Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.     

Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin

Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type betaB1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (betaB1(DeltaN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens betaB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.     

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.     

Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells

Probiotic bacteria are microorganisms that benefit the host by preventing or ameliorating disease. However, little information is known regarding the scientific rationale for using probiotics as alternative medicine. The purpose of this paper is to investigate the mechanisms of probiotic beneficial effects on intestinal cell homeostasis. We now report that one such probiotic, Lactobacillus rhamnosus GG (LGG), prevents cytokine-induced apoptosis in two different intestinal epithelial cell models. Culture of LGG with either mouse or human colon cells activates the anti-apoptotic Akt/protein kinase B. This model probiotic also inhibits activation of the pro-apoptotic p38/mitogen-activated protein kinase by tumor necrosis factor, interleukin-1alpha, or gamma-interferon. Furthermore, products recovered from LGG culture broth supernatant show concentration-dependent activation of Akt and inhibition of cytokine-induced apoptosis. These observations suggest a novel mechanism of communication between probiotic microorganisms and epithelia that increases survival of intestinal cells normally found in an environment of pro-apoptotic cytokines.     

Susceptibility of Maconellicoccus hirsutus (Homoptera: Pseudococcidae) to methyl bromide

Eggs, crawlers, early nymphs, late nymphs, and adults of the pink hibiscus mealybug, Maconellicoccus hirsutus (Green), were tested for their susceptibility to methyl bromide in 2-h laboratory fumigations at ambient conditions (25 degrees C, 95% RH). Dose-response tests indicated that the egg was the most susceptible life stage with an LC99 of 20.2 mg/liter. Based on probit analysis of dose-response data, no significant differences were observed among susceptibilities of the crawler, early stage or late stage nymphs, or adults at either the LC50 or LC99 level, but late stage nymphs were more tolerant than early stage nymphs in a separate paired comparison test. Confirmatory tests showed that a dose of 48 mg/liter methyl bromide, the USDA-Animal Plant Health Inspection Service treatment dose schedule for mealybugs at 21-26 degrees C, produced 100% mortality of all life stages. On the basis of these results, we conclude that the methyl bromide treatment schedule for mealybugs will provide quarantine security for M. hirsutus infesting commodities for export or import.     

Suitability of transgenic glyphosate-resistant soybeans to green cloverworm (Lepidoptera: Noctuidae)

Host plant suitability to green cloverworm, Hypena scabra (F.), was evaluated on two conventional soybean varieties (Stine 2499-0 and 2972-2) and two RoundUp Ready Soybeans (Stine 2506-4 and 2892-4) with and without exposure to glyphosate. No differences among treatments were detected on developmental time and survivorship. Developmental time from first instar to adult ranged from 24.7 to 25.5 d, and survivorship ranged from 86 to 96%. No sex bias was observed among treatments (proportion of females ranged from 0.41 to 0.50). Morphological differences were observed between sexes; males had a longer and wider thorax, longer wings, and longer body. Females had longer and wider abdomens. Although treatments did not affect size (calculated with principal component analyses), significant differences were observed between males and females. These results suggest that soybean genetic differences (between conventional varieties and analogous transgenic varieties) or plant stress (induced by glyphosate metabolization) do not affect the plant suitability to H. scabra.     

Persistence and efficacy of spinosad residues in farm stored wheat

Degradation and insecticidal effectiveness of spinosad residues were evaluated in Kansas during November 2000 to November 2001 in farm bins holding wheat (34-metric ton capacity). About 50 kg of hard red winter wheat from each of three bins were brought to the laboratory and treated separately with 1-ml aqueous suspensions of spinosad to provide rates of 0.1, 0.5,1, 3, 6 mg (AI)/kg of wheat. Wheat treated with distilled water served as the control treatment. Untreated and spinosad-treated wheat samples (250 g each) were placed in three plastic pouches of two different mesh sizes, and buried 2.5 cm below the grain surface. Pouches with large mesh openings were used to monitor insect infestations and kernel damage in untreated and spinosad-treated samples. Pouches with small mesh were used for extracting spinosad residues and for conducting laboratory bioassays with adults of the lesser grain borer, Rhyzopertha dominica (F.) and red flour beetle, Tribolium castaneum (Herbst) at 28 degrees C and 65% RH. Wheat temperature and relative humidity near the pouches during the 1 yr of storage ranged from -10 to 32 degrees C and 50 to 70%, respectively. Moisture of wheat samples varied from 12.4 to 13%. Observed spinosad residues on wheat samples were 25% less than the calculated rates of 0.1 to 6 mg/kg. However, these residues were stable during the 1 yr of storage, and killed all R. dominica adults exposed for 14 d in the laboratory. Mortality of T. castaneum adults increased with an increase in spinosad rate. The linear regression slope of LD50s (0.3-2.7 mg/kg) against storage time was not significantly different from zero, indicating no loss in spinosad toxicity to T. castaneum adults. Insect species, insect numbers, and kernel damage over time in wheat samples inside pouches with large mesh openings were highly inconsistent, and failed to accurately characterize spinosad performance. Laboratory bioassays with R. dominica and T castaneum adults using grain from pouches with small mesh openings accurately gauged spinosad persistence and insecticidal activity under the field conditions.     

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus

Effects of environmental salinity and 17α-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3–5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.     

Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are readily incorporated into phospholipids of smooth muscle cells (SMC) and endothelial cells (EC). Incorporation of EETs into intact porcine coronary arteries potentiates EC-dependent relaxation, but not vasorelaxation induced by agents that act solely on SMC. To explore the potential mechanisms responsible for this difference, porcine coronary artery SMC and EC preloaded with [3H]14,15-EET were treated with calcium ionophore A23187. Although the amount of EET incorporated into EC and SMC was similar, A23187 stimulated a five-fold increase in release of radioactivity from EC, but only a 21% increase in release from SMC. Thin layer chromatography (TLC) examination of cell lipids demonstrated that > 70% of the incorporated radioactivity was present in phosphatidylcholine (PC) in both SMC and BC. After treatment of EC PC with PLA2, TLC analysis indicated that approximately equal to 75% of radioactivity was present as free EET, and 25% of radioactivity was present as lyso-PC. Therefore, most of the 14,15-EET was esterified into the sn-2 position of PC in EC. However, in SMC, approximately equal to 70% of radioactivity was present as lyso-PC after PLA2 treatment, indicating that the EET was predominately esterified into the sn-1 position. In contrast, all of the 14,15-EET was esterified into the sn-2 position of PI in both EC and SMC. These results suggest that the preferential incorporation of 14,15-EET into the sn-1 position of PC in SMC may help to explain the greater retention of the compound in SMC, while incorporation into the sn-2 position of PC in EC may facilitate agonist-induced 14,15-EET release and potentiation of EC-dependent porcine coronary artery relaxation.