kD Polypeptide Research Articles

Analysis of Non-Sumoylated and Sumoylated Isoforms of Pax-6, the Master Regulator for Eye and Brain Development in Ocular Cell Lines.

Pax-6 is a master regulator for eye and brain development. Previous studies including ours have shown that Pax-6 exists in 4 major isoforms. According to their sizes, they are named p48, p46, p43 and p32 with the corresponding molecular weight of 48, 46, 43 and 32 kd, respectively. While p48 and p46 is derived from alternative splicing, p32 Pax-6 is generated through an internal translation initiation site. As for 43 kd Pax-6, two resources have been reported. In bird, it was found that an alternative splicing can generate a p43 Pax-6. In human and mouse, we reported that the p43 kd Pax-6 is derived from sumoylation: addition of a 11 kd polypeptide SUMO1 into the p32 Pax-6 at the K91 residue. Whether other Pax-6 isoforms can be sumoylated or not remains to be explored. The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. Both non-sumoylated and sumoylated isoforms of Pax-6 exist in 6 major types of ocular cells among which five are lens epithelial cells, and one is retinal pigment epithelial cell. Our results revealed that the most abundant isoforms of Pax-6 are the p32 and p46 Pax-6. These two major isoforms can be sumoylated to generate p43 (mono-sumoylated p32 Pax-6), p57 and p68 Pax-6 (mono- and di-sumoylated p46 Pax-6). In addition, the splicing-generated p48 Pax-6 is also readily detected. Our results for the first time, have determined the relative isoform abundance and also the sumoylation patterns of pax-6 in 6 major ocular cell lines.

Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy.

Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects; however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance.

Identification of seed storage protein markers for drought tolerance in mungbean

<p><strong>A set of 292 mungbean germplasm accessions including 62 popularly adapted local land races and two wild forms (<em>Vigna radiata</em> var. sublobata), important breeding lines and standard ruling varieties were screened for drought stress tolerance at seedling stage. Eight genotypes e.g., C. No. 35, OUM 14-1, OUM 49-2, Pusa 9072, OM 99-3, Banapur local B, Nipania munga, Kalamunga 1-A) have been identified to possess drought tolerance. Globulin seed storage protein profiling was carried out in 19 selected mungbean genotypes comprising eight drought tolerant, seven drought sensitive, two wild forms of mungbean (TCR 20 and TCR 213) and two standard checks (LGG 460 and T 2-1</strong><strong>)</strong><strong> to explore differentially expressed polypeptides. Seed protein profiles revealed 15 scorable polypeptide bands with molecular weights ranging from 10.0 to 102.2kD. A specific 12.8kD polypeptide band was present in all above drought tolerant test genotypes including the wild accession TCR 20. Such a polypeptide band may serve as useful biochemical marker for identification of drought tolerant genotypes in mungbean. </strong></p><p><strong> Key words: </strong>Genetic diversity, seed storage protein profile, wild and cultivated <em>Vigna radiata.</em><strong> </strong></p>

Sequential Therapy with Saratin, Bevacizumab and Ilomastat to Prolong Bleb Function following Glaucoma Filtration Surgery in a Rabbit Model.

To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control—mean survival time of 19.7 ±2.7 days—(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)—mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.

Revealing genetic variation in upland rice using seed storage protein profiling

A set of 36 upland rice varieties including 24 popular short duration ruling rice varieties and 12 mutants were electrophoresed following SDS-PAGE of total seed storage protein. The SDS-PAGE of total seed storage protein revealed altogether 23 scorable polypeptide bands with molecular weights ranging from 10.0 to 116.0 kD. Polypeptide bands at 37–39 kD and 22–23 kD were monomorphic and rest of the bands had shown polymorphism to the extent of 91.3% among the test genotypes. Rudra, Zhu 11–26, Vandana, Anjali, Annada, Saria, ORT 28 and ORT 36 had shown maximum 22 polypeptide bands, while Badami and ORT 39 revealed as low as 11 bands only. ORT 39 can be identified from rest of the test genotypes by absence of 57.0, 53.0 and 51.2 kD polypeptide bands. Similarly, Badami had unique absence of bands at 93.0, 70.0, 67.3 and 60 kD which were present in all other short duration test genotypes including mutants of Mandakini as well as Zhu 11–26. The polypeptide band at 27.5 kD was characteristically absent in cv. Sneha. Similar is the case for Badami and Mandakini which lacked 108.0 kD polypeptide band. In this context, a low mol. wt. band (16.0 kD) was specifically revealed only in five genotypes e. g. Rudra, Zhu 11–26, Kalinga-III, Vandana and Annada. Such a genotype-specific fingerprint is of immense value for varietal certification. ORT 28 and ORT 10 revealed inducement of an additional band at 65 kD, while a low mol. wt. polypeptide band at 16.0 kD was absent in both these mutants as compared to their parent (Zhu 1126). All mutants of Mandakini revealed 108.0, 47.0 and 45.0 kD polypeptide bands, but these were specifically absent in their parents. In contrast, Mandakini revealed the 13.0 kD, but it was absent in its mutants e. g. ORT 30, ORT 5, ORT 11 and ORT 22. This signifies mutational change which has direct bearing in total seed protein expression. ORT 39 and Mandakini were identified to have medium amylose content based on the staining intensity of the protein marker at 60 kD, whereas Keshari and four mutants of Mandakini (ORT 30, ORT 5, ORT 11 and ORT 22); and one mutant of Zhu 11–26 (ORT 32) were identified to be qualitatively superior for high lysine content owing to lack of the 13.0 kD prolamin polypeptide band. The test genotypes were distributed into three clusters at about 72% phenon level. Badami and ORT 39 formed two single variety divergent clusters (Clusters I and II), while cluster III was a large multivariety cluster comprising 34 upland genotypes which were further grouped into four sub-clusters (Clusters IIIA-IIID) at around 88.4% phenon level. Besides, the seed protein based clusters revealed some sort of characteristic morphological features. Genotypes comprising cluster I (Badami), cluster II (ORT 39) and cluster IIIA (ORT 30 and ORT 11) were identified as highly divergent which may serve as a valuable source genotypes in recombination breeding. The entire work was carried out during 2012–14.

Comparative Analysis and Molecular Characterization of a Gene BANF1 Encoded a DNA-Binding Protein During Mitosis from the Giant Panda and Black Bear

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.

Sodium Azide (NaN3) Induced Genetic Variation ofChlorophytum borivilianumSant. F. and Analysis of Variants Using RAPD Markers

The present research was carried out to identify the effect of sodium azide (NaN3) as an alternative method for mutation induction additionally, to find out the effect of NaN3 treatments on gene expression and morphological growth of chlorophytum borivilianum (also known as Safed musli). The experiments have revealed interesting information regarding influence of NaN3 treatments on genetic stability of Safed musli. In the protein profiling of treated leaves two polypeptides were found up regulated only in water immersed explants as compared to control. One of the polypeptides (43.3 KD) was found up-regulated in explants treated for 1 mM, 2 mM and at lesser extent in, 3 mM and 5 mM while 99.8 KD polypeptide was up regulated in all the treatments concentration of NaN3 namely 1 mM, 2 mM, 3 mM and 5 mM. A total of 19 polypeptide species were clearly resolved with a number of additional unclear bands in the gel analysis of control. The molecular weight of these polypeptides ranged from 14.8 KD to 122.2 KD. Two polypeptides were found up-regulated in pot. One of the bands of 122.2 KD was up- regulated in all the treatments namely 1 mM, 2 mM, 3 mM and 5 mM another was 58.3 KD in 2 mM treatment only.

Cloning and bioinformatics analysis of chorismate mutase gene from Salvia miltiorrhiza

Chorismate mutase catalyzes the conversion of chorismate to prephenate that is the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. A chorismate mutase gene, designated SmCM1, was isolated from Salvia miltiorrhiza by using RT-PCR. The full length of SmCM1 cDNA consists of 948 nucleotides and has an open reading frame of 765 bp. The deduced amino acid sequence of SmCM1 has 255 amino acid residues which forms a 36.0 kD polypeptide with calculated pI of 6.41 as expected. The putative polypeptide contains a CM_2 super family function domain. Blast W results showed that SmCM1 had 70% of the similarity with Petunia x hybrid CM, 72% of the similarity with Arabidopsis thaliana CM, and 64% of similarity with Populus trichocarpa CM. The transcription level of SmCM1 in root, stem and leaf was analysed by realtime quantitative PCR. The results showed the expression level of the SmCM1 in leaf was highest, and lowest in root. Yeast extract and silver ion joint induction could markedly stimulate the increase of mRNA expression of SmCM1 and its upstream 3-deoxy-7- phosphoheptulonate synthase (DAHPS) and chorismate synthase (CS). It was 7.9, 5.5 and 9.8 times of control on 8 h after induction, respectively.

