Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca2+-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca2+ storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma–style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis

Adolescent male with anorexia nervosa: a case report from Iraq

This is the first reported case of an adolescent male with anorexia nervosa in Iraq. This disorder is believed to be rare in males across cultures and uncommon for both genders in Arab countries. The patient met the DSM-IV diagnostic criteria for anorexia nervosa. He was hospitalized and received medical and psychiatric treatment at local facilities as discussed below and responded well to treatment

Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers

Epithelial cell adhesion molecule (Ep-CAM; CD326) is used as a target by many immunotherapeutic approaches, but little data are available about Ep-CAM expression in major human malignancies with respect to level, frequency, tumour stage, grade, histologic tumour type and impact on survival. We analysed by immunohistochemical staining tissue microarrays with 4046 primary human carcinoma samples from colon, stomach, prostate and lung cancers for both frequency and intensity of Ep-CAM expression under highly standardised conditions. A total of 3360 samples were analysable. High-level Ep-CAM expression was observed in 97.7% (n=1186) of colon, 90.7% of gastric (n=473), and 87.2% of prostate cancers (n=414), and in 63.9% of lung cancers (n=1287). No detectable Ep-CAM staining was found with only 0.4% of colon, 2.5% of gastric, 1.9% of prostate cancers, and 13.5% of lung cancers. The only significant correlation of Ep-CAM expression with tumour grading was observed in colon cancer where high-level Ep-CAM expression on grade 3 tumours was down to 92.1% (P<0.0001). Adenosquamous and squamous carcinomas of the lung had a lower percentage of high-level Ep-CAM expression compared to adenocarcinomas with 35.4 and 53.6%, respectively, and with 45.5 and 17.3% of tumours being Ep-CAM negative. With the exception of moderately differentiated colon carcinoma, where patients not expressing Ep-CAM on their tumours showed an inferior survival (P=0.0014), correlation of Ep-CAM expression with survival did not reach statistical significance for any of the other cancer indications and subgroups. In conclusion, the data strongly support the notion that Ep-CAM is a prime target for immunotherapies in major human malignancies. This is because the most common human cancers show (i) a low frequency of Ep-CAM-negative tumours, (ii) a high frequency of Ep-CAM expression on cells of a given tumour, and (iii) for most cancers, an insignificant influence of tumour staging, grading and histology on Ep-CAM expression

Conserved Molecular Underpinnings and Characterization of a Role for Caveolin-1 in the Tumor Microenvironment of Mature T-Cell Lymphomas

Neoplasms of extra-thymic T-cell origin represent a rare and difficult population characterized by poor clinical outcome, aggressive presentation, and poorly defined molecular characteristics. Much work has been done to gain greater insights into distinguishing features among malignant subtypes, but there also exists a need to identify unifying characteristics to assist in rapid diagnosis and subsequent potential treatment. Herein, we investigated gene expression data of five different mature T-cell lymphoma subtypes (n = 187) and found 21 genes to be up- and down-regulated across all malignancies in comparison to healthy CD4+ and CD8+ T-cell controls (n = 52). From these results, we sought to characterize a role for caveolin-1 (CAV1), a gene with previous description in the progression of both solid and hematological tumors. Caveolin-1 was upregulated, albeit with a heterogeneous nature, across all mature T-cell lymphoma subtypes, a finding confirmed using immunohistochemical staining on an independent sampling of mature T-cell lymphoma biopsies (n = 65 cases). Further, stratifying malignant samples in accordance with high and low CAV1 expression revealed that higher expression of CAV1 in mature T-cell lymphomas is analogous with an enhanced inflammatory and invasive gene expression profile. Taken together, these results demonstrate a role for CAV1 in the tumor microenvironment of mature T-cell malignancies and point toward potential prognostic implications

Co expression of SCF and KIT in gastrointestinal stromal tumours (GISTs) suggests an autocrine/paracrine mechanism

KIT is a tyrosine kinase receptor expressed by several tumours, which has for specific ligand the stem cell factor (SCF). KIT is the main oncogene in gastrointestinal stromal tumours (GISTs), and gain-of-function KIT mutations are present in 70% of these tumours. The aim of the study was to measure and investigate the mechanisms of KIT activation in 80 KIT-positive GIST patients. KIT activation was quantified by detecting phosphotyrosine residues in Western blotting. SCF production was determined by reverse transcriptase–PCR, ELISA and/or immunohistochemistry. Primary cultures established from three GISTs were also analysed. The results show that KIT activation was detected in all cases, even in absence of KIT mutations. The fraction of activated KIT was not correlated with the mutational status of GISTs. Membrane and soluble isoforms of SCF mRNA were present in all GISTs analysed. Additionally, SCF was also detected in up to 93% of GISTs, and seen to be present within GIST cells. Likewise, the two SCF mRNA isoforms were found to be expressed in GIST-derived primary cultures. Thus, KIT activation in GISTs may in part result from the presence of SCF within the tumours

Novel Crystalline SiO2 Nanoparticles via Annelids Bioprocessing of Agro-Industrial Wastes

The synthesis of nanoparticles silica oxide from rice husk, sugar cane bagasse and coffee husk, by employing vermicompost with annelids (Eisenia foetida) is reported. The product (humus) is calcinated and extracted to recover the crystalline nanoparticles. X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) show that the biotransformation allows creating specific crystalline phases, since equivalent particles synthesized without biotransformation are bigger and with different crystalline structure

Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

BACKGROUND: Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a C(γ)-exo or a C(γ)-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying C(γ)-exo puckering. METHODOLOGY/PRINCIPAL FINDINGS: While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the C(γ)-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of -4.71 kJ·mol(-1) in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub. CONCLUSIONS/SIGNIFICANCE: The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein

Identification and Characterization of Peripheral T-Cell Lymphoma-Associated SEREX Antigens

Peripheral T-cell lymphomas (PTCL) are generally less common and pursue a more aggressive clinical course than B-cell lymphomas, with the T-cell phenotype itself being a poor prognostic factor in adult non-Hodgkin lymphoma (NHL). With notable exceptions such as ALK+ anaplastic large cell lymphoma (ALCL, ALK+), the molecular abnormalities in PTCL remain poorly characterised. We had previously identified circulating antibodies to ALK in patients with ALCL, ALK+. Thus, as a strategy to identify potential antigens associated with the pathogenesis of PTCL, not otherwise specified (PTCL, NOS), we screened a testis cDNA library with sera from four PTCL, NOS patients using the SEREX (serological analysis of recombinant cDNA expression libraries) technique. We identified nine PTCL, NOS-associated antigens whose immunological reactivity was further investigated using sera from 52 B- and T-cell lymphoma patients and 17 normal controls. The centrosomal protein CEP250 was specifically recognised by patients sera and showed increased protein expression in cell lines derived from T-cell versus B-cell malignancies. TCEB3, BECN1, and two previously uncharacterised proteins, c14orf93 and ZBTB44, were preferentially recognised by patients' sera. Transcripts for all nine genes were identified in 39 cancer cell lines and the five genes encoding preferentially lymphoma-recognised antigens were widely expressed in normal tissues and mononuclear cell subsets. In summary, this study identifies novel molecules that are immunologically recognised in vivo by patients with PTCL, NOS. Future studies are needed to determine whether these tumor antigens play a role in the pathogenesis of PTCL

Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9

BACKGROUND: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. METHODS: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. RESULTS: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer's disease - outcomes for which large-scale trial data were unavailable. CONCLUSIONS: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate