The human double-stranded RNA- (dsRNA) activated protein kinase (PKR) has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). The minimal-length polypeptide required to bind dsRNA contains both dsRBMs, as determined by mobility-shift and filter-binding assays. Mobility-shift experiments indicate binding requires a minimum of 16 base pairs of dsRNA, while a minimal-length site for saturation of longer RNAs is 11 base pairs. Bulge defects in the helix disfavor binding, and single-stranded tails do not strongly influence the dsRNA length requirement. These polypeptides do not bind an RNA-DNA hybrid duplex or dsDNA as judged by either mobility-shift or competition experiments, suggesting 2'-OH contacts on both strands of the duplex stabilize binding. Related experiments on chimeric duplexes in which specific sets of 2'-OHs are substituted with 2'-H or 2'-OCH3 reveal that the 2'-OHs required for binding are located along the entire 11 basepair site. These results are supported by Fe(II) EDTA footprinting experiments that show protein-dependent protection of the minor groove of dsRNA. The dependence of dsRNA-protein binding on salt concentration suggests that only one ionic contact is made between the protein and dsRNA phosphate backbone and that at physiological salt concentrations 90% of the free energy of binding is nonelectrostatic. Thus, the specificity of PKR for dsRNA over RNA-DNA hybrids and dsDNA is largely due to molecular recognition of a network of 2'-OHs involving both strands of dsRNA and present along the entire 11 base-pair site.

BACKGROUND

Low vitamin D status has been suggested as a risk factor for type 2 diabetes. Although the epidemiologic evidence is scarce, 2 recent studies have suggested an association. The present study investigated the relation of serum vitamin D with type 2 diabetes incidence using pooled data from these 2 cohorts.

METHODS

Two nested case-control studies, collected by the Finnish Mobile Clinic in 1973-1980, were pooled for analysis. The study populations consisted of men and women aged 40-74 years and free of diabetes at baseline. During a follow-up period of 22 years, 412 incident type 2 diabetes cases occurred, and 986 controls were selected by individual matching. Serum vitamin D (serum 25(OH)D) was determined from frozen samples, stored at baseline. Pooled estimates of the relationship between serum vitamin D concentration and type 2 diabetes incidence were calculated.

RESULTS

Men had higher serum vitamin D concentrations than women and showed a reduced risk of type 2 diabetes in their highest vitamin D quartile. The relative odds between the highest and lowest quartiles was 0.28 (95% confidence interval = 0.10-0.81) in men and 1.14 (0.60-2.17) in women after adjustment for smoking, body mass index, physical activity, and education.

CONCLUSIONS

The results support the hypothesis that high vitamin D status provides protection against type 2 diabetes. Residual confounding may contribute to this association.

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.

OBJECTIVE

A protective association between higher vitamin D levels and the onset of multiple sclerosis (MS) has been demonstrated; however, its role in modulating MS clinical course has been little studied. We investigated whether higher levels of serum 25-hydroxyvitamin D (25-OH-D) were associated with a lower risk of relapses in people with MS.

METHODS

We conducted a prospective cohort study of 145 participants with relapsing-remitting MS from 2002 to 2005. Serum 25-OH-D levels were measured biannually, and the hazard of relapse was assessed using survival analysis.

RESULTS

There was an inverse linear relationship between 25-OH-D levels and the hazard of relapse over the subsequent 6 months, with hazard ratio (HR) 0.91 (95% confidence interval [CI]: 0.85-0.97) per 10 nmol/l increase in 25-OH-D level (p = 0.006). When variation due to timing of blood collection was removed by estimating 25-OH-D at the start of each season, this association persisted, with HR 0.90 (95% CI, 0.83-0.98) per 10 nmol/l increase (p = 0.016). Taking into account the biological half-life of 25-OH-D, we estimated 25-OH-D at monthly intervals, resulting in a slightly enhanced association, with HR 0.88 (95% CI, 0.82-0.95) per 10 nmol/l increase (p = 0.001). Adjusting for potential confounders did not alter these findings.

