Variant V6 Research Articles

Distinct Binding and Immunogenic Properties of the Gonococcal Homologue of Meningococcal Factor H Binding Protein

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

Methionine Adenosyltransferase 2B, HuR, and Sirtuin 1 Protein Cross-talk Impacts on the Effect of Resveratrol on Apoptosis and Growth in Liver Cancer Cells

Resveratrol is growth-suppressive and pro-apoptotic in liver cancer cells. Methionine adenosyltransferase 2B (MAT2B) encodes for two dominant variants V1 and V2 that positively regulate growth, and V1 is anti-apoptotic when overexpressed. Interestingly, crystal structure analysis of MAT2B protein (MATβ) protomer revealed two resveratrol binding pockets, which raises the question of the role of MAT2B in resveratrol biological activities. We found that resveratrol induced the expression of MAT2BV1 and V2 in a time- and dose-dependent manner by increasing transcription, mRNA, and protein stabilization. Following resveratrol treatment, HuR expression increased first, followed by SIRT1 and MAT2B. SIRT1 induction contributes to increased MAT2B transcription whereas HuR induction increased MAT2B mRNA stability. MATβ interacts with HuR and SIRT1, and resveratrol treatment enhanced these interactions while reducing the interaction between MATβ and MATα2. Because MATβ lowers the Ki of MATα2 for S-adenosylmethionine (AdoMet), this allowed steady-state AdoMet level to rise. Interaction among MATβ, SIRT1, and HuR increased stability of these proteins. Induction of MAT2B is a compensatory response to resveratrol as knocking down MAT2BV1 potentiated the resveratrol pro-apoptotic and growth-suppressive effects, whereas the opposite occurred with V1 overexpression. The same effect on growth occurred with MAT2BV2. In conclusion, resveratrol induces HuR, SIRT1, and MAT2B expression; the last may represent a compensatory response against apoptosis and growth inhibition. However, MATβ induction also facilitates SIRT1 activation, as the interaction stabilizes SIRT1. This complex interplay among MATβ, HuR, and SIRT1 has not been previously reported and suggests that these proteins may regulate each other's signaling.

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics

Glucose oxidase is an oxidoreductase exhibiting a high β-d-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-d-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4U/mg WT, 47.5U/mg V7; oxygen activity: 172.3U/mg WT, 30.1U/mg V7). V7 is still highly β-d-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications.

504 MICRORNA-124 SUPPRESSES PROSTATE TUMOR GROWTH BY DOWN-REGULATING THE EXPRESSION OF ANDROGEN RECEPTORS AND THEIR ALTERNATIVE SPLICE VARIANTS IN CWR22-RV1 IN-VIVO MODEL

