Studies on Cellular Site of Calcium Action in Promoting Pollen Growth

Studies were conducted to determine cellular site of Ca action in promoting pollen growth of Crinum asiaticum and a few other species. The following experimental results have strongly indicated that Ca binding takes place in pectins of the pollen tube walls. This appeared to increase the wall rigidity and to regulate permeability of the pollen cells thereby enhancing pollen growth. Radioactive Ca incorporation was observed exclusively in the pollen tube wall regions. The promoting action of Ca on pollen growth disappeared when pectinase was supplemented to the media. This was not the case with cellulase and other enzymes used. Methyl donors promoted pollen growth, and the promotion was more than doubled if Ca ions were present. Ethionine, on the other hand, inhibited tube elongation and exhibited no Ca effect. Growth of pollen tubes in oscillaled liquid media during elongation was poorer than growth in standing media. The Ca effect was also reduced when pollen was oscillated. The observations made of the reduced rate of ⁴⁵Ca incorporation when pollen was washed in water, and the hydroxylamineferric chloride test, have indicated that a considerable portion of these pectins are cold-water soluble.     

Structural analysis of thermosome from hyperthermophilic archaeon Thermococcus

The purification and characterization of thermostable chaperonin of the thermosome family from hyperthermophilic archaeon Thermococcus profunds are described. The purified thermosome is a homooligomeric complex and an ATPase with maximal activity at 80 degrees C. The electron micrographs obtained from negatively stained as well as frozen-hydrated specimen showed an eight-fold symmetry of chaperonin. They were about 15 nm height and 16 nm in diameter with a central cavity of 5 nm. In order to understand the ATPase cycling of thermosome, we analyzed the oligomeric structure of thermosome treated with several nucleotides.     

Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-quinones in the blood of rats treated with benzo[a]pyrene

The lipid peroxidation (as malondialdehyde, MDA), activities of superoxide dismutase (SOD) and catalase (CAT), and benzo[a]pyrene (BaP) metabolites were investigated in sera and erythrocytes of male Sprague-Dawley rats treated with BaP (20 mg per rat). MDA levels were significantly increased in sera (16.98+/-3.29 nmol/ml serum, P<0.05) 12 h after BaP treatment and persisted up to 96 h (13.80+/-1. 65 nmol/ml serum, P<0.05), but no significant change in NIDA levels was observed in erythrocytes. SOD and CAT activities were significantly increased in erythrocytes shortly after BaP exposure, and they were slightly decreased in sera, indicating an inverse correlation between lipid peroxidation and antioxidant enzyme activity. BaP and BaP-quinones (BaP-1,6-quinone and BaP-3,6-quinone) were measured in sera during the study period. A rapid increase of unmetabolized BaP was observed in sera (41.27+/-4.14 pmol/ml serum) 3 h after BaP treatment, reaching a peak at 6 h (48.56+/-4.62 pmol/ml serum) followed by a sharp decrease. Formation of the BaP-1, 6-quinone and BaP-3,6-quinone started in sera 3 h after BaP treatment, reached a peak at 24 h (7.23+/-1.02 pmol/ml serum) and 12 h (9.20+/-0.98 pmol/ml serum), respectively, and then decreased gradually. The time-dependent pattern of serum lipid peroxidation and the level of erythrocyte antioxidant enzymes were shown to be related to the concentrations of the BaP-quinone metabolites. These results suggest that BaP treatment, probably via the formation of BaP-quinones, oxidatively altered lipids and antioxidant enzymes in the blood, and might be associated with BaP-related vascular toxicity including carcinogenesis.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis

Developmentally regulated GTP-binding protein (DRG) is a new subfamily within the superfamily of GTP-binding proteins. Its expression is regulated during embryonic development. To investigate the effect of the expression of DRG2 on cell growth, we constructed a human Jurkat-T-cell line that overexpresses DRG2. Overexpression of DRG2 suppressed the growth and the aggregation of Jurkat cells but did not induce apoptotic cell death. We used cDNA microarray analysis to examine the global changes in gene expression induced by an overexpression of DRG2. DNA array analyses identified genes that may suppress cell growth at a number of levels in multiple signaling cascades in Jurkat cells and also several prosurvival genes that may protect cells from apoptosis.     

