K+ Efflux and Retention in Response to NaCl Stress Do Not Predict Salt Tolerance in Contrasting Genotypes of Rice (Oryza sativa L.)

Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K⁺) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world’s most important and salt-sensitive crop species. Using efflux analysis with ⁴²K, coupled with growth and tissue K⁺ analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K⁺ set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K⁺ release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K⁺ status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na⁺ accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K⁺ efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K⁺ status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na⁺ accumulation is the key factor in performance decline on NaCl stress.     

Role of Cysteine Residues in Human Plasma Phospholipid Transfer Protein

Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys₁₃₅ and Cys₁₇₅; these residues, which correspond to Cys₁₂₉ and Cys₁₆₈ in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV·SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys₁₂₉ Gly and PLTP Cys₁₆₈ Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.     

Application of isotopic ratio mass spectrometry for the in vitro determination of demethylation activity in human liver microsomes using N-methyl-13C-labeled substrates

The reaction of demethylation mediated by cytochrome P450 (CYP) leads to the equimolar production of demethylated metabolite and formaldehyde. From a 13C-substrate labeled on a carbon of the methyl moiety, [13C]formaldehyde (H13CHO) is liberated. A highly sensitive and specific assay involving the oxidation of H13CHO to 13CO(2) by a double-enzymatic-step reaction is reported. The 13CO(2) was quantified by the method of reverse isotopic dilution based on gas chromatography-isotope ratio mass spectrometry analysis. The method first involves the limiting step of the CYP-dependent reaction, which is stopped with a mixture of zinc sulfate 5 mM and trichloroacetic acid 100 mM. Then, the transformation of H13CHO to 13CO(2) is performed with the formaldehyde (0.2 unit) and the formate (0.2 unit) dehydrogenase NAD-dependent enzymes. The recovery of 13CO(2) from the incubation mixture was equal to 91.4 +/- 3.0%. The accuracy and the precision of the present method were within 12 and 10%, respectively. The limit of quantification was set to 25 pmol. The performance of the assay was validated on human liver microsomes with five probes: [13C]erythromycin, [1-13C]caffeine, [3-13C]caffeine, [7-13C]caffeine, and [13C(2)]aminopyrine. This method is useful for the rapid determination of N-demethylase activity of human liver microsomes from methyl-13C-substrates.     

Effects of Environmental Lead Pollution, Smoking, and Smokeless Tobacco (Maras Powder) Use on Blood Lead Level

One hundred sixty-four adult male volunteers (29 controls [Group 1] and 135 combi drivers) enrolled in the study. The combi drivers were divided into three groups as nonusers of either Maras powder or cigarette (Group 2), smokers (Group 3), and users of Maras powder (Group 4). Blood lead levels (BLLs) were analyzed by atomic absorption spectrophotometer. BLL was detected as 2.8 ± 2.3 μg/dL in Group 1 (n = 29); however, it was 3.5 ± 1.6 μg/dL in Group 2 (n = 33), 3.8 ± 2.4 μg/dL in Group 3 (n = 62), and 3.9 ± 2.4 μg/dL in Group 4 (n = 40). BLL in Group 1 was found significantly lower than other groups (p < 0.05). The use of cigarette or Maras powder by the drivers did not give rise to a marked difference on the BLLs (p > 0.05). BLL of (combi) drivers was detected to be significantly higher than nondrivers; however, it was still under the hazardous level of 10 μg/dL announced by WHO. Although there are publications reporting that usage of tobacco increases the level of lead in blood, both smoking and use of Maras powder did not affect BLL markedly in our study. Poster presented (the abstract section published in Congress Book) at the 7th Congress of Turkish Family Physicians, 23–26 May 2006, Cesme-IZMIR, Turkey.     

Evidence for a highly elastic shell-core organization of cochlear outer hair cells by local membrane indentation

Cochlear outer hair cells (OHCs) are thought to play an essential role in the high sensitivity and sharp frequency selectivity of the hearing organ by generating forces that amplify the vibrations of this organ at frequencies up to several tens of kHz. This tuning process depends on the mechanical properties of the cochlear partition, which OHC activity has been proposed to modulate on a cycle-by-cycle basis. OHCs have a specialized shell-core ultrastructure believed to be important for the mechanics of these cells and for their unique electromotility properties. Here we use atomic force microscopy to investigate the mechanical properties of isolated living OHCs and to show that indentation mechanics of their membrane is consistent with a shell-core organization. Indentations of OHCs are also found to be highly nonhysteretic at deformation rates of more than 40 microm/s, which suggests the OHC lateral wall is a highly elastic structure, with little viscous dissipation, as would appear to be required in view of the very rapid changes in shape and mechanics OHCs are believed to undergo in vivo.     

