BACKGROUND

beta-Blocker therapy has been shown to increase ejection fraction in patients with heart failure of idiopathic etiology. However, in patients with heart failure of ischemic etiology, the effects of this treatment on left ventricular function remain uncertain, as do the effects on exercise performance and symptoms.

RESULTS

This study investigated the effects of carvedilol, a beta-blocker with alpha 1-blocking properties, on left ventricular size and function, maximal and submaximal exercise performance, and symptoms in 415 patients with stable heart failure of ischemic etiology (ejection fraction < 45%). After a 2- to 3-week run-in phase on open-label low-dose carvedilol, patients were randomized to continued treatment with carvedilol (up to 25 mg BID) or to matching placebo. After 6 months, left ventricular ejection fraction measured by radionuclide ventriculography had increased by 5.2% (2P < .0001) in the carvedilol group compared with the placebo group, and left ventricular end-systolic and end-diastolic dimensions measured by two-dimensionally guided M-mode echocardiography had decreased by 2.6 mm (2P = .0005) and 1.3 mm (2P = .05), respectively. There were no significant changes in either treadmill exercise duration or 6-minute walk distance between carvedilol and placebo groups (both 2P>> .1); in the carvedilol group, exercise performance was therefore maintained with a 23% lower rate-pressure product. Symptoms assessed by the New York Heart Association (NYHA) scale and the Specific Activity Scale (SAS) were unchanged in two thirds of patients in both groups, but there was a small excess of patients whose symptoms worsened and a deficit of patients whose symptoms improved among those assigned carvedilol (NYHA, 2P = .05; SAS, 2P = .02).

CONCLUSIONS

In patients with heart failure of ischemic etiology, 6-month treatment with carvedilol improved left ventricular function and maintained exercise performance at a lower rate-pressure product, but symptoms assessed by functional class were slightly worsened. A larger-scale trial is now required to determine whether this treatment will reduce serious morbidity and mortality from heart failure.

OBJECTIVE

To investigate in vitro if P-glycoprotein (P-gp) transport can differentiate between antibiotic drugs exhibiting increased active renal clearance (CL(r)) in cystic fibrosis (CF) patients (i.e., dicloxacillin, trimethoprim) and drugs that do not exhibit this phenomenon (i.e.. cefsulodin, sulfamethoxazole).

METHODS

Transport studies were carried out in MDCK (wild type) and MDR1-MDCK (P-gp overexpressing) cells that were grown to confluence on Transwell inserts. [14C]-mannitol transport and transepithelial electrical resistance (TEER) were measured to validate the integrity of the cells. Drug concentrations were analyzed using HPLC.

RESULTS

Dicloxacillin and trimethoprim are substrates of P-gp (B->>A/A->>B ratios in MDR1-MDCK cells are 32 and 50, respectively). P-gp inhibitors (i.e., GG918, cyclosporine, ketoconazole, vinblastine) decreased the B->>A transport of dicloxacillin and trimethoprim and increased the A->>B transport of trimethoprim while non-P-gp inhibitors (e.g., PAH) had no effect. In contrast, cefsulodin and sulfamethoxazole are not substrates of P-gp (B->>sA/A->>B values in MDCK and MDR1-MDCK cells are -1).

CONCLUSIONS

Our in vitro studies suggest that P-glycoprotein may play a role in increasing renal clearance of drug substrates in CF patients. Dicloxacillin and trimethoprim. which are both substrates of P-gp, show increased active renal clearance in CF patients while cefsulodin and sulfamethoxazole, which are not P-gp substrates, do not show increased active renal clearance in CF patients.

Various mechanisms can contribute to cellular resistance to doxorubicin. These include expression of the multidrug transporter P-glycoprotein (product of the mdr1 gene [also known as PGY1], Mrp (multidrug resistance-associated protein), the p110 major vault protein, altered glutathione metabolism, and altered levels or activity of topoisomerase II (Topo II). We reported recently that single-step treatment of human MES-SA sarcoma cells with 40 nM doxorubicin resulted in selection of spontaneous mutants at a rate of 1.8 x 10(-6) per cell generation. All individually selected mutants manifested the multidrug-resistant phenotype, related to activation of the mdr1 gene.