Expression of metallothionein in liver and kidney of freshwater fish Cyprinus carpio var. communis (Linn) exposed to arsenic trioxide

In the present study, the toxic effects of arsenic trioxide (Ar2O3) to freshwater fish C. carpio were estimated at different median lethal concentration levels and its influences on the expression of metallothionein (MT) activity in liver and kidney were also observed. During this study, LC50 values were found to be 25.7, 27.4, 29.5, and 32 ppm for the period of 24, 48, 72, and 96 hrs respectively and LC16 and LC84 values of ArO3 also measured. The MT activity was estimated in liver and kidney of fish treated with different concentrations of ArO3 (5, 15, and 20 ppm) at different time intervals (48, 96, and 144 hrs) by using SDS-PAGE technique. From the results, 5 KD and 6 KD polypeptides were known to be metallothionein in liver and kidney respectively when the fishes were treated with different concentrations of arsenic arsenic trioxide. In the present investigation, ArO3 induced significant expression on metallothionein activity and it can be useful biomarker in environmental biomonitoring of arsenic contamination

Allosteric Akt Inhibitor MK2206 Synergizes with Bendamustine in Promoting the Apoptosis of Chronic Lymphocytic Leukemia Cells and Selectively Targets B-Cell Receptor Mediated Cytokine Production

AbstractAbstract 3928 Background:Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods:Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results:MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion:MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments:This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures:Shanafelt:Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

Cloning and characterization of a g-tocopherol methyltransferase from Chinese cabbage

α-Tocopherol, with antioxidant properties, is synthesized by photosynthetic organisms and play important roles in human and animal nutrition. The final step of the α-tocopherol biosynthetic pathway is catalyzed by γ-tocopherol methyltransferase (γ-TMT). In the major oilseed crops, γ-tocopherol, the biosynthetic precursor of α-tocopherol, is the predominant form. The full-length cDNA of γ-TMT was obtained from Chinese cabbage ( Brassica. rapa , Pekinensis Group), named BoTMT, by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicates that, BoTMT gene consisted of the open reading frame of 1041 nucleotides encoding a protein of 39 kD polypeptide. A BoTMT whole cell system was developed for the production of α-tocopherol through the expression of BoTMT in Escherichia coli BL21 (DE3) strain. Keywords: γ-Tocopherol methyltransferase, Chinese cabbage, Perilla frustescens , tocopherol, high performance liquid chromatography (HPLC)

CDNA, genomic sequence cloning and over-expression of ribosomal protein P2 gene (RPLP2) from the Giant Panda (Ailuropoda melanoleuca)

RPLP2 is a component of the 60S large ribosomal subunit encoded by RPLP2 gene and directly participate in protein synthesis, which is located in the cytoplasm. The cDNA and genomic sequence ofRPLP2 were cloned successfully from the Giant Panda using RT-PCR and Touchdown-PCR technology, respectively. The results showed that the cDNA fragment cloned is 394 bp in size, containing an open reading frame of 348 bp. The length of the genomic sequence is 1838 bp, with four exons and three introns. The deduced protein is composed of 115 amino acids with 11.66 KD of estimated molecular weight and 4.14 of pI. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species reported, including Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus. The homologies for nucleotide sequences of Giant PandaPRLP2 are 90.80, 87.64, 88.79 and 87.64% to those of the four species, while the homologies for amino acid sequences are 88.27, 85.20, 86.31 and 85.20%. The RPLP2 gene was overexpressed inEscherichia coli BL21, and the result indicated that fusing RPLP2 with the N-terminally His-tagged form gave rise to the accumulation of an expected 17.5 KD polypeptide, in accordance with the predicted protein, which could be used to purify and investigate the function of this protein. Key words: cDNA cloning, overexpression, ribosomal protein P2 gene (RPLP2), Giant Panda (Ailuropoda melanoleuca).