You have accessJournal of UrologyProstate Cancer: Basic Research (III)1 Apr 2013504 MICRORNA-124 SUPPRESSES PROSTATE TUMOR GROWTH BY DOWN-REGULATING THE EXPRESSION OF ANDROGEN RECEPTORS AND THEIR ALTERNATIVE SPLICE VARIANTS IN CWR22-RV1 IN-VIVO MODEL Hao G. Nguyen, Xu-Bao Shi, Lingru Xue, Joy C. Yang, Allen C. Gao, Christopher P. Evans, and Ralph W. DeVere White Hao G. NguyenHao G. Nguyen Sacramento, CA More articles by this author , Xu-Bao ShiXu-Bao Shi Sacramento, CA More articles by this author , Lingru XueLingru Xue Sacramento, CA More articles by this author , Joy C. YangJoy C. Yang Sacramento, CA More articles by this author , Allen C. GaoAllen C. Gao Sacramento, CA More articles by this author , Christopher P. EvansChristopher P. Evans Sacramento, CA More articles by this author , and Ralph W. DeVere WhiteRalph W. DeVere White Sacramento, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1898AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The androgen receptor (AR) and its spliced variants play a central role in the progression to castrate-resistant prostate cancer (CRPC). The unique ability of microRNA to modulate the expression of genes important to prostate cancer progression can be exploited for therapeutic intervention. We hypothesize that microRNA-124 (miR124), unlike the androgen receptor signaling inhibitors (Enzalutamide and Biclutamide), targets both AR and it's alternatively splice variant V7 (AR-V7) in CWR22-Rv1 cells, thus delaying the development of CRPC. METHODS miR124 was introduced into CWR22-Rv1 cells transiently using Lipofectamine 2000 and stably using lenti-virus system. To determine the expression of AR and AR-V7, we used Western Blot analysis with specific antibodies to full-length AR and the variant. Real-time quantitative PCR was employed to evaluate the mRNA level of AR and alternate spliced variants AR-V1, AR-V3, AR-V4, and AR-V7. Xenograft mouse model using subcutaneous injection with CWR22-Rv1 cells stably over-expressing miR-124 was used to evaluate the effect of miR124 on tumor growth in-vivo. RESULTS miR124 was identified as key regulators of AR spliced variants AR-V7 and AR-V3. We showed that miR124 significantly down-regulated the expression of AR-V7 at the post-translational level (P=0.001) and AR-V3 at the post-transcriptional level. It modestly down-regulated full-length AR expression in addition to suppressing the AR signaling targets, including the anti-apoptosis protein Survivin. Xenograft tumors with enhanced expression of miR124 showed significant reduced levels of both full-length AR and AR-V7, resulting in reduced tumor growth (P=0.001). CONCLUSIONS Our study demonstrated, in the pre-clinical setting, the novel finding that miR124 significantly suppresses the full length AR expression and its alternative spliced variants, AR-V7 and AR-V3 and their downstream targets. Thus, miR124 is a potential therapeutic opportunity to delay the development of CRPC in patients treated with Enzalutamide. We have shown that miRNA 124 can be delivered systematically, a prerequisite for its clinical use. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e206-e207 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hao G. Nguyen Sacramento, CA More articles by this author Xu-Bao Shi Sacramento, CA More articles by this author Lingru Xue Sacramento, CA More articles by this author Joy C. Yang Sacramento, CA More articles by this author Allen C. Gao Sacramento, CA More articles by this author Christopher P. Evans Sacramento, CA More articles by this author Ralph W. DeVere White Sacramento, CA More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Researches on the Behaviour of Certain Genotypes of Vegetable Marrow (Cucurbita pepo L. convar. giromontia Alef.) at the Thermic and Hydric Stress

For assessing the behaviour of the vegetable marrow to the abiotic factors, six genotypes ofvegetable marrow were studied (L-201, L-189, L-190, Perfect, Horizon and Happy organized in 3technological variants: V1- culture irrigated by irrigation standards assuring optimal conditions for theplants' growing and development; V2- culture for which small irrigation standards are administered, incritical moments; V3- non-irrigated culture, as of bedding the culture. In the conditions oftechnological variant V3, the plants went through the first phenophases due to the precipitationsaccumulated in May-June; the excessive temperatures and lack of precipitations from June-Julydetermined significant differences between the technological variants and between the genotypes, withrespect to fruits' quality and production potential.

Special green works influence on yield quantity and quality at grape varieties for flavoured and semi-flavored wines from S.C.D.V.V. Blaj.