CONCLUSIONS

In this prospective population-based cohort study, in a cohort largely on immunomodulatory therapy, higher 25-OH-D levels were associated with a reduced hazard of relapse. This occurred in a dose-dependent linear fashion, with each 10 nmol/l increase in 25-OH-D resulting in up to a 12% reduction in risk of relapse. Clinically, raising 25-OH-D levels by 50 nmol/l could halve the hazard of a relapse.

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.

OBJECTIVE

To estimate the intraocular pressure (IOP) reduction achieved by the most frequently prescribed glaucoma drugs and a placebo in a meta-analysis of randomized clinical trials.

METHODS

Meta-analysis of randomized clinical trials.

METHODS

Twenty-seven articles reporting on 28 randomized clinical trials. These articles reported 6953 participants for the trough and 6841 for the peak.

METHODS

Articles published up to December 2003 were identified in the following data sources: Medline, Embase, and the Cochrane Controlled Trials Register, and references from relevant articles. Over 85% of the patients had to be diagnosed with primary open-angle glaucoma (POAG) or ocular hypertension (OH), and articles had to be written in English, German, French, or Dutch. Quality of trials was assessed by a Delphi list with additions. The pooled 1-month IOP-lowering effect from baseline at peak and trough was calculated by performing meta-analysis using the random effects model.

METHODS

Absolute and relative change in IOP from baseline, for peak and trough moments.

RESULTS

Relative IOP reductions from baseline [mean (95% confidence interval)] were -23% (-25% to -22%) for a peak and -20% (-23% to -17%) for a trough for 0.5% betaxolol; peak, -27% (-29% to -25%), and trough, -26% (-28% to -25%), for 0.5% timolol; peak, -22% (-24% to -20%), and trough, -17% (-19% to -15%), for 2.0% dorzolamide; peak, -17% (-19% to -15%), and trough, -17% (-19% to -15%) for 1.0% brinzolamide; peak, -25% (-28% to -22%), and trough, -18% (-21% to -14%) for 0.2% brimonidine; peak, -31% (-33% to -29%), and trough, -28% (-30% to -26%) for 0.005% latanoprost; peak, -31% (-32% to -29%), and trough, -29% (-32% to -25%) for 0.004% travoprost; peak, -33% (-35% to -31%), and trough, -28% (-29% to -27%) for 0.03% bimatoprost; and peak, -5% (-9% to -1%), and trough, -5% (-10% to -0%) for the placebo. The difference in absolute IOP reduction from baseline between timolol and prostaglandin analogs or prostamide varied from -0.4 to 0.1 mmHg at trough and from 1.0 to 1.5 mmHg at peak. Quality scores of included studies were generally high, a mean of 14.2 on a scale from 0 to 20 (interquartile range, 13-16).

CONCLUSIONS

This meta-analysis suggests that bimatoprost, travoprost, latanoprost, and timolol are the most effective intraocular pressure-reducing agents in POAG and OH patients.

Estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium. Their role as breast carcinogens has long been suspected and recently confirmed by epidemiological studies. Three major mechanisms are postulated to be involved in their carcinogenic effects: stimulation of cellular proliferation through their receptor-mediated hormonal activity, direct genotoxic effects by increasing mutation rates through a cytochrome P450-mediated metabolic activation, and induction of aneuploidy. Recently it has been fully demonstrated that estrogens are carcinogenic in the human breast by testing in an experimental system the natural estrogen 17beta-estradiol (E(2)) by itself or its metabolites 2-hydroxy, 4-hydroxy, and 16-a-hydroxy-estradiol (2-OH-E(2), 4-OH-E(2), and 16-alpha-OH E(2)), respectively, by inducing neoplastic transformation of human breast epithelial cells (HBEC) MCF-10F in vitro to a degree at least similar to that induced by the chemical carcinogen benz(a)pyrene (BP). Neither Tamoxyfen (TAM) nor ICI-182,780 abrogated the transforming efficiency of estrogen or its metabolites. The E(2) induced expression of anchorage independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, is associated with the loss of 9p11-13 and only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-alpha and progesterone receptor negative adenocarcinomas that expressed keratins, EMA and E-cadherin. The E(2) induced tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of the human breast epithelial cell MCF-10F in vitro confirms the carcinogenicity of E(2), supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.