Sequence analysis of β-esterase isoenzymes related to fertility changeover in TsCMS7311 of Chinese cabbage ( Brassica rapa L. ssp. pekinensis )

In order to explore the mechanism of temperature regulating fertility transformation in the thermosensitive male sterile of Chinese cabbage (TsCMS7311) ( Brassica rapa L. ssp. pekinensis ), the paper studied the related β-esterase isoenzymes from the gene expression. The β-esterase isoenzymes related to fertility changeover in TsCMS7311 were purified by one-dimensional and two-dimensional gel electrophoresis. Two polypeptides, whose molecular weights were 57.1 and 62 kD, were analyzed with Q-TOF mass spectrometry. We obtained three sequences of short peptides from the 57.1 kD polypeptide and two sequences of short peptides from the 62 kD polypeptide. The two short peptides sequences of 62 kD polypeptide were the same as that from the 57.1 kD polypeptide. This result indicated that the sequences of the two polypeptides were highly similar. By comparing the sequence with protein database of NCBI GenBank, it was found that the three short peptides were highly homologous (60 to 100%) to protein p27 SJ . This study would lay the theoretic foundation for the research of TsCMS7311. Keywords: Chinese cabbage, β-esterase isoenzymes, sequence analysis

Cadmium을 처리하여 기내 배양한 담배 식물의 생장, 엽록소 함량 및 rubisco와 rubisco activase의 함량과 활성에 미치는 질산염의 영향

Influence of nitrate on growth, chlorophyll content, content and activity of rubisco and rubisco activase of tobacco plant cultured on MS medium treated with cadmium in vitro was studied. In vitro growth and chlorophyll content reduced at 0.2 mM Cd was recovered by nitrate and this recovery was most significant at 80 mM nitrate. Rubisco content at 80 mM nitrate was more increased compared to that at other concentrations. A similar change was also shown in rubisco activity. These resultsindicate that the activation and induction of rubisco reduced by Cd were recovered by nitrate. The degree of intensity of 55 and 15 kD polypeptides identified as the large and small subunits of rubisco by SDS-PAGE analysis at 80 mM nitrate was significantly higher than that at other concentrations. The content and activity of rubisco activase at 80 mM nitrate was significantly increased than that at other concentrations. These data suggest that the recovery effects of rubisco by nitrate may be associated with rubisco activase.

Genetic Variants In Platelet Supervillin but Not Archvillin Are Associated with PFA-100 Closure Times In African Americans

AbstractAbstract 4311Myocardial infarction typically occurs after atherosclerotic plaque rupture that exposes collagen under high shear stress, favoring platelet adhesion, activation with ADP release, aggregation and platelet thrombus formation. There is a well-established genetic contribution to myocardial infarction, and we have previously shown a shear-dependent assay of platelet function is heritable (JTH 2007;5:1617). Collagen-induced platelet aggregation is highly heritable in African Americans (AAs) but not in European Americans, which may be relevant since AAs have the highest overall coronary heart disease mortality rate of all ethnic groups in the US. The mechanisms for these ethnic differences in platelet function are not clear. The goal of the current study was to identify genetic variants in AAs affecting the Platelet Function Analyzer (PFA)-100 (ADP cartridge) that is both collagen- and shear-dependent. We recruited healthy AA subjects and measured closure times in the PFA-100. Subjects were excluded if there was evidence of exposure to anti-platelet therapy (absent response to arachidonic acid). Closure times showed a normal distribution (mean CT = 96 sec +/− 20 sec [SD]). Genome-wide genotyping with the Infinium II Assay using the Illumina BeadStation 500 system was performed. After controlling for population admixture and excluding markers with a less than 95% call rate, 131 subjects passed criteria for ethnicity and successful genotyping of 956,604 tagSNPs. Statistical analysis identified a locus on chromosome 10 that appeared to be associated with PFA-100 closure times. Of the 7 most significantly associated SNPs, 5 were in SVIL, the gene encoding supervillin. The closest flanking genes to the SNP with the best P value were >77 kb upstream (2 pseudogenes) and >214 kb downstream (LTZ1; lysozyme-like 1). After adjusting for VWF and fibrinogen levels, rs10826650 in SVIL was associated with PFA-100 closure times (P = 5.92E–07). We pursued SVIL (chr 10p11.23) because it is known to form a high-affinity link between the actin cytoskeleton and the plasma membrane during cell spreading and disassembly of focal adhesions. Supervillin slows the rate of cell spreading and increases myosin contractility by linking the myosin II heavy chain with myosin light chain kinase. Supervillin also decreases the stability of focal adhesions and co-localizes with signaling molecules in cholesterol-rich lipid rafts. Prior work has identified 2 transcript variants encoding different isoforms of supervillin. Isoform 1 (supervillin) is reported to be most abundantly expressed in muscle, bone marrow, thyroid gland and salivary gland; isoform 2 (archvillin) is reported as muscle specific. Because neither isoform has been characterized in platelets, we performed gene expression analysis with 23 leukocyte depleted platelet (LDP) RNA samples (<1 leukocyte per 5 million platelets) using the Sentrix BeadChip and BeadStation system from Illumina, Inc. A probe recognizing both isoforms was highly expressed in these samples, whereas a probe specific for the archvillin isoform was barely detectable. The level of SVIL mRNA expression was higher than PECAM1 but less than GPIbα. These microarray data were confirmed by RT-PCR using LDP RNA and PCR primers specific for supervillin. Notably, SVIL was not detected in megakaryocyte lines, HEL or Meg-01. Anti-supervillin antisera recognized the appropriate sized ∼205 kD polypeptide by immunoblotting. PFA-100 closure times were found to directly correlate with supervillin (r = 0.481, P = 0.02) but not with archvillin (r = 0.11, P=n.s.) mRNA levels. In summary, we have demonstrated for the first time that platelets contain supervillin but not the alternately spliced isoform archvillin, and that expression is associated with PFA-100 closure times in AAs. Because higher supervillin expression was associated with longer closure times, this finding is consistent with an inhibitory role for supervillin in regulating the rapid actin cytoskeletal rearrangement required to form platelet thrombi on collagen under shear stress. Disclosures:No relevant conflicts of interest to declare.