This paper was done in order to establish the influence of special green works (grapesthinning, partial deleafing and grapes bagging) on yield quantity and quality at the varieties: MuscatOttonel, Neuburger, Traminer roz and Sauvignon blanc, in the ecoclimatic conditions of TA¢rnaveVineyard. Harvesting took place at end of September. To assess the quantity of harvest was set theaverage number of grapes per vine, average weight of a grape and the vine production. Knowing thenumber of vines per hectare the yield per hectare was calculated, expressed in t/ha. For thesedeterminations were made direct measurements in the field. Quality of wine varieties can be expressedby mechanical analysis of the grape, determining composition, structure and grain indices. The qualityof grapes from the vine represents a very important feature of varieties. This refers to the accumulationof reducing sugars and acidity in the grapes. Carbohydrate concentration, performed at full maturity, isa characteristic of grapes variety, vineyard and harvest year. Analyzing production in terms of variety,there is a superior significant difference at Sauvignon blanc compared to the other three varieties,followed by Muscat Ottonel and Traminer roz. Analyzing the interaction of the two factors (variety,special green works), the highest yields were obtained from Sauvignon blanc V3, followed atinsignificant difference by Sauvignon Blanc variants Mt and V2. For all varieties is noted that inrelation to the applied green work, the smallest productions were obtained at V1, this being due to thesmaller number of grapes per vine. The greatest influence on yield differences were exerted by varietyand variety-green work interaction, while the green work has the smallest influence. Thus, SauvignonBlanc stands out with the highest yields, regardless of the green work used. The amount of sugar atMuscat Ottonel does not differ significantly from any other variety, being very close to the othervarieties. Following the accumulation of sugar under the influence of green works, differs significantlysuperior variant V2, which confirms that the partial defoliation favorises the sugar accumulation.Under the combined action of the two factors, stand out also variant V2 of Traminer roz. Consideringthe total acidity of the must, after execution of the green works, there is a decrease of acidity to specialgreen work variants. Cumulative influence variety-green work leads to the highest values of allvariants, Neuburger, followed by Sauvignon Blanc and Muscat Ottonel.

1266P - Retrospective Study of Clinicopathologic Factors Associated with Alk Rearrangement and Survival Outcome in Chinese Patients (PTS) with NSCLC

ABSTRACT Background We tested 400 NSCLC pts for ALK, EGFR and KRAS status and correlated clinical factors with ALK status and overall survival (OS). Methods ALK status was assessed with RACE-PCR, IHC and FISH; EGFR and KRAS status were assessed by direct DNA sequencing. OS was estimated by the Kaplan-Meier method. Results Consecutive, unselected cases from southern (n = 294) and northern (n = 106) China were tested; 31 (7.7%), 105 (26.2%) and 33 (8.2%) pts were ALK + , EGFR+ and KRAS + , respectively. ALK and EGFR aberrations were largely exclusive (P = 0.009); 2 cases were ALK + /EGFR+. ALK+ status was associated with female sex (P = 0.023), non/light-smoking (P = 0.001) and adenocarcinoma (P = 0.017), and was more prevalent in southern (9.2%; 27/294) than northern (3.8%; 4/106) pts. The ALK fusion partner was EML4 in all 31 cases; predominantly variants V1–3 (90.3%, 28/31). In 326 modeled pts, female sex (HR = 2.84; P = 0.022), wild type EGFR (HR = 15.87; P = 0.001), non/light-smoking (HR = 3.95; P = 0.021) and adenocarcinoma (HR = 4.12; P = 0.031) were associated with ALK+. In 187 cases evaluated by IHC and RACE-PCR, IHC sensitivity was 100% (19/19; [5 IHC + ; 10 IHC + +; 4 IHC + ++ ]) and specificity was 79.8% (134/168). All 19 ALK+ cases by RACE-PCR were also ALK+ by FISH. In the first 303 pts, there were no significant OS differences between ALK + , EGFR + , KRAS+ and triple-negative pts, between ALK+ and ALK– pts, or between ALK+ cases matched with non-TKI-treated controls balanced for disease stage, sex, histology and EGFR/KRAS status. In ALK+ pts, non/light-smoking status (P = 0.016) and prior surgery (P = 0.024) were associated with increased OS by log-rank test. In a Cox model, disease stage (P ≤ 0.001 for both stage I and II), and surgical treatment (P = 0.001) were prognostic for OS. Conclusion ALK fusion prevalence was 7.7% overall, and was higher in southern pts. ALK+ was associated with non/light-smoking, adenocarcinoma and female sex. ALK fusion variants were mostly V1–3, which may inform RT-PCR screening. IHC for ALK protein and FISH for ALK rearrangement may be useful for patient selection. ALK+ was not a favorable prognostic factor, although disease stage and smoking history may influence OS in ALK+ NSCLC. Disclosure C. Blanckmeister: Employment: Pfizer. Myself. Compensated. Stock ownership: Pfizer. Myself. J. Chen: Employment: Senior Medical Affairs Therapeutic Area Manager. Pfizer. Myself. Compensated. All other authors have declared no conflicts of interest.

