20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.     

Tyrosine kinase, p56lck-induced cell motility, and urokinase-type plasminogen activator secretion involve activation of epidermal growth factor receptor/extracellular signal regulated kinase pathways

We have recently reported that tyrosine kinase, p56(lck) regulates cell motility and nuclear factor kappaB-mediated secretion of urokinase-type plasminogen activator (uPA) through tyrosine phosphorylation of IkappaBalpha following hypoxia/reoxygenation (Mahabeleshwar, G. H., and Kundu, G. C. (2003) J. Biol. Chem. 278, 52598-52612). However, the role of hypoxia/reoxygenation (H/R) on ERK1/2-mediated uPA secretion and cell motility and the involvement of p56(lck) and EGF receptor in these processes in breast cancer cells is not well defined. We provide here evidence that H/R induces Lck kinase activity and Lck-dependent tyrosine phosphorylation of EGF receptor in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. H/R also stimulates MEK-1 and ERK1/2 phosphorylations, and H/R-induced phosphorylations were suppressed by the dominant negative form of Lck (DN Lck, K273R) as well as pharmacological inhibitors of EGF receptor and Lck indicating that EGF receptors and Lck are involved in these processes. Transfection of these cells with wild type Lck or Lck F505 (Y505F) but not with Lck F394 (Y394F) induced phosphorylations of EGF receptor followed by MEK-1 and ERK1/2, suggesting that Lck is upstream of EGF receptor and Tyr-394 of Lck is crucial for these processes. H/R also induced uPA secretion and cell motility in these cells. DN Lck and inhibitors of Lck, EGF receptor, and MEK-1 suppressed H/R-induced uPA secretion and cell motility. To our knowledge, this is the first report that p56(lck) in presence of H/R regulates MEK-1-dependent ERK1/2 phosphorylation and uPA secretion through tyrosine phosphorylation of EGF receptor, and it further demonstrates that all of these signaling molecules ultimately control the motility of breast cancer cells.     

Senescence and hyperspectral reflectance of cotton leaves exposed to ultraviolet-B radiation and carbon dioxide

The objectives of this study were to determine the effects of UV-B radiation and atmospheric carbon dioxide concentrations ([CO(2)]) on leaf senescence of cotton by measuring leaf photosynthesis and chlorophyll content and to identify changes in leaf hyperspectral reflectance occurring due to senescence and UV-B radiation. Plants were grown in controlled-environment growth chambers at two [CO(2)] (360 and 720 micro mol mol(-1)) and three levels of UV-B radiation (0, 7.7 and 15.1 kJ m(-2) day(-1)). Photosynthesis, chlorophyll, carotenoids and phenolic compounds along with leaf hyperspectral reflectance were measured on three leaves aged 12, 21 and 30 days in each of the treatments. No interaction was detected between [CO(2)] and UV-B for any of the measured parameters. Significant interactions were observed between UV-B and leaf age for photosynthesis and stomatal conductance. Elevated [CO(2)] enhanced leaf photosynthesis by 32%. On exposure to 0, 7.7 and 15.1 kJ of UV-B, the photosynthetic rates of 30-day-old leaves compared with 12-day-old leaves were reduced by 52, 76 and 86%, respectively. Chlorophyll pigments were not affected by leaf age at UV-B radiation of 0 and 7.7 kJ, but UV-B of 15.1 kJ reduced the chlorophylls by 20, 60 and 80% in 12, 21 and 30-day-old leaves, respectively. The hyperspectral reflectance between 726 and 1142 nm showed interaction for UV-B radiation and leaf age. In cotton, leaf photosynthesis can be used as an indicator of leaf senescence, as it is more sensitive than photosynthetic pigments on exposure to UV-B radiation. This study revealed that, cotton leaves senesced early on exposure to UV-B radiation as indicated by leaf photosynthesis, and leaf hyperspectral reflectance can be used to detect changes caused by UV-B and leaf ageing.     