The genomic revolution has identified therapeutic targets for a plethora of diseases, creating a need to develop robust technologies for combination drug therapy. In the present work, we describe a self-assembled polymeric nanoparticle (NP) platform to target and control precisely the codelivery of drugs with varying physicochemical properties to cancer cells. As proof of concept, we codelivered cisplatin and docetaxel (Dtxl) to prostate cancer cells with synergistic cytotoxicity. A polylactide (PLA) derivative with pendant hydroxyl groups was prepared and conjugated to a platinum(IV) [Pt(IV)] prodrug, c,t,c-[Pt(NH(3))(2)(O(2)CCH(2)CH(2)COOH)(OH)Cl(2)] [PLA-Pt(IV)]. A blend of PLA-Pt(IV) functionalized polymer and carboxyl-terminated poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer in the presence or absence of Dtxl, was converted, in microfluidic channels, to NPs with a diameter of ∼100 nm. This process resulted in excellent encapsulation efficiency (EE) and high loading of both hydrophilic platinum prodrug and hydrophobic Dtxl with reproducible EEs and loadings. The surface of the NPs was derivatized with the A10 aptamer, which binds to the prostate-specific membrane antigen (PSMA) on prostate cancer cells. These NPs undergo controlled release of both drugs over a period of 48-72 h. Targeted NPs were internalized by the PSMA-expressing LNCaP cells via endocytosis, and formation of cisplatin 1,2-d(GpG) intrastrand cross-links on nuclear DNA was verified. In vitro toxicities demonstrated superiority of the targeted dual-drug combination NPs over NPs with single drug or nontargeted NPs. This work reveals the potential of a single, programmable nanoparticle to blend and deliver a combination of drugs for cancer treatment.

Ions of low molecular weight like metal cations and basic amines are present in lipopolysaccharides regardless of the isolation procedure employed. They are present in salt form with the acidic groups of the molecule and, partly, bound by chelation. Electrodialysis which removed a large proportion of these basic materials led to acidic lipopolysaccharides often with reduced solubility. Electrodialyzed lopopolysaccharides could be rendered soluble by neutralizing with alkali or with a basic amine. Depending on the base employed for neutralization preparations were obtained which showed in water distinct differences in solubility, viscosity and opalescence. These differences were related to differences in the sedimentation coefficients of the various salt forms. Neutralization with triethylamine led in all cases to highly soluble preparations with low sedimentation coefficients, while, on the other hand, neutralization with Mg(OH)2 led in most cases to insoluble preparations. The acidic lipopolysaccharides obtained by electrodialysis deteriorate on storing in a freeze-dried form. On heating in distilled water autohydrolysis occurs and free lipid A is liberated. The lipid A which is so far known as a water-insoluble material showed increased solubility when prepared from electrodialyzed lipopolysaccharides.