Differential Protein Stability and ALK Inhibitor Sensitivity of EML4-ALK Fusion Variants

ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown. By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied. Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity. Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.

Abstract 4504: Retrospective study of clinicopathologic factors associated with ALK rearrangement and survival outcome in Chinese patients with NSCLC

Abstract Background: ALK rearrangement and EGFR mutation are significant molecular subtypes of non-small cell lung cancer (NSCLC) that are targets for different tyrosine kinase inhibitors (TKIs). We tested a large cohort of NSCLC patients for ALK, EGFR and KRAS abnormalities and correlated clinical factors with the ALK fusion gene and overall survival (OS). Methods: ALK status was detected by RACE sequencing; EGFR and KRAS status were also detected by direct DNA sequencing. OS was estimated by the Kaplan-Meier method. Results: 294 consecutive, unselected, banked NSCLC cases were tested; 27 (9.2%), 79 (29.2%) and 26 (9.6%) patients were ALK-, EGFR- and KRAS-positive, respectively. ALK rearrangement and EGFR mutation were largely exclusive (r=−0.15, P=0.007) although two cases were both ALK- and EGFR-positive. In non-parametric tests, ALK-positivity was associated with female gender (χ2=5.189; P=0.023), non/light-smoking (χ2=6.067; P=0.014) and adenocarcinoma (χ2=4.301; P=0.038). The ALK fusion partner was EML4 in all 27 cases; EML4-ALK fusion variants V1, V2 and V3 (V3a/V3b/V3c) made up the majority (24/27) of EML4-ALK fusions. There were no significant OS differences between ALK-positive, EGFR-mutant, KRAS-mutant and triple-negative groups, between ALK-positive and -negative patients, or when ALK-positive cases were matched 1:2 or 1:1 with (non-TKI-treated) controls balancing for disease stage, gender, histology and EGFR/KRAS status. In ALK-positive patients, bivariate analysis found that non- or light-smoking status (P=0.016) and prior surgery (P=0.024) were associated with increased OS and a multivariate Cox regression model found that disease stage and surgical treatment were prognostic for OS. In a multivariate logistic regression model, female gender (HR=2.793; P=0.041), wild type EGFR (HR=15.147; P=0.001), non/light-smoking (HR=3.572; P=0.020) and adenocarcinoma (HR=5.713; P=0.024) were significantly associated with ALK-rearrangement. Conclusion: Overall, the ALK fusion gene occurred in 9.2% of NSCLC patients, and was associated with non/light-smoking history, adenocarcinoma histology and female gender. ALK fusion variants were mostly V1-3, which may inform ALK screening by RT-PCR. ALK rearrangement was not a favorable prognostic factor in NSCLC, although smoking history may influence OS in ALK-positive patients. A TKI (crizotinib) now exists for ALK-positive NSCLC and may improve OS in these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4504. doi:1538-7445.AM2012-4504

Abstract 4537: Interaction with wild-type (wt) androgen receptor (AR) alters the function of AR splice-variants and response to AR-ligand binding domain inhibition