Morphogenetic status of somatic embryos of Citrus sinensis from mature polyembryonic seeds and those produced in vitro

In tissue culture of sweet orange (Citrus sinensis (L.) Osbeck, cv. Tarocco), we obtained mass regeneration of somatic embryos with two morphologically distinct cotyledons about 3 mm in length, their numbers amounting to 110–150 embryos per petri dish and 60 to 80% of the population. The morphogenetic state of somatic embryos was compared using the embryos with the cotyledons of different size (from 3 to 10 mm) from mature polyembryonic seeds as a test system and the cell number, size, and ultrastructural organization, and the number of protein bodies in the cotyledon cells as morphological and biochemical criteria. Cell number in the cotyledons of different size was related to the content of protein bodies therein. Typical protein bodies where 33 kD polypeptide of storage proteins was identified were detected only in the cotyledons, which size was identical to that of embryonic cotyledons from monoembryonic seeds of citrus plants. In the cells of smaller cotyledons, we detected protein-accumulating vacuoles with electron-dense inclusions that irrespective of their size, shape and structure accumulated the gold label. The number of the cells with protein depositions in vacuoles decreased when the cotyledons became smaller. Irrespective of the origin of embryos (in vivo or in vitro), lipids were the major storage metabolites in the cells of 3-mm cotyledons. As the cotyledon-forming cells became smaller and less numerous, their metabolic activity tended to decrease in line with the fragmentation of endoplasmic reticulum, the absence of polysomic complexes, and indistinct inner organization of mitochondria and plastids. It was concluded that somatic embryos developing in vivo and in vitro were physiological dwarfs, that is, the structures with diminutive storage organ with characteristically incomplete cell differentiation. Apparently such forms emerged due to the suppression of cotyledon growth at the initial stages of their organogenesis; as a result, the cell population could not properly realize both organo- and histogenesis.

Genotyping the BDNF rs6265 (val66met) polymorphism by one-step amplified refractory mutation system PCR

The brain derived neutrophic factor (BDNF), a 27 kD polypeptide, is one of the most widely expressed neurotrophins in the brain, regulating neural development and plasticity. The BDNF gene contains a functional single-nucleotide polymorphism (rs6265), which results in a valine to methionine substitution (val66met), leading to reduced mature BDNF expression. This polymorphism has been widely implicated in a host of psychiatric disorders and is a focus of many ongoing psychiatric genetic studies. To develop an efficient and rapid method to detect the val66met polymorphism in a one-step PCR reaction. We have designed four PCR primers that amplify the BDNF gene region containing rs6265. The specificity of the four primers in a single PCR reaction amplifies two allele-specific amplicons (253 and 201 bp) and the entire region (401 bp) as an internal control, which are easily distinguished on a polyacrylamide gel. The effectiveness and efficiency of the results are validated by traditional NlaIII restriction enzyme digestion, sequencing of resulting bands and confirmation on 308 genomic DNA samples. This new method describes a rapid, sensitive, cost effective and high throughput genotyping of the BDNF val66met polymorphism, ideal for large-scale genotyping studies.

