Increased skeletal muscle expression of PKC-theta but not PKC-alpha mRNA in type 2 diabetes: inverse relationship with in-vivo insulin sensitivity.

BACKGROUND: Increases in PKC-theta (the major isoenzymic form of PKC in skeletal muscle) protein and isozyme activity have been reported in skeletal muscle from patients with type 2 diabetes mellitus (T2DM) and dietary-induced rodent models of insulin resistance, but the underlying biochemical mechanism is unclear and muscle PKC-theta mRNA expression has not been previously reported in patients with T2DM or in relation to in-vivo measurements of insulin sensitivity. METHODS: Diet-only treated patients with T2DM (n=7) and healthy nondiabetic controls (n=7) of similar BMI attended the clinical research unit on two occasions, 1 week apart, for a skeletal muscle biopsy 2 h after a 75-g oral glucose load and measurement of whole-body insulin sensitivity using the euglycaemic hyperinsulinaemic clamp. RESULTS: Type 2 DM patients were insulin resistant (M-value 3.0 +/- 0.4 vs. 8.6 +/- 0.8 mg glucose kg(-1) min(-1)) with fasting hyperinsulinaemia (306 +/- 116 vs. 34 +/- 9 pmol L(-1), P<0.001) and hypertriglyceridaemia (3.6 +/- 0.7 vs. 1.3 +/- 0.3 mmol L(-1), P<0.01) relative to controls. Semi-quantitative RT-PCR showed that expression of PKC-theta mRNA (relative to GAPDH mRNA) was 6-fold higher in T2DM subjects (0.63 + 0.25% vs. 0.09 + 0.07%, P<0.001), whereas there was no difference in expression of PKC-alpha mRNA between the two groups. Expression of PKC-theta mRNA was inversely correlated with insulin sensitivity (M) and positively correlated with fasting serum insulin concentration (P<0.02). CONCLUSIONS: This is the first clinical study of PKC-theta mRNA expression in human diabetic skeletal muscle. The results indicate that transcriptional up-regulation of PKC-theta may at least partly contribute to the increased muscle PKC-theta signalling in T2DM, and that PKC-theta mRNA may be inversely related to in-vivo insulin sensitivity.

Acute hyperhomocysteinaemia affects pulse pressure but not microvascular vasodilator function.

AIMS: Raised homocysteine (hcy) levels are associated with premature coronary artery disease, but the underlying vascular mechanism and the extent to which hcy affects small vessel vasodilator responses (especially non-nitric oxide mediated pathways) are unclear. METHODS: This double-blind, placebo-controlled crossover study in 14 healthy male subjects evaluated the effects of single-dose oral methionine 15 g (to induce acute hyperhomocysteinaemia) on cutaneous microvascular vasodilator responses to incremental-dose iontophoretic administration of acetylcholine (Ach) and sodium nitroprusside (SNP) using laser Doppler fluximetry (LDF), and the effects on von Willibrand factor (vWF) levels and systemic haemodynamics. RESULTS: Methionine administration produced a three fold rise in plasma hcy levels at 8 h, which was accompanied by a significant increase in pulse pressure (53 vs 49 mmHg, P < 0.05) but no change in heart rate. Acute hyperhomocysteinaemia had no significant effect on incremental microvascular vasodilator dose-response curves to Ach and SNP, or circulating levels of vWF. CONCLUSIONS: The present study shows that acute hyperhomocysteinaemia increases pulse pressure (a marker of large vessel stiffness) but has no effect on endothelial-dependent (non-NO-mediated) microvascular vasodilation.

Lignification induced by pseudomonads harboring avirulent genes on Arabidopsis.