Luria and Delbrück fluctuation analysis was performed with MES-SA cells to determine the mutation rate and the nature and mechanisms of resistance after single-step selection with doxorubicin in the presence of the cyclosporin PSC 833, a potent modulator of multidrug resistance.

Ten flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 8 x 10(6) cells. After reseeding in 96-well plates, the populations were treated with 40 nM doxorubicin and 2 microM PSC 833 for 3 weeks. Surviving colonies were scored, individually harvested, and propagated. The drug-resistant phenotype was assessed by the tetrazolium dye (MTT) cytotoxicity assay and by monitoring cellular glutathione content and radiolabeled drug accumulation. Coupled reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate mdr1, MRP, Topo II alpha, and Topo II beta gene expression. Topo II, P-glycoprotein, and p110 levels were examined by immunoblotting or immunocytochemistry. Topo II activity was assessed by decatenation of kinetoplast DNA, and etoposide-induced cleavable complex formation was studied by the potassium-sodium dodecyl sulfate precipitation assay.

Mutations were detected at a rate of 2.5 x 10(-7) per cell generation. Analysis of variance indicates that spontaneous mutations, rather than changes in cellular function, conferred resistance to doxorubicin and PSC 833. None of the isolated clones expressed mdr1 messenger RNA or P-glycoprotein, and none exhibited an increase in MRP expression. No alterations were found in cellular glutathione content, intracellular accumulations of daunorubicin and etoposide, levels of p110 protein, or levels of Topo II beta transcripts. However, a significant decrease in Topo II alpha messenger RNA and protein was found in all examined clones, as well as decreased Topo II catalytic activity and reduced cleavable complex formation in the presence of etoposide.

PSC 833 co-selection reduced the mutation rate for doxorubicin-selected resistance by 10-fold and suppressed the emergence of mdr1 mutants. Survival of cells exposed to doxorubicin and PSC 833 occurs by selection of spontaneously arising mutants that exhibit altered Topo II alpha expression.

Our results suggest that treatment with multidrug resistance modulators such as PSC 833 together with multidrug resistance-related cytotoxins may suppress the activation of mdr1 and prevent the emergence of resistant cancer cell clones with the multidrug-resistant phenotype.

The prevalence of obesity and related disease risk is high in black South African (SA) women, possibly influenced by the dietary transition associated with urbanization. This study explored interactions between dietary fat intake and the tumor necrosis factor-alpha (TNFA) -308 G/A polymorphism on obesity, insulin resistance, and serum lipid concentrations in urbanized black SA women. Normal-weight (n = 105) and obese (n = 118) women underwent measurements of body composition, fat distribution, fasting serum lipids, glucose and insulin concentrations, and dietary intake. Participants were genotyped for the functional TNFA -308 G/A polymorphism. The genotype or allele frequency of the TNFA -308 G/A polymorphism did not differ between the BMI groups. However, when dietary fat intake was 30% of total energy intake [percentage energy (%E)], the odds of being obese with the TNFA GA+AA genotype was only 12% of that with GG, but increasing intake of dietary fat (%E) was associated with a significantly faster rate of increase in obesity risk in women with the TNFA GA+AA genotype compared with those with the GG genotype (P = 0.036). There were significant diet-gene interactions between alpha-linolenic acid (%E) and the total cholesterol:HDL-cholesterol ratio (P = 0.036), and PUFA (%E) and LDL cholesterol levels (P = 0.026), with participants with the A allele being more responsive to changes in relative fat intake. The TNFA -308 G/A polymorphism modified the relationship between dietary fat intake, obesity risk, and serum lipid concentrations in black SA women.