Abstract Introduction: Androgen ablation induces splicing of AR pre-mRNA to produce constitutively active AR-variants. However, these variants usually co-exist in a cell with wtAR and may form heterodimers with the wtAR. This dimerization results in the activity of the variant being directed via ligand activated AR. In contrast, the AR splice variants may act independent of their association with wtAR. Clinically differentiating these two potential activities is crucial since the former activity would respond to agents targeting the ligand-binding domain (LBD) while the latter would not respond. Purpose: To determine if wtAR accounts for the activity of AR splice variants in cells and the effect co-existence of both variants has on therapy directed at the AR LBD. Methods: We expressed AR splice variants V7 or ARv567es along with wtAR at various ratios in M12 AR-null cells or the variants alone at graded concentrations in LNCaP or CWR22Rv1 cells. AR activity was determined by probasin AR-luciferase in all cells and PSA and TMPRSS2 mRNA in LNCaP and CWR22Rv1 cells. Response to LBD inhibition was determined by the addition of MDV-3100. Expression ratios of wt and variant AR were determined by qt-RTPCR and Western immunoblot. Results: WtAR or AR variants were expressed at ratios ranging from 1:10 to 10:1 in M12 cells. In the absence of wtAR, there was marked elevation ARE-reporter activity when either V7 or ARv567es were expressed and there were no differences in reporter activity when either DHT or MDV-3100 were added_consistent with these variants being constitutively active. WtAR showed minimal ARE-LUC activity until DHT was added. MDV-3100 blocked the DHT-induced ARE-LUC activity in wtAR transfected cells by >95%. As increased concentrations of wtAR were expressed in cells transfected with AR variants, the ligand-independent activity of AR variants was suppressed. However, MDV-3100 could not suppress the constitutive AR activity. Similar results were seen in LNCaP cells. In contrast, for CWR22Rv1 cells, which express primarily the V7 AR variant but have no ligand-independent AR activity and transactivate AR with DHT, MDV-3100 completely suppressed ARE-LUC activity. However, transfection with ARv567es resulted in a >100 fold increase in basal ARE-LUC activity that was not affected by MDV-3100. Summary: These data indicate that the presence of wtAR suppresses AR V7 and ARv567es ligand-independent activity by heterodimerization as previously described. This effect imbues partial responsiveness to MDV-3100. However, as the proportion of AR variant to wtAR is increased, response to MDV-3100 is lost. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4537. doi:10.1158/1538-7445.AM2011-4537

Versican V0 and V1 Direct the Growth of Peripheral Axons in the Developing Chick Hindlimb

Peanut agglutinin-binding disaccharides and chondroitin sulfate mark transient mesenchymal barriers to advancing motor and sensory axons innervating the hindlimbs during chick development. Here we show that the vast majority of these carbohydrates are at the critical stage and location attached to the versican splice variants V0 and V1. We reveal that the isolated isoforms of this extracellular matrix proteoglycan suppress axon extension at low concentrations and induce growth cone collapse and rapid retraction at higher levels. Moreover, we demonstrate that versican V0 and/or V1, recombinantly expressed in collagen-I gels or ectopically deposited in the hindlimbs of chicken embryos in ovo, cause untimely defasciculation and axon stalling. Consequently, severe disturbances of nerve patterning are observed in the versican-treated embryos. Our experiments emphasize the inhibitory capacity of versicans V0 and V1 in axonal growth and evidence for their function as basic guidance cues during development of the peripheral nervous system.

PET Tracers Based on Zirconium-89

Positron emission tomography (PET) imaging with radiolabeled monoclonal antibodies has always been a dynamic area in molecular imaging. With decay half-life (3.3 d) well matched to the circulation half-lives of antibodies (usually on the order of days), (89)Zr has been extensively studied over the last decade. This review article will give a brief overview on (89)Zr isotope production, the radiochemistry generally used for (89)Zr-labeling, and the PET tracers that have been developed using (89)Zr. To date, (89)Zr-based PET imaging has been investigated for a wide variety of cancer-related targets, which include human epidermal growth factor receptor 2, epidermal growth factor receptor, prostate-specific membrane antigen, splice variant v6 of CD44, vascular endothelial growth factor, carbonic anhydrase IX, insulin-like growth factor 1 receptor, among others. With well-developed radiochemistry, commercial availability of chelating agents for (89)Zr labeling, increasingly widely available isotope supply, as well as successful proof-of-principle in pilot human studies, it is expected that PET imaging with (89)Zr-based tracers will be a constantly evolving and highly vibrant field in the near future.

Neointima Formed by Arterial Smooth Muscle Cells Expressing Versican Variant V3 Is Resistant to Lipid and Macrophage Accumulation

Extracellular matrix (ECM) of neointima formed following angioplasty contains elevated levels of versican, loosely arranged collagen, and fragmented deposits of elastin, features associated with lipid and macrophage accumulation. ECM with a low versican content, compact structure, and increased elastic fiber content can be achieved by expression of versican variant V3, which lacks chondroitin sulfate glycosaminoglycans. We hypothesized that V3-expressing arterial smooth muscle cells (ASMC) can be used to form a neointima resistant to lipid and macrophage accumulation associated with hypercholesterolemia. ASMC transduced with V3 cDNA were seeded into ballooned rabbit carotid arteries, and animals were fed a chow diet for 4 weeks, followed by a cholesterol-enriched diet for 4 weeks, achieving plasma cholesterol levels of 20 to 25 mmol/L. V3 neointimae at 8 weeks were compact, multilayered, and elastin enriched. They were significantly thinner (57%) than control neointimae; contained significantly more elastin (118%), less collagen (22%), and less lipid (76%); and showed significantly reduced macrophage infiltration (85%). Mechanistic studies demonstrated that oxidized low-density lipoprotein stimulated the formation of a monocyte-binding ECM, which was inhibited in the presence of V3 expressing ASMC. These results demonstrate that expression of V3 in vessel wall creates an elastin-rich neointimal matrix that in the presence of hyperlipidemia is resistant to lipid deposition and macrophage accumulation.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Modelling of crop yields and N 2O emissions from silty arable soils with differing tillage in two long-term experiments

The choice of tillage system affects crop growth and soil nitrogen dynamics. Models help us to better understand these systems and the interaction of the processes involved. Objectives were to test a calibration and validation scheme for applications of the denitrification–decomposition (DNDC) model to describe a long-term field experiment with conventional tillage (CT) and reduced tillage (RT) at two sites (G and H, silty Haplic Luvisols) near Göttingen, Germany (G-CT, G-RT, H-CT, H-RT). Crop growth of field bean ( Vicia faba L.) and winter wheat ( Triticum aestivum L.) as well as soil water dynamics and nitrous oxide (N 2O) emissions were determined for two subsequent years. A model test was performed based on a model parameterization to best describe the case G-CT. This parameterization was then applied to the other cases as a retrospective simulation. Results of model variant v1 (no parameter optimization) indicated that soil water contents were not accurately simulated using the DNDC default values for a silt loam. After successful calibration of the soil water flow model using modified water-filled pore spaces at field capacity and wilting point and a modified hydro-conductivity that led to a good fit of the measured water content data, grain yields were markedly underestimated and modelled N 2O emissions were too large (v2). An optimization of the crop properties (maximum grain yield, N fixation index, thermal degree days, transpiration coefficient) was essential for a better match of measured yields (v3). Further adjustments in the model (v4) were required to better match cumulative N 2O emissions: reducing the initial soil organic carbon content and mineralization rates. Predictions of crop yields and annual cumulative N 2O emissions using model variant v4 were fairly accurate for the reduced tillage system G-RT and also for the second field experiment H-CT and H-RT, but annual distributions of N 2O emissions were not. Overall our results indicate that site specific calibration was an essential requirement for the silty German sites, and that the pedotransfer functions and denitrification sub-model of DNDC may need further improvement.

The Effectiveness of the Fertilizers Applied in the Plum and Cherry Orchards at the Fruit Research Station Cluj-Napoca

SUMMARY Fertilizing is an important step of the agrotechnical complex in the plum and cherry orchards (Koskela et al., 2010; Botu et al., 2010). This ensures high fruit productions every year (Davidescu et al., 1981; Rusu, 1993). This study focuses on the effects of applying different fertilizers on plum and cherry species. During 2010 these effects were monitored for the ‘Carpatin’ plum varietie and ‘Germersdorf’ cherry varietie. The planting distance for cherry trees is 6/3 m and for plum trees it's 4/3m. The experience was done on 6 variants each consisting of 6 randomly chosen trees: V1 – untreated, the control; V2 – fertilized with 20 t/ha of manure + N60 + P50 + K60/ha; V3 – fertilized N80 + P60 + K80/ha ;V4 – fertilized with 30 t/ha of manure + N80 + P50 + K125/ha; V5 – fertilized cu N90 + P60 + K120/ha;V6 – fertilized with 40 t/ha of manure + P70 + K100/ha. In order to determine the effectiveness of the fertilizers had been applied growth dynamics, phenological observations, average productions and the quality of the fruit were monitored. The biggest growth sprout for the plum trees was observed at the V6 variant, where the trees' total height increased by 2,95 m, the crown diameter increased by 2,10 m and the trunk diameter increased by 23 cm. In comparison to the control these dimensions show an increase of 18%, 12% and respectively 15%. For the cherry trees the biggest height, crown and trunk diameter development were also observed for the V6 variant. Experiments have proven the effect that organic fertilizers have in the orchards. Fertilizing improves vegetative growth and therefore an increased fruit production.

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin

Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium.

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin

Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium.

The Impact of Temperature and Humidity on Germination Energy and Length of Embryonic Roots at Sweet Corn

SUMMARY Actual climatic conditions affect agricultural harvests reason for farmers have to be direct to those crops which are most adapted at conditions of low humidity and high temperatures. Sweet corn presented in the last period a growing interest both thanks to its chemical composition as well as nutritive value and to the various possibilities for utilization in the alimentary industry. Besides these features sweet corn became interesting as well for its drought resistance in the first vegetation stages (Bors, 2011). The evolution of crop depends by the evolution of specie during germination process, reasons for it was considered as necessary to study the germination energy and the length of embryonic roots. Seeds were put to germinate both in standard conditions (AOSA and ISTA rules, V1 – 16 hours at 20°C and 8 hours at 30°C during 7 days – as control) and in less favorable conditions, similar with some conditions which can occur in field immediately after sowing: V2 – damping in water for 24 h at 25°C followed by 7 days at 25°C; V3 – 7 days at 25°C; V4 – damping in water for 24 h at 25°C followed by thermal shock (2 h at 50°C) and germination during 7 days at 25°C. Among the tested variants the highest germination energy (362.1%), very significant in comparison with control (standard conditions), was recorded by the variants V3 with seeds put to germinate in constant conditions of temperature (25˚C) and humidity. The germination energy was distinctly significant higher in comparison with control in case of variants V4 (241.4%) and V2 (244.8%) which for the seeds were preliminary damped in water for 24 hours. The lenght of embryonic roots was higher for all variants: V2 (6.43 cm), V3 (6.58 cm) and V4 (7.50 cm) in comparison with control V1 (6.10 cm). The result of statistical analysis indicated a distinct significant increase of lenght of embryonic roots in comparison with control just in variant V4 (1.4 cm), which for the seeds were damped with water for 24 h and then exposed at a thermal shock (50°C) for 2 h. Based on the obtained results it can be concluded that sweet corn has good odds to resist at a short period of drought imediately after sowing if the sowing is done in conditions of good humidity of soil.

Neutrophil Migration across Intestinal Epithelium: Evidence for a Role of CD44 in Regulating Detachment of Migrating Cells from the Luminal Surface

The migration of polymorphonuclear leukocytes (PMNs) across the intestinal epithelium is a histopathological hallmark of many mucosal inflammatory diseases including inflammatory bowel disease. The terminal transmigration step is the detachment of PMNs from the apical surface of the epithelium and their subsequent release into the intestinal lumen. The current study sought to identify epithelial proteins involved in the regulation of PMN migration across intestinal epithelium at the stage at which PMNs reach the apical epithelial surface. A panel of Abs reactive with IFN-γ-stimulated T84 intestinal epithelial cells was generated. Screening efforts identified one mAb, GM35, that prevented PMN detachment from the apical epithelial surface. Microsequencing studies identified the GM35 Ag as human CD44. Transfection studies confirmed this result by demonstrating the loss of the functional activity of the GM35 mAb following attenuation of epithelial CD44 protein expression. Immunoblotting and immunofluorescence revealed the GM35 Ag to be an apically expressed v6 variant exon-containing form of human CD44 (CD44v6). ELISA analysis demonstrated the release of soluble CD44v6 by T84 cells during PMN transepithelial migration. In addition, the observed release of CD44v6 was blocked by GM35 treatment, supporting a connection between CD44v6 release and PMN detachment. Increased expression of CD44v6 and the GM35 Ag was detected in inflamed ulcerative colitis tissue. This study demonstrates that epithelial-expressed CD44v6 plays a role in PMN clearance during inflammatory episodes through regulation of the terminal detachment of PMNs from the apical epithelial surface into the lumen of the intestine.