The responses of Arabidopsis thaliana ecotypes to the bacterial pathogen Pseudomonas syringae pv. maculicola 4326 (Psm4326) harboring cloned avirulence genes avrB and avrRpt2 from P. syringae pv. glycinea were examined. Psm4326 containing avirulent genes, avrB and avrRpt2 induced lignification and peroxidase activities in the bacteria infiltrated leaves of Col-O only and not in Mt-O, Bla-2 and Po-1. However, Arabidopsis ecotypes infiltrated with Psm4326 harboring with and without avirulent genes all showed differential induction of mRNA for peroxidase gene and lignin accumulation up to 24 h after infiltration. Only avrB gene in Col-O showed strong corelationship between peroxidase mRNA expression as well as lignification gradually up to 36 h after infiltration. These results extend previous observations that avirulence genes from pathogens of one host plant can be recognized by non-host plants and provide the genetic framework for analysis of the plant-specific response to the bacterial avirulent gene products in A. thaliana.

Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants.

With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of approximately 2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

Microvascular vasodilator response to acetylcholine is increased in women with pre-eclampsia.

OBJECTIVES: To evaluate in vivo microvascular responses to incremental doses of the endothelial-dependent vasodilator, acetylcholine, and the endothelial-independent vasodilator, sodium nitroprusside, in women with pre-eclampsia and gestation-matched normotensive pregnant controls. DESIGN: Prospective clinical study. SETTING: Southern Derbyshire Acute Hospitals Trust, and University of Nottingham Division of Vascular Medicine. POPULATION: Thirteen women with pre-eclampsia and 16 normotensive pregnant controls. METHODS: Cutaneous microvascular vasodilator responses to iontophoretic administration of incremental doses of acetylcholine and sodium nitroprusside (25-100 microC) were evaluated under temperature-controlled conditions using laser Doppler fluximetry. RESULTS: Resting skin temperature and blood flow were similar among 13 women with pre-eclampsia [mean blood pressure 151/93 mmHg (4/3); mean gestation 35.6 weeks (1.0); and mean proteinuria 1.1 g/24h (0.2)] and 16 normotensive pregnant controls [mean blood pressure 111/63 mmHg (2/2); mean gestation 34.3 weeks (0.9)]. Peak vasodilator responses to acetylcholine were increased in women with pre-eclampsia (median flux ratio 15.1 [IQR 12.3-17.6] vs 11.7 [IQR 8.4-12.6], P < 0.05), whereas sodium nitroprusside responses were not different between the two groups: 11.4 [IQR 8.6-13.4] vs 9.5 [IQR 8.0-12.3]. CONCLUSION: In vivo microvascular vasodilator responses to acetylcholine are increased in women with pre-eclampsia, while endothelial-independent vasodilation is unchanged. Although the mechanism of acetylcholine induced vasodilation in small vessels is unclear, this study confirms previous animal data and provides in vivo evidence of altered microvascular endothelial cell function in pre-eclampsia.

Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

Ozone sensitivity in hybrid poplar correlates with insensitivity to both salicylic acid and jasmonic acid. The role of programmed cell death in lesion formation.

Our earlier studies demonstrated that the ozone-sensitive hybrid poplar clone NE-388 displays an attenuated level of ozone-, wound-, and phytopathogen-induced defense gene expression. To determine if this reduced gene activation involves signal transduction pathways dependent on salicylic acid (SA) and/or jasmonic acid (JA), we compared the responses of NE-388 and an ozone-tolerant clone, NE-245, to these signal molecules. JA levels increased in both clones in response to ozone, but only minimal increases in SA levels were measured for either clone. Treatment with SA and methyl jasmonate induced defense gene expression only in NE-245, indicating that NE-388 is insensitive to these signal molecules. DNA fragmentation, an indicator of programmed cell death (PCD), was detected in NE-245 treated with either ozone or an avirulent phytopathogen, but was not detected in NE-388. We conclude that these clones undergo two distinct mechanisms of ozone-induced lesion formation. In NE-388, lesions appear to be due to toxic cell death resulting from a limited ability to perceive and subsequently activate SA- and/or JA-mediated antioxidant defense responses. In NE-245, SA-dependent PCD precedes lesion formation via a process related to the PCD pathway activated by phytopathogenic bacteria. These results support the hypothesis that ozone triggers a hypersensitive response.