Female rats exhibit greater intake and motivation to self-administer cocaine. In females but not males, isolation by itself is a stressor, which could lead to increased drug intake. Therefore, we hypothesized that social housing would buffer against stress and reduce the motivation to self-administer cocaine primarily in females. Male and female Sprague-Dawley rats were housed individually or in same-sex pairs. The individually housed rats and one of each pair were allowed to self-administer (SA) a low dose of cocaine (0.2 mg/kg/inf) on a fixed ratio (FR1) schedule for one week. Motivation for cocaine SA was measured for an additional 2 weeks on a progressive ratio schedule. Isolated females had greater cocaine-intake on the FR1 schedule and greater motivation to take cocaine than males. Pair-housing in females, but not males, attenuated the motivation to take cocaine. Isolated females, but not males, showed escalation of their motivation to take cocaine, which was attenuated by pair housing of females. Concluding, the motivation to take cocaine escalates in females but not males, and pair-housing of females attenuates this escalation.

The objective of this study was to assess family physicians' knowledge, opinions and practices regarding oral cancers in the state of Maryland, USA. A 40-item, self-administered questionnaire was mailed to all members of the Maryland Academy of Family Physicians. Unweighted data (n=240) were analyzed using SAS and SUDAAN software; results were evaluated using an alpha < or =0.05. Family physicians (FPs) were aware of the major risk factors for oral cancers, but misinformation existed about the non-risk factors. Approximately 77% asked their patients the eight questions related to risk factors for oral cancer when taking a medical history but less than 24% provided an oral cancer examination to patients 40 years of age and over. Nearly 64% were interested in a continuing education course about oral cancer. This survey identified gaps in knowledge and practices among FPs but it is encouraging that they expressed interest in continuing education courses on this topic.

ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, we have undertaken biochemical and structural studies of fragments of ZFY. We describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal beta--hairpin and N-terminal alpha-helix (beta beta alpha motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the "jumping-linker" model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX4HX3-hydrophobic residue-X3. Analogous use of a hydrophobic residue in a linker that spans the minor groove has recently been described in crystallographic and 3D NMR studies of homeodomain-DNA complexes. The proposed model of ZFY is supported in part by the hydroxyl radical footprint of the TFIIIA-DNA complex [Churchill, M.E.A., Tullius, T.D., & Klug, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5528-5532].

OBJECTIVE

Our objectives were to study the extent of docetaxel binding to plasma in the presence and absence of its excipient, polysorbate 80 (Tween 80; Imperial Chemical Industries PLC, London, United Kingdom), in vitro and to evaluate the pharmacokinetics of unbound docetaxel in vivo.

METHODS

Equilibrium dialysis was used for determination of the fraction unbound (f(u)) docetaxel and was applied to study the pharmacokinetic behavior of unbound docetaxel in 23 patients with cancer receiving an intravenous infusion of the drug formulated in polysorbate 80 (Taxotere; Aventis Pharma SA, Vitry-sur-Seine Cedex, France).

RESULTS

Polysorbate 80, added at clinically relevant concentrations (up to 1.0 microL/mL), increased f(u) in vitro by 13% (7.84% +/- 0.0752% versus 6.95% +/- 0.0678%, P <.00001). Similarly, f(u) calculated on the basis of the observed area under the plasma concentration-time curve (AUC) values [f(u)(AUC)] in vivo was 12% higher than f(u) in pretreatment samples [f(u)(pre)] (6.00% +/- 1.03% versus 5.49% +/- 1.01%, P =.038). Of various serum proteins evaluated, only alpha(1)-acid glycoprotein was significantly related to f(u) (P <.0018), with higher f(u) in the presence of lower protein levels. Total docetaxel clearance was related to alpha(1)-acid glycoprotein (R(2) = 0.13, P =.058), f(u)(pre) (R(2) = 0.15, P =.039), and f(u)(AUC) (R(2) = 0.29, P =.0048).

CONCLUSIONS

This study demonstrates that the plasma binding of docetaxel is influenced by both alpha(1)-acid glycoprotein and its formulation vehicle. Further investigation is required to resolve the potential clinical significance of these observations.

The SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis) syndrome (SaS) includes different skeletal manifestations such as recurrent multifocal osteomyelitis, osteitis and arthritis, which are frequently associated with different forms of skin pustulosis (palmoplantar pustulosis, pustular psoriasis and severe acne). This syndrome is strictly related to the spondyloarthopathies (particularly to psoriatic arthritis) and many SaS cases fulfil the classification criteria for the spondyloarthopathies. Because SaS is an uncommon disease, current knowledge regarding its therapy is based on limited experiences gained by treating mainly small groups of patients. As a consequence, its treatment is still empiric. Several drugs (including NSAIDs, corticosteroids, sulfasalazine, methotrexate, ciclosporin, leflunomide, calcitonin and so on) have been administered and obtained conflicting results. The use of antibiotics, due to the isolation of Propionibacterium acnes from the bone biopsies of several subjects with SaS, has not represented a turning point in therapy, although some patients are responsive to this treatment. Initial reports concerning the administration of bisphosphonates (pamidronate and zoledronic acid) and of an anti-TNF-alpha agent (infliximab) are very promising for the future. In any case, larger, multi-centre, controlled, double-blind studies are required to emerge from the present pioneering phase.

BACKGROUND

High-throughput sequencing technologies place ever increasing demands on existing algorithms for sequence analysis. Algorithms for computing maximal exact matches (MEMs) between sequences appear in two contexts where high-throughput sequencing will vastly increase the volume of sequence data: (i) seeding alignments of high-throughput reads for genome assembly and (ii) designating anchor points for genome-genome comparisons.

RESULTS

We introduce a new algorithm for finding MEMs. The algorithm leverages a sparse suffix array (SA), a text index that stores every K-th position of the text. In contrast to a full text index that stores every position of the text, a sparse SA occupies much less memory. Even though we use a sparse index, the output of our algorithm is the same as a full text index algorithm as long as the space between the indexed suffixes is not greater than a minimum length of a MEM. By relying on partial matches and additional text scanning between indexed positions, the algorithm trades memory for extra computation. The reduced memory usage makes it possible to determine MEMs between significantly longer sequences.

BACKGROUND

Source code for the algorithm is available under a BSD open source license at http://compbio.cs.princeton.edu/mems. The implementation can serve as a drop-in replacement for the MEMs algorithm in MUMmer 3.

Kv4.2 is the major voltage-gated K(+) (Kv) channel alpha subunit responsible for the somatodendritic transient or A-type current I(SA) that activates at subthreshold membrane potentials. Stable association of Kv4.2 with diverse auxiliary subunits and reversible Kv4.2 phosphorylation regulate I(SA) function. Two classes of auxiliary subunits play distinct roles in modulating the biophysical properties of Kv4.2: dipeptidyl-peptidase-like type II transmembrane proteins typified by DPPX-S, and cytoplasmic Ca(2+) binding proteins known as K(+) channel interacting proteins (KChIPs). Here, we characterize the convergent roles that DPPX-S and KChIPs play as component subunits of Kv4.2 channel complexes. We coexpressed DPPX-S with Kv4.2 in heterologous cells and found a dramatic redistribution of Kv4.2, releasing it from intracellular retention and allowing plasma membrane expression, as well as altered Kv4.2 phosphorylation, detergent solubility, and stability. These changes are remarkably similar to those obtained upon coexpression of Kv4.2 with the structurally distinct KChIPs1-3 auxiliary subunits. KChIP4a, which negatively affects the impact of other KChIPs on Kv4.2, also inhibits the effects of DPPX-S, consistent with the formation of a ternary complex of Kv4.2, DPPX-S, and KChIPs early in channel biosynthesis. Tandem MS analyses reveal that coexpression with DPPX-S or KChIP2 leads to a pattern of Kv4.2 phosphorylation in heterologous cells similar to that observed in brain, but lacking in cells expressing Kv4.2 alone. In conclusion, transmembrane DPPX-S and cytoplasmic KChIPs, despite having distinct structures and binding sites on Kv4.2, exert similar effects on Kv4.2 trafficking, but distinct effects on Kv4.2 gating.

BACKGROUND

Mental health problems and risk behaviours among young people are of great public health concern. Consequently, within the VII Framework Programme, the European Commission funded the Saving and Empowering Young Lives in Europe (SEYLE) project. This Randomized Controlled Trial (RCT) was conducted in eleven European countries, with Sweden as the coordinating centre, and was designed to identify an effective way to promote mental health and reduce suicidality and risk taking behaviours among adolescents.

OBJECTIVE

To describe the methodological and field procedures in the SEYLE RCT among adolescents, as well as to present the main characteristics of the recruited sample.