Expression of IL-10 in human normal and cancerous ovarian tissues and cells

IL-10 is an 18-kd polypeptide that has been shown to be secreted by multiple cell types, including T and B cells, monocytes and some human tumors. However, which cell population is responsible for the elevated IL-10 levels in the serum and ascites of ovarian cancer patients, whether ovarian carcinoma cells produce IL-10, and how IL-10 influences the development and progression of ovarian carcinoma are issues that remain unclear. The aim of our study was to examine IL-10 production and secretion by ovarian carcinoma tissues and cells, and to determine its possible role in the cell and tumor micro-environment. The mean IL-10 protein levels expressed in normal ovarian tissue homogenates were significantly higher compared to cancerous ovarian tissue (p = 0.002). Yet, the IL-10 mRNA expression was significantly higher in cancerous ovarian tissues as compared to normal tissues (p = 0.021). The IL-10 receptor mRNA expression levels of the cancerous ovarian tissue homogenates were slightly, but not significantly, higher than the normal tissues. IL-10 immunostaining revealed that in both normal and cancerous ovarian tissues, IL-10 expression could be detected mainly in epithelial cells. In normal ovarian tissues, similar levels of IL-10R were demonstrated in epithelial and stromal cells. However, in cancerous ovarian tissues, epithelial cells expressed higher levels of IL-10R than the stroma. Primary normal and cancerous ovarian cell cultures and SKOV-3 cells secreted similar amounts of IL-10 after 24 hours of incubation. Our results suggest that epithelial cells are the main source of IL-10 in the ovary. Nevertheless, the target cells for IL-10 are different in normal and cancerous ovarian cells. Thus, IL-10 and its receptor could be involved in the pathogenesis of ovarian carcinoma.

The Source and Secretion of Immunoactive Relaxin in Rat Milk

The milk of many mammalian species contains hormones and growth factors in addition to nutrients and immunocompetent substances. These factors can be absorbed into the circulation of suckling neonates to exert important effects on metabolism and promote tissue and organ growth. Frequently, there is uncertainty as to whether such substances are gene products of the mammary glands themselves or are produced elsewhere and concentrated from the systemic circulation. The 6 kD polypeptide, relaxin, appears in milk of several mammalian species, including that of the rat, but proof of its source of secretion (corpus luteum vs. mammary gland) is so far lacking. The specific monoclonal anti-rat relaxin antibody MCA1 has previously been utilized successfully to investigate many of relaxin's actions in the rat, including those affecting the development of the mammary apparatus. In this report, MCA1 was utilized to aid in the identification of the source of relaxin in rat milk. Treatment of lactating rats with MCA1 completely neutralized the luteal relaxin circulating in serum but did not decrease the concentration of immunoactive relaxin secreted in milk. Moreover, the antibody did not appear to reach the mammary epithelium. The evidence thus supports the view that in the rat, the relaxin secreted in milk is primarily a product of the mammary glands and not concentrated from the systemic circulation.

Anti-angiogenic effects of tamoxifen in breast cancer by decreased secretion and reduced nuclear accumulation of angiogenin.

Abstract Abstract #1033 Angiogenin, is a 14.2 kD polypeptide member of the RNase A superfamily of enzymes with potent angiogenic activity. Angiogenin binds to endothelial cells and induces proliferation, cell migration, and invasion. Nuclear accumulation of angiogenin is essential for its angiogenic activity. In breast cancer, increased angiogenin expression has been associated with the transition from normal to invasive breast carcinoma and to ER expression. We have previously reported a pro-angiogenic action of estradiol and anti-angiogenic activity of tamoxifen in breast cancer by regulation of VEGF secretion and generation of endostatin. In this paper we investigated if estradiol and/or tamoxifen affected angiogenin. We show that estradiol increased and tamoxifen decreased angiogenin secretion of human breast cancer cells in culture. In vivo, in solid human breast cancers in nude mice, tamoxifen therapy decreased angiogenin extracellularly, sampled using microdialysis. Moreover, estradiol-induced angiogenin derived from cancer cells significantly increased human endothelial cell (HUVEC) proliferation. Adding tamoxifen to the estradiol treatment as well as an antibody against angiogenin, reversed this increase. Additionally, exposure of tamoxifen to HUVEC in culture significantly decreased the endogenous secretion of angiogenin as well as the translocation of angiogenin to the nucleus. We conclude that angiogenin expression is estrogen-regulated in human breast cancer cells. In addition, decreased secretion of angiogenin by cancer and endothelial cells, and a restrained nuclear accumulation of angiogenin, may suggest novel anti-angiogenic mechanisms of tamoxifen. This may be a target for anti-estrogen therapeutic options in the future. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1033.