterase, with a preference for 2-O-acetyl-beta-D-xylopyranoside.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (～1/100 of the exciting wavelength).     

Pancreatic anticancer activity of a novel geranylgeranylated coumarin derivative

A series of hydroxycoumarin derivatives has been synthesized and evaluated against human pancreatic PANC-1 cancer cells under nutrient-deprived conditions. Several compounds exhibited 100% preferential cytotoxicity at low micromolar concentrations under nutrition starvation, and showed no cytotoxicity under nutrient-rich conditions. In this study, a novel geranylgeranylated ether coumarin derivative 9 was found to exhibit the highest cytotoxic activity of 6.25 μM within 24h. The preferential anti-tumor activity exhibited by compound 9 against PANC-1 under low oxygen and nutrient environment illustrates its great potential as a promising lead structure for the development of novel agents to combat pancreatic cancer.     

Biodegradation of low-molecular-weight halogenated hydrocarbons by methanotrophic bacteria

Low-molecular-weight halogenated hydrocarbons are susceptible to degradation by anaerobic and aerobic bacteria. The methanotrophic bacterium Methylosinus trichosporium 0B3b degrades trichloroethylene more rapidly than other bacteria examined to date. Expression of soluble methane monooxygenase (MMO) is correlated with high rates of biodegradation. An analysis of 16 S rRNA sequences of 11 ribosomal RNAs from type I, type II and type X methanotrophs and methanol-utilizing bacteria have revealed four clusters of phylogenetically related methylotrophs. This information may be useful for the identification and enumeration of methylotrophs in bioreactors and other environments during remediation of contaminated waters.     

Marine actinomycetes as a source of novel secondary metabolites

A set of 600 actinomycetes strains which were isolated from marine sediments from various sites in the Pacific and Atlantic Oceans were screened for the production of bioactive secondary metabolites. Marine streptomycete strains were found to be producers of well known chemically diverse antibiotics isolated from terrestrial streptomycetes, as in the case of marine Micromonospora strains. New marine members of the rare genus Verrucosispora seem to be a promising source for novel bioactive secondary metabolites as shown in the case of the abyssomicin producing strain AB-18-032.     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.