METHODS

Analyses were conducted to determine: 1) representativeness of study sites compared to respective national data; 2) response rate of schools and pupils, drop-out rates from baseline to 3 and 12 month follow-up, 3) comparability of samples among the four Intervention Arms; 4) properties of the standard scales employed: Beck Depression Inventory, Second Edition (BDI-II), Zung Self-Rating Anxiety Scale (Z-SAS), Strengths and Difficulties Questionnaire (SDQ), World Health Organization Well-Being Scale (WHO-5).

RESULTS

Participants at baseline comprised 12,395 adolescents (M/F: 5,529/6,799; mean age=14.9±0.9) from Austria, Estonia, France, Germany, Hungary, Ireland, Israel, Italy, Romania, Slovenia and Spain. At the 3 and 12 months follow up, participation rates were 87.3% and 79.4%, respectively. Demographic characteristics of participating sites were found to be reasonably representative of their respective national population. Overall response rate of schools was 67.8%. All scales utilised in the study had good to very good internal reliability, as measured by Cronbach's alpha (BDI-II: 0.864; Z-SAS: 0.805; SDQ: 0.740; WHO-5: 0.799).

CONCLUSIONS

SEYLE achieved its objective of recruiting a large representative sample of adolescents within participating European countries. Analysis of SEYLE data will shed light on the effectiveness of important interventions aimed at improving adolescent mental health and well-being, reducing risk-taking and self-destructive behaviour and preventing suicidality.

BACKGROUND

US National Institute of Health (NIH) clinical trial registry (NCT00906620) and the German Clinical Trials Register (DRKS00000214).

Dyslexia is a common learning disability exhibited as a delay in acquiring reading skills despite adequate intelligence and instruction. Reading single real words (real-word reading, RWR) is especially impaired in many dyslexics. We performed a genome scan, using variance components (VC) linkage analysis and Bayesian Markov chain Monte Carlo (MCMC) joint segregation and linkage analysis, for three quantitative measures of RWR in 108 multigenerational families, with follow up of the strongest signals with parametric LOD score analyses. We used single-word reading efficiency (SWE) to assess speed and accuracy of RWR, and word identification (WID) to assess accuracy alone. Adjusting SWE for WID provided a third measure of RWR efficiency. All three methods of analysis identified a strong linkage signal for SWE on chromosome 13q. Based on multipoint analysis with 13 markers we obtained a MCMC intensity ratio (IR) of 53.2 (chromosome-wide P < 0.004), a VC LOD score of 2.29, and a parametric LOD score of 2.94, based on a quantitative-trait model from MCMC segregation analysis (SA). A weaker signal for SWE on chromosome 2q occurred in the same location as a significant linkage peak seen previously in a scan for phonological decoding. MCMC oligogenic SA identified three models of transmission for WID, which could be assigned to two distinct linkage peaks on chromosomes 12 and 15. Taken together, these results indicate a locus for efficiency and accuracy of RWR on chromosome 13, and a complex model for inheritance of RWR accuracy with loci on chromosomes 12 and 15.

OBJECTIVE

Immortalized cell lines representing fibroblast cells from corneal stroma would facilitate studies of corneal cell biology and injury response.

METHODS

Primary cultures of cells derived from mouse corneal stroma were transfected with a human telomerase reverse transcriptase (hTERT) expression construct to maximize chances of cellular immortalization. A resulting cell line was analyzed for telomerase activity, cell growth characteristics, senescence and gene expression patterns. Specific responses to transforming growth factor beta (TGF-beta) were also analyzed.

RESULTS

An immortalized cell line was derived and was named MK/T-1. MK/T-1 cells show no signs of cellular senescence or transformation at over 100 passages. Telomerase activity was significantly higher in MK/T-1 cells as compared to the parental cell cultures. However, relative telomere length (RTL) in the MK/T-1 and parental cells was not significantly different. Senescence associated beta-galactosidase (SA-beta-Gal) activity was not detected in late passage MK/T-1 cells while the parental cells had already upregulated SA-beta-Gal at high levels by passage 9. The MK/T-1 cells express vimentin, tubulin, lumican, mimecan, decorin and collagen I, but not keratocan. Exposure of the MK/T-1 cells to TGF-beta induces the expression of smooth muscle alpha-actin (ASMA), the activation of MAP Kinase (p38-MAPK) and morphological changes consistent with cytoskeletal reorganization.