The effectiveness of condoms in reducing heterosexual transmission of HIV.

CONTEXT: It is not established whether the condom is as effective at preventing heterosexual transmission of HIV as it is for preventing conception. An overall estimate of condom effectiveness for HIV prevention is needed. METHODS: Information on condom usage and HIV serology was obtained from 25 published studies of serodiscordant heterosexual couples. Condom usage was classified as always (in 100% of acts of intercourse), sometimes (1-99%, 0-99% or 1-100%) or never (0%). Studies were stratified by design, direction of transmission and condom usage group. Condom efficacy was calculated from the HIV transmission rates for always-users and never-users. RESULTS: For always-users, 12 cohort samples yielded a consistent HIV incidence of 0.9 per 100 person-years (95% confidence interval, 0.4-1.8). For 11 cohort samples of never-users, incidence was estimated at 6.8 per 100 person-years (95% confidence interval, 4.4-10.1) for male-to-female transmission, 5.9 per 100 (95% confidence interval, 1.5-15.1) for female-to-male transmission and 6.7 per 100 (95% confidence interval, 4.5-9.6) in samples that specified the direction of transmission. Generally, the condom's effectiveness at preventing HIV transmission is estimated to be 87%, but it may be as low as 60% or as high as 96%. CONCLUSIONS: Consistent use of condoms provides protection from HIV. The level of protection approximates 87%, with a range depending upon the incidence among condom nonusers. Thus, the condom's efficacy at reducing heterosexual transmission may be comparable to or slightly lower than its effectiveness at preventing pregnancy

PIP: This cross-sectional and longitudinal study examines the overall effectiveness of condoms in reducing heterosexual transmission of HIV. Data on condom usage and HIV serology was obtained from 25 published studies of serodiscordant heterosexual couples. Condom usage was rated as ¿always¿ (in 100% of acts of intercourse), ¿sometimes¿ (1-99%, 0-99%, or 1-100%), or ¿never¿ (0%). Studies were stratified by design, direction of transmission and condom usage group. Condom efficacy was calculated from the HIV transmission rates for always-users and never-users. Results showed that 12 cohort samples gave a consistent HIV incidence of 0.9 per 100 person-years. For 11 cohort samples of never-users, incidence was estimated at 6.8 per 100 person-years for male-to-female transmission and 5.9 per 100 for female-to-male transmission. Overall, the condom's effectiveness at preventing HIV transmission is estimated to be 87%, but it may vary between 60% and 96%.

p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB.

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid.

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O3-induced H2O2 levels and the O3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.

Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast.

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.

Aluminum induces oxidative stress genes in Arabidopsis thaliana.

Changes in gene expression induced by toxic levels of Al were characterized to investigate the nature of Al stress. A cDNA library was constructed from Arabidopsis thaliana seedlings treated with Al for 2 h. We identified five cDNA clones that showed a transient induction of their mRNA levels, four cDNA clones that showed a longer induction period, and two down-regulated genes. Expression of the four long-term-induced genes remained at elevated levels for at least 48 h. The genes encoded peroxidase, glutathione-S-transferase, blue copper-binding protein, and a protein homologous to the reticuline:oxygen oxidoreductase enzyme. Three of these genes are known to be induced by oxidative stresses and the fourth is induced by pathogen treatment. Another oxidative stress gene, superoxide dismutase, and a gene for Bowman-Birk protease inhibitor were also induced by Al in A. thaliana. These results suggested that Al treatment of Arabidopsis induces oxidative stress. In confirmation of this hypothesis, three of four genes induced by Al stress in A. thaliana were also shown to be induced by ozone. Our results demonstrate that oxidative stress is an important component of the plant's reaction to toxic levels of Al.