Characterization of stanniocalcin 2, a novel target of the mammalian unfolded protein response with cytoprotective properties

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a highly conserved homeostatic response in all eukaryotic cells, termed the unfolded-protein response (UPR). Here we describe the characterization of stanniocalcin 2 (STC2), a mammalian homologue of a calcium- and phosphate-regulating hormone first identified in fish, as a novel target of the UPR. Expression of STC2 gene is rapidly upregulated in cultured cells after exposure to tunicamycin and thapsigargin, by ATF4 after activation of the ER-resident kinase PERK. In addition, STC2 expression is also activated in neuronal cells by oxidative stress and hypoxia but not by several cellular stresses unrelated to the UPR. In contrast, expression of another homologue, STC1, is only upregulated by hypoxia independent of PERK or ATF4 expression. In vivo studies revealed that rat cortical neurons rapidly upregulate STC2 after transient middle cerebral artery occlusion. Finally, siRNA-mediated inhibition of STC2 expression renders N2a neuroblastoma cells and HeLa cells significantly more vulnerable to apoptotic cell death after treatment with thapsigargin, and overexpression of STC2 attenuated thapsigargin-induced cell death. Consequently, induced STC2 expression is an essential feature of survival component of the UPR.     

Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC_17b) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins. aorta; mesenteric vessels; calponin; caldesmon     

What is the ultimate goal in neural regulation of cardiovascular function?

We used the following multiple-choice question after a series of lectures in cardiovascular physiology in the first year of an undergraduate medical curriculum (n = 66) to assess whether students had understood the neural regulation of cardiovascular function. In health, neural cardiovascular mechanisms are geared toward maintaining A) cardiac output, B) total peripheral resistance (TPR), C) arterial blood pressure (BP), D) tissue blood flow. The same question was administered to 275 graduates preparing for postgraduate exams (but not following the same series of lectures as the undergraduates). In both groups, we found a large proportion of incorrect answers (70% in undergraduates and 85% in graduates) and sorted this out by offering a step-by-step explanation and two examples and found it successful: 1) What happens to BP and heart rate (HR) when a person loses 500 ml of blood ( approximately 10% of blood volume) in one minute? 2) What happens to your BP and HR as you get out of bed after a night's sleep? Flow = perfusion pressure/resistance to flow; cardiac output = BP/TPR; BP = cardiac output x TPR = [stroke volume (SV) x HR] x TPR. In both examples, BP decreases and is rapidly brought into the normal range by the arterial baroreflex mechanism. TBF is regulated chiefly by varying local vascular resistance (autoregulation). In summary, the ultimate goal of all neural cardiovascular reflex mechanisms is to maintain arterial BP within a range in which tissues can regulate their own blood flows. Cardiovascular control during exercise was used as an example to emphasize these facts. A discussion of this kind triggered interest in the minds of students and graduates, helping them get rid of a major misconception in about 20-40 minutes.     

Pharmaceutical development and manufacturing of a parenteral formulation of a novel antitumor agent, VNP40101M

The objective of this study was to develop and manufacture a stable parenteral formulation for Phase I clinical trials of VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2-methylamino)carbonyl] hydrazine), a novel antitumor agent. The solubility and stability of the drug was determined. Solubility studies suggested that VNP40101M exhibited poor aqueous solubility but showed appreciable solubility in nonaqueous solvents. The aqueous solubility of the drug could not be increased by adjusting the pH. At a pH above 7, basecatalyzed decomposition of VNP40101M occurred. The low octanol-water partition coefficient of 0.75 suggested poor solubility in lipophilic solvents. Based on these preformation observations, a parenteral formulation containing 10 mg/mL of VNP40101M was prepared in a solvent system consisting of 30% ethyl alcohol and 70% polyethylene glycol-300 (PEG-300). To minimize base-catalyzed hydrolytic degradation. citric acid at 0.6% concentration was included to acidify the formulation. Rubber closures, filter membranes, and liquid transfer tubing were selected on the basis of compatibility studies and absence of loss of drug the of adsorption of these components. The formulation was subjected to accelerated stability studies and dilution studies with large volume parenteral (LVP) solutions, normal saline, and 5% dextrose injection (D5W). The results of the dilution study indicated that the formulation could be diluted in these solutions up to 2 mg/mL for 8 hours without drug precipitation and degradation. Accelerated stability studies suggested that the product should be kept at 2°C to 8°C for long-term storage. The developed formulation was successfully scaled up and manufactured for use in clinical trials. Published: August 26, 2001.     