CONCLUSIONS

MK/T-1 cells represent an immortalized fibroblast cell line derived using cultures from corneal stroma cell preparations. Expression of hTERT may contribute to immortalization of the MK/T-1 cells by a mechanism other than increases in RTL. MK/T-1 cells may be a useful model in which to study the responses of corneal fibroblast cells to cytokines and other diverse environmental factors in vitro.

Influenza viruses of contrasting receptor specificity have been examined for their ability to infect receptor-modified MDCK cells containing sialyloligosaccharide receptor determinants of defined sequence. Cells were treated with sialidase to remove sialic acid and render them resistant to infection and were then incubated with sialyltransferase and CMP-sialic acid to restore sialic acid in the SA alpha 2,6Gal or SA alpha 2,3Gal linkages. The viruses A/RI/5 + /57 and A/duck/Ukraine/1/63, previously shown to exhibit preferential binding of SA alpha 2,6Gal and SA alpha 2,3Gal linkages, respectively, were found to exhibit differential infection of the receptor-modified cells in accord with their receptor specificity. Coinfection of SA alpha 2,3Gal derivatized cells with a mixture of the two viruses resulted in selective propagation of the SA alpha 2,3Gal-specific A/duck/Ukraine/1/63 virus. The results demonstrate the potential for cell surface receptors to mediate selection of receptor-specific variants of influenza virus.

Small molecules that dimerize proteins in living cells provide powerful probes of biological processes and have potential as tools for the identification of protein targets of natural products. We synthesized 7-alpha-substituted derivatives of beta-estradiol tethered to the natural product biotin to regulate heterodimerization of estrogen receptor (ER) and streptavidin (SA) proteins expressed as components of a yeast three-hybrid system. Addition of an estradiol-biotin chimera bearing a 19-atom linker to yeast expressing DNA-bound ER-alpha or ER-beta LexA fusion proteins and wild-type SA protein fused to the B42 activation domain activated reporter gene expression by as much as 450-fold in vivo (10 muM ligand). Comparative analysis of lower affinity Y43A (biotin Kd approximately 100 pM) and W120A (biotin Kd approximately 100 nM) mutants of SA indicated that moderate affinity interactions can be readily detected with this system. Comparison of a 7-alpha-substituted estradiol-biotin chimera with a structurally similar dexamethasone-biotin chimera revealed that yeast expressing ER proteins can detect cognate ligands with up to 5-fold greater potency and 70-fold higher activity than yeast expressing analogous glucocorticoid receptor (GR) proteins. This approach may facilitate the identification of protein targets of biologically active small molecules screened against genetically encoded libraries of proteins expressed in yeast three-hybrid systems.

Chick embryo fibroblast (CEF)-grown stocks of the WSN strain of influenza A(HINI) contain two variants which were designated F and C for fuzzy and clear plaque morphology on Madin-Darby bovine kidney (MDBK) cells. During growth in MDBK cells plaque-isolated F virus was completely replaced by C virus (L. Noronha-Blob and I.T. Schulze (1976), Virology 69, 314-322). The parental (F) and the mutant (C) viruses contain hemagglutinins which differ in their ability to bind to host cells. In addition, the host cells from which the purified viruses are obtained affect their binding properties. Thus, as compared to MDBK-grown F virus (FBK), MDBK-grown C virus (CBK) produced high amounts of mRNA and high virus yields in MDBK cells. CBK had greater affinity for SA alpha 2,3Gal and SA alpha 2,6Gal linkages on derivatized human erythrocytes than did FBK, independent of whether neuraminidase was present on the virions. CBK was also resistant to components of calf serum which inhibited FBK hemagglutination at 37 degrees. As compared to FBK, CBK had increased ability to bind to both MDBK cells and CEF at 37 degrees in the presence or absence of an inhibitor of neuraminidase. In addition, when cells with virus bound at 0 degrees were transferred to 37 degrees, CBK remained cell associated whereas about 80% of FBK dissociated from both cells. Thus, mutation from F to C increased the ability of the virus to associate with MDBK cell receptors. Studies carried out with F and C viruses from both cells indicated that the expression of the mutation depended in part on the host cells in which the virus was grown and in part on the cells used to measure the binding properties. A model relating these observations to selection of HA variants in nature is presented.

Auxin is a key plant growth regulator that also impacts plant-pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole-3-acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector-triggered immunity was active in YUC1-overexpressing plants, and we observed only minor effects on SA levels and SA-mediated responses. Furthermore, a plant line carrying both the YUC1-overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA-mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA-mediated defenses.

We have examined the expression of MIP-3alpha/CCL20 in oral squamous cell carcinoma (SCC) in vivo and in vitro. In addition, we have investigated whether the expression of MIP-3alpha/CCL20 is regulated by bacterial infection and inflammatory cytokines. In order to determine the mRNA level of MIP-3alpha, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with LightCycler using the double-stranded DNA dye, SYBR Green I. Oral epithelial cells and six SCC cell lines (SCC-9, SAS, BSC-OF, HSC-4, HSC, Ca9-22) were found to express MIP-3alpha mRNA. The expression of MIP-3alpha was upregulated by infection with Actinobacillus actinomycetemcomitans and by stimulation with lipopolysaccharide and TNF-alpha. By in situ hybridization, the detectable MIP-3alpha expression in SCC was localized primarily at the epithelial pearls corresponding to the spinous layer. These results suggest that MIP-3alpha contributes to the oral immunoresponse to bacterial infection, and may be involved in the growth of SCC.

OBJECTIVE

Patients with gliomas frequently present with seizures, but the factors associated with seizure development are still poorly understood. In this study, we assessed peritumoral synaptic network activity in a glioma animal model and tested the contribution of aberrant glutamate release from gliomas on glioma-associated epileptic network activity.

METHODS

In vitro brain slices were made from glioma-implanted mice. Using extracellular field recordings, we analyzed peritumoral epileptiform activity induced by Mg(2+)-free medium in slices from tumor-bearing animals and sham-operated controls. We assessed the effect of sulfasalazine (SAS), a blocker of system and glutamate release, on spontaneous and evoked activity in tumor-associated slices.

RESULTS

Tumor-associated cortical networks were hyperexcitable. The onset latency of Mg(2+)-free-induced epileptiform activity was significantly shorter in tumor-bearing slices, and the incidence of Mg(2+)-free-induced ictal-like events was higher. Block of glutamate release from system decreased the response area of evoked activity and completely blocked Mg(2+)-free-induced ictal-like, but not interictal-like events.

CONCLUSIONS

Control of seizures in patients with gliomas is an essential component of clinical management; therefore, understanding the origin of seizures is vital. This work provides evidence that peritumoral synaptic network activity is disrupted by tumor masses resulting in network excitability. We show that blocking glutamate release via system with SAS, a drug already approved by the U.S. Food and Drug Administration (FDA), can inhibit Mg(2+)-free-induced ictal-like epileptiform events similar to other chemicals used to decrease seizure activity. We, therefore, suggest that further studies should consider SAS a promising agent to aid in the treatment of seizures associated with gliomas.

A new nonadentate ligand, N, N, N1, N1-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA) for a Tb3+ fluorescent complex was synthesized. The Tb3+ complex is strongly fluorescent, having a large fluorescence quantum yield of 1.00 and very long fluorescence lifetime of 2.681 ms in 0.05 M borate buffer of pH 9.1. Streptavidin (SA) was labeled with BPTA by using its succinimidyl monoester, and the BPTA-Tb(3+)-labeled SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb(3+)-labeled SA was also used in competitive-type TR-FIA of bensulfuron-methyl (BSM) in water. The detection limits of these assays are 42 pg/mL for AFP, 70 pg/mL for CEA, and 0.4 ng/mL for BSM. In addition, a new simultaneous measurement method for AFP and CEA in a human serum sample was developed by using 4,4'-bis(1",1",2",2",3",3" -heptafluoro-4",6"-hexanedion-6"-ly)chlorosulfo- o-terphenyl (BHHCT)-Eu(3+)-labeled anti-AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb(3+)-labeled SA. The concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AFP and CEA by TR-FIA with a single-label method. A good correlation was obtained with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.