Vitamin D inhibits the development and growth of prostate cancer cells. Epidemiologic results on serum vitamin D levels and prostate cancer risk have, however, been inconsistent. We conducted a longitudinal nested case-control study on Nordic men (Norway, Finland and Sweden) using serum banks of 200,000 samples. We studied serum 25(<em>OH</em>)-vitamin D levels of 622 prostate cancer cases and 1,451 matched controls and found that both low (</=19 nmol/l) and high >>/=80 nmol/l) 25(<em>OH</em>)-vitamin D serum concentrations are associated with higher prostate cancer risk. The normal average serum concentration of 25(<em>OH</em>)-vitamin D (40-60 nmol/l) comprises the lowest risk of prostate cancer. The U-shaped risk of prostate cancer might be due to similar 1,25-dihydroxyvitamin D(3) availability within the prostate: low vitamin D serum concentration apparently leads to a low tissue concentration and to weakened mitotic control of target cells, whereas a high vitamin D level might lead to vitamin D resistance through increased inactivation by enhanced expression of 24-hydroxylase. It is recommended that vitamin D deficiency be supplemented, but too high vitamin D serum level might also enhance cancer development.

OBJECTIVE

The objective of the present study was to examine the cross-sectional relation between serum 25-hydroxyvitamin D [25-(OH) D] levels and depression in overweight and obese subjects and to assess the effect of vitamin D supplementation on depressive symptoms.

METHODS

Cross-sectional study and randomized double blind controlled trial of 20,000 or 40,000 IU vitamin D per week versus placebo for 1 year.

METHODS

A total of 441 subjects (body mass index 28-47 kg m(-2), 159 men and 282 women, aged 21-70 years) recruited by advertisements or from the out-patient clinic at the University Hospital of North Norway.

METHODS

Beck Depression Inventory (BDI) score with subscales 1-13 and 14-21.

RESULTS

Subjects with serum 25(OH)D levels < 40 nmol L(-1) scored significantly higher (more depressive traits) than those with serum 25(OH)D levels>> or = 40 nmol L(-1) on the BDI total [6.0 (0-23) versus 4.5 (0-28) (median and range)] and the BDI subscale 1-13 [2.0 (0-15) versus 1.0 (0-29.5)] (P < 0.05). In the two groups given vitamin D, but not in the placebo group, there was a significant improvement in BDI scores after 1 year. There was a significant decrease in serum parathyroid hormone in the two vitamin D groups without a concomitant increase in serum calcium.

CONCLUSIONS

It appears to be a relation between serum levels of 25(OH)D and symptoms of depression. Supplementation with high doses of vitamin D seems to ameliorate these symptoms indicating a possible causal relationship.

OBJECTIVE

To explore associations between serum 25-hydroxyvitamin D(3) [25(OH)D] concentrations and liver histology in patients with non-alcoholic fatty liver disease (NAFLD).

RESULTS

We studied 60 consecutive patients with biopsy-proven NAFLD, and 60 healthy controls of comparable age, sex and body mass index (BMI). NAFLD patients had a marked decrease in winter serum 25(OH)D concentrations (51.0+/-22 vs. 74.5+/-15 nmol/L, P<0.001) compared with controls. Metabolic syndrome (MetS; as defined by the Adult Treatment Panel III criteria) and its individual components occurred more frequently among NAFLD patients. The marked differences in 25(OH)D concentrations observed between the groups were little affected by adjustment for age, sex, BMI, creatinine, calcium, homeostasis model assessment (HOMA)-insulin resistance, and the presence of the MetS. Interestingly, among NAFLD patients, decreased 25(OH)D concentrations were closely associated with the histological severity of hepatic steatosis, necroinflammation and fibrosis (P<0.001 for all) independent of age, sex, BMI, creatinine, calcium, HOMA-insulin resistance, and presence of the MetS.

CONCLUSIONS

Compared with controls, NAFLD patients have a marked decrease in serum 25(OH)D concentrations, which is closely associated with histopathological features of NAFLD. Further investigation into whether vitamin D(3) may play a role in the development and progression of NAFLD appears to be warranted.

We previously demonstrated that AKT2, a member of protein kinase B family, is activated by a number of growth factors via Ras and PI 3-kinase signaling pathways. Here, we report the frequent activation of AKT2 in human primary ovarian cancer and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase (PI 3-kinase)/Akt pathway. In vitro AKT2 kinase assay analyses in 91 ovarian cancer specimens revealed elevated levels of AKT2 activity (>3-fold) in 33 cases (36.3%). The majority of tumors displaying activated AKT2 were high grade and stages III and IV. Immunostaining and Western blot analyses using a phospho-ser-473 Akt antibody that detects the activated form of AKT2 (AKT2 phosphorylated at serine-474) confirmed the frequent activation of AKT2 in ovarian cancer specimens. Phosphorylated AKT2 in tumor specimens localized to the cell membrane and cytoplasm but not the nucleus. To address the mechanism of AKT2 activation, we measured in vitro PI 3-kinase activity in 43 ovarian cancer specimens, including the 33 cases displaying elevated AKT2 activation. High levels of PI 3-kinase activity were observed in 20 cases, 15 of which also exhibited AKT2 activation. The remaining five cases displayed elevated AKT1 activation. Among the cases with elevated AKT2, but not PI 3-kinase activity (18 cases), three showed down-regulation of PTEN protein expression. Inhibition of PI 3-kinase/AKT2 by wortmannin or LY294002 induces apoptosis in ovarian cancer cells exhibiting activation of the PI 3-kinase/AKT2 pathway. These findings demonstrate for the first time that activation of AKT2 is a common occurrence in human ovarian cancer and that PI 3-kinase/Akt pathway may be an important target for ovarian cancer intervention.

Atypical antipsychotic drugs (APDs), all of which are relatively more potent as serotonin (5-HT)(2A) than dopamine D(2) antagonists, may improve negative symptoms and cognitive dysfunction in schizophrenia, in part, via increasing cortical dopamine release. 5-HT(1A) agonism has been also suggested to contribute to the ability to increase cortical dopamine release. The present study tested the hypothesis that clozapine, olanzapine, risperidone, and perhaps other atypical APDs, increase dopamine release in rat medial prefrontal cortex (mPFC) via 5-HT(1A) receptor activation, as a result of the blockade of 5-HT(2A) and D(2) receptors. M100907 (0.1 mg/kg), a 5-HT(2A) antagonist, significantly increased the ability of both S:(-)-sulpiride (10 mg/kg), a D(2) antagonist devoid of 5-HT(1A) affinity, and R:(+)-8-OH-DPAT (0.05 mg/kg), a 5-HT(1A) agonist, to increase mPFC dopamine release. These effects of M100907 were abolished by WAY100635 (0.05 mg/kg), a 5-HT(1A) antagonist, which by itself has no effect on mPFC dopamine release. WAY100635 (0.2 mg/kg) also reversed the ability of clozapine (20 mg/kg), olanzapine (1 mg/kg), risperidone (1 mg/kg), and the R:(+)-8-OH-DPAT (0.2 mg/kg) to increase mPFC dopamine release. Clozapine is a direct acting 5-HT(1A) partial agonist, whereas olanzapine and risperidone are not. These results suggest that the atypical APDs via 5-HT(2A) and D(2) receptor blockade, regardless of intrinsic 5-HT(1A) affinity, may promote the ability of 5-HT(1A) receptor stimulation to increase mPFC DA release, and provide additional evidence that coadministration of 5-HT(2A) antagonists and typical APDs, which are D(2) antagonists, may facilitate 5-HT(1A) agonist activity.

Calcitriol, or 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D3) is a well-known endocrine regulator of calcium homeostasis. More recently, local calcitriol production by immune cells was shown to exert autocrine or paracrine immunomodulating effects. Immune cells that produce calcitriol also express the vitamin D receptor (VDR) and the enzymes needed to metabolize vitamin D3 (1α-, 25-, and 24-hydroxylases). Studies of animal models and cell cultures showed both direct and indirect immunomodulating effects involving the T cells, B cells, and antigen-presenting cells (dendritic cells and macrophages) and affecting both innate and adaptive immune responses. The overall effect is a switch from the Th1/Th17 response to the Th2/Treg profile. The immunomodulating effects of vitamin D may explain the reported epidemiological associations between vitamin D status and a large number of autoimmune and inflammatory diseases. Such associations have been suggested by observational studies not only in rheumatoid arthritis, lupus, inflammatory bowel disease, and type 1 diabetes; but also in infections, malignancies, transplant rejection, and cardiovascular disease. In animal models for these diseases, vitamin D supplementation has been found to produce therapeutic effects. Thus, vitamin D is a key focus for public health efforts and may hold promise for the treatment of dysimmune diseases.