Incidence and severity of frogeye leaf spot and associated yield losses in soybeans in agroecological zone II of Zambia

The incidence and severity of frogeye leaf spot of soybean (Glycine max (L.) Merr.) was studied in agroecological region II of Zambia during the 1997/98 crop growing season. A survey was conducted on farmers' fields on SCSI Kaleya, Magoye and Hernon-147 cultivars. Disease incidence and severity was assessed by monitoring disease increments at two weeks interval (beginning of January to April) from nine fields, three from each province. Soybean cultivars were evaluated for yield losses resulting from frogeye leaf spot. Field plots of each cultivar were either sprayed twice with benomyl (benlate) or not sprayed at all. The results showed that the incidence of frogeye leaf spot was highest in Southern province (5.1), followed by Lusaka province (4.9) while Central province had the lowest disease incidence (1.8). Values for area under disease progress curve (AUDPC) were significantly greater (P < 0.05) for Lusaka and Southern provinces than for Central province. Yields in benomyl protected plots ranged from 1444 kg ha⁻¹ to 2320 kg ha⁻¹ and were significantly different among the cultivars. Average yields of non protected plants were reduced by 30.5% for Kaleya, 35.6% for Hernon-147 and 37.2% for SCS1. Incidence and severity increased with time and varied depending on weather parameters and susceptibility of cultivars to the disease. Yield losses due to frogeye leaf spot occurred through a reduction in seed size. Differences in weather conditions and amount of inocula are believed to contribute to the observed variation in incidence and severity of the disease at different locations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hydrogeochemical characterization of Yercaud lake southern India: Implications on lake water chemistry through multivariate statistics

In this paper, a study on the surface and deeper waters of the Yercaud Lake, Tamil Nadu, South India, was carried out to understand the geochemistry of the lake waters and also to determine its utility for agricultural purposes. Totally, 50 surface and deep water samples were collected from Yercaud Lake. Major ion and heavy metals were measured. The data obtained were interpreted using the lake water composition. The mean concentration of physicochemical parameters and heavy metals for the surface and deep waters have the following values, pH (7.6), EC (263.4), Ca^2 +(16.3), Mg^2 +(7.4), Na⁺(19.2), K⁺(1.5), Cl^?(18.2), NO₃^?(1.5), SO₄^2 ?(1.5), HCO₃^– (97.9), Fe (1.3), Mn (0.1), Cr (0.4), Cu (0.005), Pb (0.31), Zn (0.01), Co (0.095) and Ni (0.075). The data generated reflects that the water samples are dominated by recharge process, especially due to the monsoonal rains and natural springs within the lake. The geochemical data reveals that the lake water is suitable for the agricultural purpose and the chemistry of water is mainly influenced by the weathering of bedrock, especially the charnockites bedrock. The sodium adsorption ratio and sodium percentage (%Na) values indicate that the lake water is suitable for irrigation. Dominant heavy metals in the lake waters are mainly because of the lithogenic as well as through minor anthropogenic inputs. Based on our data it is noted that proper management plans are required to monitor the pollution source in the lake, with strict policy measures.     

Exploration of the expeditious potential of Pseudomonas fluorescens lipase in the kinetic resolution of racemic intermediates and its validation through molecular docking

A profoundly time‐efficient chemoenzymatic method for the synthesis of (S)‐3‐(4‐chlorophenoxy)propan‐1,2‐diol and (S)‐1‐chloro‐3‐(2,5‐dichlorophenoxy)propan‐2‐ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic alcohols, respectively. Docking of the R‐ and S‐enantiomers of the intermediates demonstrated stronger H‐bond interaction between the hydroxyl group of the R‐enantiomer and the key binding residues of the catalytic site of the lipase, while the S‐enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost‐effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs.