Catalytic domain crystal structure of protein kinase C-theta (PKCtheta)

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads.     

Effect of attract and kill formulations and application rates on trap catches of European pine shoot moth (Lepidoptera: Tortricidae) and shoot damage in Scots pine saplings

Attract and kill technology was tested for management of European pine shoot moth, Rhyacionia buoliana (Denis & Schiffermüller), in 4-6-yr-old Scots pine, Pinus sylvestris L., plantations managed by Jablonna and Pultusk Forest Districts, Poland. In 2001, two formulations based on ricinoleic acid and hydrocarbon fraction (petroleum jelly) in combination with (E)-9-dodecenyl acetate, the sex pheromone of the pine shoot moth; permethrin as a contact insecticide; and Tinuvin UV absorber were used. In 2002, different formulations and application rates of the attracticide based on petroleum jelly were tested. Significantly reduced trap catches occurred in plots treated with three attracticide formulations [Rhykil-1 (with Tinuvin UV absorber), Rhykil-2 (with a new UV absorber, 3,3'-dihydroxy-2,2'-bipyridyl), and Rhykil-3 (without the insecticide)] at 3,000 droplets per hectare in comparison with those in control plots, suggesting that all formulations were highly effective. Significantly lower catches than in control plots also were observed when Rhykil-1 was applied at 1000, 2,000, and 3,000 droplets per hectare. However, only slight reduction of shoot damage in treated plots was observed in both experiments. The formulation without the insecticide had similar efficacy to that of the formulation combined with the insecticide. In 2003, the Rhykil-2 attracticide was tested at 250, 500, and 1000 droplets per hectare. Although there were no significant differences in trap catches between treated and control plots, shoot damage level was reduced substantially in all treated plots. These results suggest that attract and kill technology may be used at rates lower than 1000 droplets per hectare for management of R. buoliana; however, its "kill" effect should be confirmed in further studies.     

Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.     

Growth and development of copepodite stages of Pseudocalanus spp.

Quantitative expressions are presented describing the effectsof temperature and food concentration on the growth and developmentof the copepodite stages of Pseudocalanus spp. The calculationswere made on the basis of experimental data from the literaturefor two geographically separate populations of Pseudocalanusfrom Puget Sound (Washington, USA) and from the southern NorthSea. Relationships were obtained between the growth parametersfor Pseudocalanus sp. from Puget Sound and temperature in the8–15.5°C range and food concentrations from 10 mgC m^-3 to excess, as well as for Pseudocalanus elongatus fromthe southern North Sea at high food concentrations and in the4–15°C temperature range. Also computed was the totaldevelopment time of P. elongatus for the final four copepodidstages as a function of food concentration from 50 mg C m^-3to excess and temperature from 5 to 20°C. Empirical modelscomputed here may be used with good precision in mathematicalmodels of pelagic communities.     

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.     

Analysis of CYP1A1 exon 7 polymorphisms by PCR-SSCP in a Brazilian population and description of two novel gene variations

CYP1A1 polymorphisms have been associated with a higher risk to develop lung cancer, particularly in Japanese. The type and the frequency of the polymorphisms can vary according to the ethnicity. In the present study, we aimed to determine the frequency of CYP1A1(*)2B and (*)4, and to look for other possible polymorphisms that may happen in exon 7 in individuals from Rio de Janeiro, an ethnic mixed population from Brazil. We developed a PCR-SSCP method for screening the genomic polymorphic region from 2289 to 2645 bp. Seven different migration patterns were found among 405 individuals, 130 healthy blood donors and 275 outpatients from Hospital Universitário Pedro Ernesto located in Rio de Janeiro. Five of the migration patterns corresponded to the genotypes: (*)1/(*)1 (the wild type); (*)1/(*)2B; (*)1/(*)4, (*)2B/(*)4 (heterozygous polymorphic) and (*)2B/(*)2B (homozygous polymorphic). Two other patterns corresponded to gene alterations not yet published: a C > T transition localized at the position 2461, and a C > T transition localized at position 2445. The genotype frequencies of the studied polymorphisms were: for CYP1A1(*)2B - 83.7% to (*)1/(*)1, 15.1% to (*)1/(*)2B and 1.2% to (*)2B/(*)2B; for CYP1A1(*)4 - 93.1% to (*)1/(*)1, 6.9% to (*)1/(*)4. The distribution of CYP1A1(*)2B and (*)4 genotypes combined were similar between white and non-white individuals. However, when the non-white individuals were stratified between blacks and mulattos, and then compared with white, black individuals showed a higher frequency of the wild type genotype (P = 0.008) and a lower frequency of genotype (*)1/(*)4 (P = 0.026). Additionally, when black and mulatto individuals were compared, blacks had a higher frequency of the wild type genotype (P = 0.008) and a lower frequency of the(*)1/(*)2B genotype (P = 0.0008), showing a different ethnic distribution of CYP1A1 polymorphisms.     

Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models

Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A high-sensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLa-luc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging.     

Gap junctions

Electrical coupling through gap junctions constitutes a mode of signal transmission between neurons (electrical synaptic transmission). Originally discovered in invertebrates and in lower vertebrates, electrical synapses have recently been reported in immature and adult mammalian nervous systems. This has renewed the interest in understanding the role of electrical synapses in neural circuit function and signal processing. The present review focuses on the role of gap junctions in shaping the dynamics of neural networks by forming electrical synapses between neurons. Electrical synapses have been shown to be important elements in coincidence detection mechanisms and they can produce complex input-output functions when arranged in combination with chemical synapses. We postulate that these synapses may also be important in redefining neuronal compartments, associating anatomically distinct cellular structures into functional units. The original view of electrical synapses as static connecting elements in neural circuits has been revised and a considerable amount of evidence suggests that electrical synapses substantially affect the dynamics of neural circuits.     

Candida ciferrii in an immunocompromised patient

A case of possible infection due to Candida ciferrii in an immunocompromised patient is presented. This fungal species has been rarely reported as cause of human infection. The isolate showed in vitro resistance to fluconazole.