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid hormone that renders dendritic cells (DCs) tolerogenic, favoring the induction of regulatory T cells. Induction of DCs with tolerogenic properties by 1,25(OH)(2)D(3) is associated with increased selective expression of immunoglobulin-like transcript 3 (ILT3), suggesting its involvement in the immunoregulatory properties of this hormone. Here we show an in vivo correlate of the increased ILT3 expression on DCs in healing psoriatic lesions following topical treatment with the 1,25(OH)(2)D(3) analog calcipotriol. Analysis of DC subsets reveals a differential regulation of ILT3 expression by 1,25(OH)(2)D(3), with a marked up-regulation in myeloid DCs but no effect on its expression by plasmacytoid DCs. A regulatory role for ILT3 expressed on DCs is indicated by the increased interferon-gamma (IFN-gamma) secretion promoted by anti-ILT3 addition to cultures of DCs and T cells, but this effect is blunted in 1,25(OH)(2)D(3)-treated DCs, suggesting ILT3-independent mechanisms able to regulate T-cell activation. Although ILT3 expression by DCs is required for induction of regulatory T cells, DC pretreatment with 1,25(OH)(2)D(3) leads to induction of CD4(+)Foxp3(+) cells with suppressive activity irrespective of the presence of neutralizing anti-ILT3 monoclonal antibody (mAb), indicating that ILT3 expression is dispensable for the capacity of 1,25(OH)(2)D(3)-treated DCs to induce regulatory T cells.

Of 133 patients with advanced urothelial tract cancer given methotrexate (MTX), vinblastine (VBL), Adriamycin (ADR) (doxorubicin; Adria Laboratories, Columbus, OH), and cisplatin (DDP) (M-VAC regimen), significant tumor regression occurred in 72% +/- 8% of 121 with transitional cell carcinoma (TCC) evaluable for response. Complete remission (CR) was achieved in 36% +/- 9% of patients, of whom 11% required the addition of surgical resection of residual disease. Although 68% of CR patients have relapsed, CR median survival will exceed 38 months compared with 11 months for partial (36%) and minor (6%) responders, and 8 months for nonresponders: 2-year and 3-year survivals were 68% and 55%, respectively, versus 0% to 7% for the remaining patients. Sixteen percent of responders developed brain lesions, half of whom had no systemic relapse at the time of progression. Three patients with non-TCC histologies did not respond. In 32 patients who had pathologic restaging, the clinical (T) understaging (T less than pathologic [P] restaging) error was 35%. Although all metastatic sites showed evidence of tumor regression, CR was noted more frequently in lung, in intraabdominal lymph nodes and masses, and in bone (24% to 35%); the rate for hepatic lesions was 15%. There were 52% of 21 N3-4M0 patients who achieved CR versus 33% of 100 with N0-+M+ lesions. Toxicity was significant with 4 (3%) drug-related deaths, 25% incidence of nadir sepsis, 58% greater than or equal to 3+ myelosuppression, and 49% with mucositis. Responsiveness of metastasis in various sites, patterns of relapse, and the usefulness of the new CR response criteria are reported, as is the current status of cisplatin and methotrexate combination regimens.

BACKGROUND

The development of plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in Alzheimer's disease (AD). Alterations in ceramides and sphingomyelins have been postulated to play a role in amyloidogensis and inflammatory stress related neuronal apoptosis; however few studies have conducted a comprehensive analysis of the sphingolipidome in AD plasma using analytical platforms with accuracy, sensitivity and reproducibility.