DAPk protein family and cancer

The Death-Associated Protein kinase (DAPk) family contains three closely related serine/threonine kinases, named DAPk, ZIPk and DRP-1, which display a high degree of homology in their catalytic domains. The recent discovery of protein-protein interactions and kinase/substrate relationships among these family members suggests that the three kinases may form multi-protein complexes capable of transmitting apoptotic or autophagic cell death signals in response to various cellular stresses including the misregulated expression of oncogenes in pre-malignant cells. Several lines of evidence indicate that the most studied member of the family, DAPk, has tumor and metastasis suppressor properties. Here we present an overview of the data connecting the DAPk family of proteins to cell death and malignant transformation and discuss the possible involvement of the autophagic cell death-inducing capacity of DAPk in its tumor suppressor activity.     

Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.     

Synthesis and analgesic activity of some acetamide derivatives

In the present study, some acetamide derivatives were synthesized and their potential analgesic activities were investigated. N-(benzothiazol-2-yl)-2-[(1-substituted-1H-tetrazol-5-yl)thio]acetamide derivatives were obtained by the nucleophilic substitution reaction of 2-chloro-N-(benzothiazole-2-yl)acetamides with appropriate tetrazol-5-thioles. The chemical structures of the compounds were elucidated by IR, 1H-NMR, 13C-NMR and FAB?-MS spectral data and elemental analyses. The prepared compounds were investigated for their potential analgesic properties against thermal, mechanical and chemical nociceptive stimuli using hot-plate, tail-clip and acetic acid-induced writhing tests, respectively. The assessment of motor coordination was carried out using Rota-Rod test. Tested compounds applied at 100 mg/kg doses caused significant decrease in acetic acid-induced writhing responses and increase in hot-plate and tail-clip latencies. None of the compounds exhibited destructive effect on motor coordination of the mice in Rota-Rod performance.     

miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3' UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.     

Transient activation of tumor-associated T-effector/memory cells promotes tumor eradication via NK-cell recruitment: minimal role for long-term T-cell immunity in cure of metastatic disease

Local delivery of IL-12 and GM-CSF to advanced primary tumors results in T- and NK-cell-dependent cure of disseminated disease in a murine spontaneous lung metastasis model. Post-therapy functional dynamics of cytotoxic T- and NK-cells were analyzed in primary and metastatic tumors to determine the specific roles of each subset in tumor eradication. Time-dependent depletion of CD8+ T and NK-cells demonstrated that CD8+ T-cells were critical to eradication of metastatic tumors within 3 days of treatment, but not later. In contrast, NK-cells were found to be essential to tumor regression for at least 10 days after cytokine delivery. Analysis of tumor-infiltrating lymphocyte populations in post-therapy primary tumors demonstrated that treatment resulted in the activation of tumor-associated CD8+ T-cells within 24 h as determined by IFNγ and perforin production. T-cell activity peaked between days 1 and 3 and subsided rapidly thereafter. Activation was not accompanied with an increase in cell numbers suggesting that treatment mobilized pre-existing T-effector/memory cells without inducing proliferation. In contrast, therapy resulted in a ≥3-fold enhancement of both the quantity and the cytotoxic activity of NK-cells in primary and metastatic tumors on day 3 post-therapy. NK-cell activity was also transient and subsided to pre-therapy levels by day 5. Depletion of CD4+ and CD8+ T-cells prior to treatment completely abrogated NK-cell infiltration into primary and metastatic tumors demonstrating the strict dependence of NK-cell recruitment on pre-existing T-effector/memory cells. Treatment failed to induce significant NK-cell infiltration in IFNγ-knockout mice establishing the central role of IFNγ in NK-cell chemotaxis to tumors. These data show that transient activation of tumor-associated T-effector/memory and NK-cells, but not long-term CD8+ T-cell responses, are critical to suppression of metastatic disease in this model; and reveal a novel role for pre-existing adaptive T-cell immunity in the recruitment of innate effectors to tumors. This work was supported by NIH/NCI grant R01-CA100656-01A1 to N.K.E.