Little attention has been given to measuring symptoms of separation anxiety (SA) in adulthood. The development of an Adult Separation Anxiety Questionnaire (ASA-27) is described and compared to a previously derived Adult Separation Anxiety Semistructured Interview (ASA-SI). Principal components analysis revealed a coherent construct of SA with high internal consistency (Cronbach's alpha =.95) and sound test-retest reliability (r =.86; P <.001). A receiver operation characteristic (ROC) analysis against the semistructured interview yielded a high area under the curve index (AUC = 0.9) suggesting that the questionnaire is an adequate alternative measure of SA. Results of this study support previous research suggesting that a construct of SA may be readily measured in adults.

Sleep apnea (SA) is defined as intermittent respiratory arrest during sleep and affects up to 20% of the adult population. SA is also associated with an increased incidence of hypertension and peripheral vascular disease. Exposing rodents to intermittent hypoxia during sleep mimics the cyclical hypoxia/normoxia of SA. We have previously shown that in mice and rats intermittent hypoxia induces ET-1 upregulation and systemic hypertension. Furthermore, intermittent hypoxia (IH) in mice increases nuclear factor of activated T cells isoform 3 (NFATc3) transcriptional activity in aorta and mesenteric arteries, whereas the calcineurin/NFAT inhibitor cyclosporin A prevents IH-induced hypertension. More importantly, NFATc3 knockout (KO) mice do not develop IH-induced hypertension. The goals of this study were to determine the role of NFATc3 in IH-induced arterial remodeling and whether IH-induced NFATc3 activation is mediated by ET-1. Oral administration of both a dual (bosentan) and a selective endothelin receptor type A antagonist (PD155080) during 2 days of IH exposure attenuated NFAT activation in aorta and mesenteric arteries. Rho kinase inhibition with fasudil also prevented IH-induced NFAT activation. Mesenteric artery cross-sectional wall thickness was increased by IH in wild-type (WT) and vehicle-treated mice but not in bosentan-treated and NFATc3 KO mice. The arterial remodeling in mesenteric arteries after IH was characterized by increased expression of the hypertrophic NFATc3 target smooth muscle-alpha-actin in WT but not in KO mice. These results indicate that ET-1 is an upstream activator of NFATc3 during intermittent hypoxia, contributing to the resultant hypertension and increased wall thickness.

We previously proposed that, during the infusion of either labeled leucine or its alpha-ketoacid, alpha-ketoisocaproate (KIC), the plasma specific activity (SA) of the transaminated product of the infused tracer ("reciprocal pool SA") may better reflect the intracellular leucine SA than the plasma SA of either infused tracer ("primary pool SA"). To test this hypothesis, 14 dogs were simultaneously infused intravenously with [3H]leucine and [14C]KIC, and blood and tissue compartments were sampled. The ratios of [3H]-leucine to [14C]leucine [( 3H]/[14C]leucine) in mixed tissue proteins and in the intracellular space of striated muscle were the same as the ratio of the isotope infusion rates and similar, although slightly lower (P less than 0.01), than [3H]KIC/[14C]leucine SA (ratio of reciprocal pool SA) in plasma. Plasma [3H]KIC/[14C]leucine SA were essentially identical to the [3H]/[14C] of leucine in 1) mixed liver proteins, 2) intrahepatic free leucine, and 3) fibrin. The [3H]/[14C]leucine in mixed renal proteins and in the intracellular space of kidney and erythrocytes were similar to those of the venous plasma [3H]/[14C]leucine SA. The plasma [3H]KIC and [14C]leucine SA (the reciprocal pool SA) were similar to the SA of [3H]- and [14C]leucine in the intracellular space of all organs investigated with the exception of kidney. Therefore, in postabsorptive dogs, the plasma SA of the transaminated product of the infused labeled KIC or leucine is an excellent predictor of the intracellular leucine SA in all tissues investigated with the exception of kidney.