RESULTS

We prospectively analyzed plasma from 26 AD patients (mean MMSE 21) and 26 cognitively normal controls in a non-targeted approach using multi-dimensional mass spectrometry-based shotgun lipidomics to determine the levels of over 800 molecular species of lipids. These data were then correlated with diagnosis, apolipoprotein E4 genotype and cognitive performance. Plasma levels of species of sphingolipids were significantly altered in AD. Of the 33 sphingomyelin species tested, 8 molecular species, particularly those containing long aliphatic chains such as 22 and 24 carbon atoms, were significantly lower (p<0.05) in AD compared to controls. Levels of 2 ceramide species (N16:0 and N21:0) were significantly higher in AD (p<0.05) with a similar, but weaker, trend for 5 other species. Ratios of ceramide to sphingomyelin species containing identical fatty acyl chains differed significantly between AD patients and controls. MMSE scores were correlated with altered mass levels of both N20:2 SM and OH-N25:0 ceramides (p<0.004) though lipid abnormalities were observed in mild and moderate AD. Within AD subjects, there were also genotype specific differences.

CONCLUSIONS

In this prospective study, we used a sensitive multimodality platform to identify and characterize an essentially uniform but opposite pattern of disruption in sphingomyelin and ceramide mass levels in AD plasma. Given the role of brain sphingolipids in neuronal function, our findings provide new insights into the AD sphingolipidome and the potential use of metabolomic signatures as peripheral biomarkers.

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.

Fluid shear stress alters the morphology and function of the endothelium by activating several kinases. Furthermore, shear stress potently inhibits apoptosis of endothelial cells. Since activation of Akt kinase has been shown to prevent cell death, we investigated the effects of shear stress on Akt phosphorylation. To test the hypothesis that shear stress interacts with the Akt kinase pathway, human umbilical venous endothelial cells were exposed to laminar shear stress (15 dyne/cm2). Western blotting with specific antibodies against the phosphorylated Akt demonstrated a time-dependent stimulation of Akt phosphorylation by shear stress with a maximal increase up to 6-fold after 1 hour of shear stress exposure. The stimulation of Akt phosphorylation by shear stress thereby seemed to be mediated by the phosphoinositide 3-OH kinase (PI3K), as evidenced by the significant inhibition of shear stress-induced Akt phosphorylation by the PI3K inhibitors wortmannin (20 nmol/L) and Ly294002 (10 micromol/L). In addition, pharmacological inhibition of P13K reduced the antiapoptotic effect of shear stress against growth factor depletion-induced apoptosis. Most important, overexpression of a dominant-negative Akt mutant significantly inhibited the apoptosis-suppressive effect of shear stress against serum depletion-induced apoptosis, thus indicating the direct involvement of shear stress-induced Akt phosphorylation for inhibition of endothelial cell apoptosis. These results define a novel shear stress-stimulated signal transduction pathway, namely, activation of the serine/threonine kinase Akt, which may contribute to the profound changes in endothelial morphology and function by shear stress.

BACKGROUND

Current unitage for the calciferols suggests that equimolar quantities of vitamins D(2) (D2) and D(3) (D3) are biologically equivalent. Published studies yield mixed results.

OBJECTIVE

The aim of the study was to compare the potencies of D2 and D3.

METHODS

The trial used a single-blind, randomized design in 33 healthy adults. Calciferols were dosed at 50,000 IU/wk for 12 wk. Principal outcome variables were area under the curve for incremental total 25-hydroxyvitamin D [25(OH)D] and change in calciferol content of sc fat.

