The NAD(P)H:Quinone Oxidoreductase 1 induces cell cycle progression and proliferation of melanoma cells

The oxidoreductase NQO1 plays a prominent role in maintaining the cellular homeostasis. NQO1 is mainly a cytosolic enzyme which catalyzes the metabolism of quinones and is present in almost all tissue types providing protection against different stresses including xenobiotics, oxidants, UV light, and ionizing radiation. This enzyme is overexpressed in many cancerous tissues and its function in carcinogenesis remains unclear. Due to the relative lack of information on the role of NQO1 in melanoma pathogenesis, we attempted to determine the expression and basic function of NQO1 in melanoma cell proliferation. We found that NQO1 is overexpressed in most melanoma cell lines with respect to melanocytes. Furthermore, the expression of this oxidoreductase significantly induces cell cycle progression by upregulating the expression of cyclins A2, B1 and D1, leading to the proliferation of melanoma cells. Our results also indicate that NQO1 is an upstream regulator of NF-κB p50, a factor linked to melanoma progression and poor patient prognosis. Interestingly, we found that NQO1 stabilizes the transactivator BCL3, which in turn upregulates NF-κB p50. More importantly, our results also indicate that NF-κB p50 correlates with the expression of NQO1 and mediates its role in the proliferation of melanoma cells.     

The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions

Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.     

Treatment of Kienbock's disease with capitohamate arthrodesis: pain relief with minimal morbidity

Despite the large number of procedures available for treatment of Kienbock's disease, no single method has emerged as being clearly superior. Ultimately, the goal of treatment must be the relief of pain and maintaining wrist range of motion. The authors' experience with 45 consecutive wrists that had undergone capitohamate fusion for treatment of Lichtman's stage 1, 2, or 3 Kienbock's disease is presented. Average follow-up was 32 months (range, 4 to 107 months). All arthrodeses healed with an average time to fusion of 1.9 months. Postoperatively, 93 percent of patients had either no pain or less pain than they had preoperatively, with preservation of wrist range of motion and improved grip strength (52 percent of normal preoperatively to 72 percent of normal postoperatively). The authors conclude that capitohamate arthrodesis relieves pain in 93 percent of patients with stage 1, 2, or 3 Kienbock's disease and is an effective treatment for this disease.     

Deletion of mtDNA disrupts mitochondrial function and structure, but not biogenesis

The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function.     

Relationships between gas-exchange characteristics and stomatal structural modifications in some desert grasses under high salinity

Two populations, one from lesser saline Derawar Fort (DF) and the other from highly saline Ladam Sir (LS) in the Cholistan desert, for each of the five grass species, Aeluropus lagopoides, Cymbopogon jwarancusa, Lasiurus scindicus, Ochthochloa compressa, and Sporobolus ioclados were examined to investigate the influence of salinity on structural and functional characteristics of stomata. Salinity tolerance in A. lagopoides mainly depended on controlled transpiration rate (E) and high water-use efficiency (WUE), which was found to be regulated by fewer and smaller stomata on both leaf surfaces as well as stomatal encryption by epidermal invaginations. C. jwarancusa had sunken stomata on the abaxial surface only, which largely reflected a reduced E, but less affected stomatal conductance (g _s) or WUE. L. scindicus had fewer but larger stomata along with hairs/trichomes which may function to avoid water loss through transpiration, and hence, to attain a high WUE. In O. compressa stomata were found only on the abaxial surface and these were completely encrypted by epidermal invaginations as well as a dense covering of microhairs, which was associated with a low E and high WUE under salinity stress. In S. ioclados, the traits of increased stomatal density and decreased stomatal area may be critical for stomatal regulation under salt-prone environments. High stomatal regulation depended largely on stomatal density, area, and degree of encryption under salinity, which is of great ecophysiological significance for plants growing under osmotic stresses.     

Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat

AIMS: Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants for improving the growth and yield of agricultural crops, however screening for the selection of effective PGPR strains is very critical. This study focuses on the screening of effective PGPR strains on the basis of their potential for in vitro auxin production and plant growth promoting activity under gnotobiotic conditions. METHODS AND RESULTS: A large number of bacteria were isolated from the rhizosphere soil of wheat plants grown at different sites. Thirty isolates showing prolific growth on agar medium were selected and evaluated for their potential to produce auxins in vitro. Colorimetric analysis showed variable amount of auxins (ranging from 1.1 to 12.1 mg l-1) produced by the rhizobacteria in vitro and amendment of the culture media with l-tryptophan (l-TRP), further stimulated auxin biosynthesis (ranging from 1.8 to 24.8 mg l-1). HPLC analysis confirmed the presence of indole acetic acid (IAA) and indole acetamide (IAM) as the major auxins in the culture filtrates of these rhizobacteria. A series of laboratory experiments conducted on two cv. of wheat under gnotobiotic (axenic) conditions demonstrated increases in root elongation (up to 17.3%), root dry weight (up to 13.5%), shoot elongation (up to 37.7%) and shoot dry weight (up to 36.3%) of inoculated wheat seedlings. Linear positive correlation (r = 0.99) between in vitro auxin production and increase in growth parameters of inoculated seeds was found. Based upon auxin biosynthesis and growth-promoting activity, four isolates were selected and designated as plant growth-promoting rhizobacteria (PGPR). Auxin biosynthesis in sterilized vs nonsterilized soil inoculated with selected PGPR was also monitored that revealed superiority of the selected PGPR over indigenous microflora. Peat-based seed inoculation with selected PGPR isolates exhibited stimulatory effects on grain yields of tested wheat cv. in pot (up to 14.7% increase over control) and field experiments (up to 27.5% increase over control); however, the response varied with cv. and PGPR strains. CONCLUSIONS: It was concluded that the strain, which produced the highest amount of auxins in nonsterilized soil, also caused maximum increase in growth and yield of both the wheat cv. SIGNIFICANCE AND IMPACT OF STUDY: This study suggested that potential for auxin biosynthesis by rhizobacteria could be used as a tool for the screening of effective PGPR strains.     

Influence of ethylene produced by soil microorganisms on etiolated pea seedlings

There is indirect evidence that soil microorganisms producing ethylene (C(2)H(4)) can influence plant growth and development, but unequivocal proof is lacking in the literature. A laboratory study was conducted to demonstrate the validity of this speculation. Four experiments were carried out to observe the characteristic "triple" response of etiolated pea seedlings to C(2)H(4) microbially derived from l-methionine as a substrate in the presence or absence of Ag(I), a potent inhibitor of C(2)H(4) action. In two experiments, the combination of l-methionine and Acremonium falciforme (as an inoculum) was used, while in another study the indigenous soil microflora was responsible for C(2)H(4) production. A standardized experiment was conducted with C(2)H(4) gas to compare the contribution of the microflora to plant growth. In all cases, etiolated pea seedlings exhibited the classical triple response, which includes reduction in elongation, swelling of the hypocotyl, and a change in the direction of growth (horizontal). The presence of Ag(I) afforded protection to the pea seedlings against the microbially derived C(2)H(4). This study demonstrates that microbially produced C(2)H(4) in soil can influence plant growth.