Compost and compost water extract-induced systemic acquired resistance in cucumber and Arabidopsis.

ABSTRACT A biocontrol agent-fortified compost mix, suppressive to several diseases caused by soilborne plant pathogens, induced systemic acquired resistance (SAR) in cucumber against anthracnose caused by Colletotrichum orbiculare and in Arabidopsis against bacterial speck caused by Pseudomonas syringae pv. maculicola KD4326. A peat mix conducive to soilborne diseases did not induce SAR. The population size of P. syringae pv. maculicola KD4326 was significantly lower in leaves of Arabidopsis plants grown in the compost mix compared to those grown in the peat mix. Autoclaving destroyed the SAR-inducing effect of the compost mix, and inoculation of the autoclaved mix with nonautoclaved compost mix or Pantoea agglomerans 278A restored the effect, suggesting the SAR-inducing activity of the compost mix was biological in nature. Topical sprays with water extract prepared from the compost mix reduced symptoms of bacterial speck and the population size of pathogenic KD4326 in Arabidopsis grown in the peat mix but not in the compost mix. The peat mix water extract applied as a spray did not control bacterial speck on plants grown in either mix. Topical sprays with salicylic acid (SA) reduced the severity of bacterial speck on plants in the peat mix but did not further reduce the severity of symptoms on plants in the compost mix. The activity of the compost water extract was heat-stable and passed through a 0.2-mum membrane filter. beta-1,3-Glucanase activity was low in cucumber plants grown in either mix, but when infected with C. orbiculare, this activity was induced to significantly higher levels in plants grown in the compost mix than in plants grown in the peat mix. Similar results were obtained for beta-D-glucuronidase (GUS) activity driven by a PR2 (beta-1,3-glucanase) gene promoter in transgenic Arabidopsis plants grown in the compost or peat mix. GUS activity was induced with topical sprays of the compost water extract or SA in plants not inoculated with the pathogen, suggesting that compost-induced disease suppression more than likely involved the potentiation of resistance responses rather than their activation and that compost-induced SAR differed from SAR induced by pathogens, SA, or compost water extract.

Association of polymorphisms of dopamine D2 receptor (DRD2), and dopamine transporter (DAT1) genes with schizoid/avoidant behaviors (SAB).

The dopaminergic system, and in particular the dopamine D2 receptor, has been implicated in reward mechanisms in the brain. Dysfunction of the D2 dopamine receptors leads to aberrant substance-seeking behaviors (ethanol, drugs, tobacco, and food) and other related behaviors (pathological gambling, Tourette's disorder, attention-deficit/hyperactivity disorder). This is the first study supporting a strong association between the dopamine D2 receptor Taq A1 allele with schizoid/avoidant behavior (SAB). Additionally, an albeit weaker association between the 480-bp VNTR 10/10 allele of the dopamine transporter (DAT1) gene with SAB was similarly found.

The effects of ozone on antioxidant responses in plants.

The response of plants to ozone exposure includes a number of physiological and biochemical changes that are the direct result of selective increases or decreases in gene expression and the resulting changes in the accumulation of the corresponding protein products. Major classes of ozone-induced proteins include antioxidant enzymes and a number of stress-related proteins associated with other biotic and abiotic stresses. In particular, there is a significant overlap in the pattern of gene induction observed in ozone-treated plants and plants exhibiting pathogen defense responses. Current knowledge concerning the specific molecular events associated with the alterations of gene expression caused by ozone and the precise roles of ozone-induced proteins in conferring tolerance to ozone is rather limited. This review summarizes some of the recent results that have been obtained concerning the molecular basis of ozone-induced responses in plants, with an emphasis on studies of the model plant system, Arabidopsis thaliana. These studies demonstrate that ozone-induced responses are caused in part by the activation of a salicylic acid dependent signaling pathway that is also required for the expression of resistance to microbial pathogens.