RESULTS

Incremental mean (sd) 25(OH)D area under the curve at 12 wk was 1366 ng · d/ml (516) for the D2-treated group and 2136 (606) for the D3 (P < 0.001). Mean (sd) steady-state 25(OH)D increments showed similar differences: 24 ng/ml for D2 (10.3) and 45 ng/ml (16.2) for D3 (P <0.001). Subcutaneous fat content of D2 rose by 50 μg/kg in the D2-treated group, and D3 content rose by 104 μg/kg in the D3-treated group. Total calciferol in fat rose by only 33 ng/kg in the D2-treated, whereas it rose by 104 μg/kg in the D3-treated group. Extrapolating to total body fat D3, storage amounted to just 17% of the administered dose.

CONCLUSIONS

D3 is approximately 87% more potent in raising and maintaining serum 25(OH)D concentrations and produces 2- to 3-fold greater storage of vitamin D than does equimolar D2. For neither was there evidence of sequestration in fat, as had been postulated for doses in this range. Given its greater potency and lower cost, D3 should be the preferred treatment option when correcting vitamin D deficiency.

The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101. The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein. DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region. Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA. Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending. Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin. Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.

The upstream cleavage site of group I self-splicing introns is identified by an absolutely conserved U.G base-pair within a double helix. Mutant introns with a wobble C.A substitute are catalytically active, but all other combinations of nucleotides at these positions abolish splicing, suggesting that an unusual RNA structure generated by the wobble pair is recognized by the catalytic intron core. The solution structure of a 20-mer oligonucleotide containing a UUCG tetraloop hairpin and a U.G wobble pair within a double helix was determined by NMR spectroscopy without any assumptions on RNA conformation. Isotopically (15N/13C)-labelled RNA was used to collect an unusually large number of experimental constraints (703 in total, corresponding to approximately 35 constraints per nucleotide) leading to the determination of a structure with very high precision (overall root-mean-square-deviation (rmsd) between 20 converged structures 1.22 A, local rmsd 0.6 A for the tetraloop and 0.85 A for the stem). Analysis of the double helical structure at the conserved U.G wobble pair reveals local distortions from the regular A-form pattern, that may constitute the characteristic feature of U.G wobble pair recognized by the group I intron core and by amino acyl tRNA synthetases. Re-examination of the previously determined tetraloop structure reveals a novel U.G base-pair with a syn guanosine and hydrogen bonding contacts involving both base protons and a sugar 2'-OH. This explains the great stability of RNA UUCG loops when compared with DNA loops of identical sequence, and is one of the first NMR observations of RNA 2'-OH resonances.

Poliovirus RNA that has been derivatized at the 3'-end with NaIO(4)-NaB(3)H(4) yields, after hydrolysis with alkali or RNase T2, predominantly labeled residues of modified adenosine; no labeled nucleoside derivative is produced by digestion with RNase A or RNase T1. The 3'-terminal bases of the RNA are, therefore,...ApA(OH). Hydrolyzates of poliovirus [(32)P]RNA, after exhaustive digestion with RNase T1 or RNase A, contain, besides internal oligonucleotides, polynucleotides resistant to further action of ribonucleases T1 and A, respectively; these polynucleotides were isolated by membrane-filter binding or ion-exchange chromatography. The sequence of the T1-resistant polynucleotide was determined to be (Ap)(n)A(OH), that of the RNase A-resistant polynucleotide was GpGp(Ap)(n)A(OH). The chain length (n) of the polyadenylic acid, as analyzed by different methods, averages 89 nucleotides. Gel electrophoresis revealed heterogeneity of the size of poly(A). Poliovirus RNA, when labeled in vitro at the 3'-end, contains [3'-(3)H]poly(A); when labeled in vivo with [(3)H]A, it contains [(3)H](Ap)(n)A(OH). The data establish that... YpGpGp(Ap)([unk])A(OH) is the 3'-terminal sequence of poliovirus RNA, Type 1 (Mahoney). Since this mammalian virus reproduces in the cell cytoplasm, these observations may modify prior interpretations of the function of polyadenylate ends on messenger RNAs.