Cellular changes resulting from forced expression of glypican-3 in hepatocellular carcinoma cells

Glypican-3 (GPC3) is a member of the glypican family, which encodes cell-surface heparan-sulfate proteoglycans, and is frequently upregulated in hepatocellular carcinoma (HCC). We have recently reported that blocking endogenous GPC3 expression promotes the growth of HCC cell lines, suggesting that GPC3 plays a negative role in HCC cell proliferation. Here, we report that forced expression of GPC3 reduced the growth of HCC cells. We also found that FGF2-mediated cell proliferation was inhibited by GPC3. In addition, we observed that the adhesion of HCC cells to collagen type I and fibronectin was decreased by GPC3, whereas cellular migration and invasiveness were stimulated. Collectively, these results suggest that progression of hepatocellular carcinoma is associated with upregulation of GPC3.     

Association of GNLY Genetic Polymorphisms with Chronic Liver Disease in a Korean Population

Granulysin (GNLY) is found in cytotoxic granules of cytolytic T lymphocytes and natural killer (NK) cells, which are critical for hepatitis B virus (HBV) clearance. GNLY cytotoxicity plays an important role in the defense against viruses or intracellular bacteria. We hypothesized that genetic variation in the GNLY gene could affect the resistance of hosts against HBV infection. We compared the distribution frequencies of GNLY polymorphisms between an HBV-induced chronic liver disease (CLD) group and a spontaneous recovery (SR) control group to determine whether GNLY polymorphisms play a role in HBV clearance. A total of 117 patients in the SR group and 230 patients in the CLD group were enrolled. Samples derived from complex infections, including hepatitis C and human immunodeficiency virus, and those associated with insufficient clinical information (10 samples in SR and 24 samples in CLD) were excluded from the study. The final analysis included 107 SR and 206 CLD samples. DNA was extracted from peripheral blood, and GNLY genotypes were determined by the GoldenGate(?) method. The genotype distribution of the single-nucleotide polymorphisms (SNPs) rs2886767 (C>T), rs1561285 (G>C), and rs11127 (T>C) were significantly different between the SR and CLD groups in a recessive model (p<0.015). These three SNPs were in a complete linkage disequilibrium (LD) block. Diplotype distributions of haplotype (HT) 1 (C-G-T) and HT2 (T-C-C) were significantly different between the SR and CLD groups in a recessive model (p=0.025) and a dominant model (p=0.008). All p-values remained significant after multiple comparisons. GNLY polymorphism genotypes and diplotypes were associated with the chronicity of HBV. These data suggested that genetic variation of GNLY may be an important factor in HBV clearance through the CD8+ T or NK cell-mediated removal of HBV-infected cells from the host.     

THE ESSENTIAL ROLE OF CALCIUM ION IN POLLEN GERMINATION AND POLLEN TUBE GROWTH

Brewbaker, James L., and Beyoung H. Kwack. (U. Hawaii, Honolulu.) The essential role of calcium ion in pollen germination and pollen tube growth. Amer. Jour. Bot. 50(9): 859–865. Illus. 1963.—A pollen population effect occurs whenever pollen grains are grown in vitro. Small pollen populations germinate and grow poorly if at all, under conditions which support excellent growth of large pollen populations. The pollen population effect is overcome completely by a growth factor obtained in water extracts of many plant tissues. This factor is shown to be the calcium ion, and its action confirmed in 86 species representing 39 plant families. Other ions (K⁺, Mg⁺⁺, Na⁺) serve in supporting roles to the uptake or binding of calcium. The high requirement of calcium (300–5000 ppm, as Ca (NO₃)₂·4H₂O, for optimum growth) and low calcium content of most pollen may conspire to give calcium a governing role in the growth of pollen tubes both in vitro and in situ. It is suspected that ramifications of this role extend to the self-incompatibilities of plants and to the curious types of arrested tube growth distinguishing, for example, the orchids. A culture medium which proved its merit in a wide variety of pollen growth studies included, in distilled water, 10% sucrose, 100 ppm H₃BO₃, 300 ppm Ca (NO₃)₂·4H₂O, 200 ppm MgSO₄·7H₂O and 100 ppm KNO₃.     

Hemodynamic Analysis in an Idealized Artery Tree: Differences in Wall Shear Stress between Newtonian and Non-Newtonian Blood Models

Development of many conditions and disorders, such as atherosclerosis and stroke, are dependent upon hemodynamic forces. To accurately predict and prevent these conditions and disorders hemodynamic forces must be properly mapped. Here we compare a shear-rate dependent fluid (SDF) constitutive model, based on the works by Yasuda et al in 1981, against a Newtonian model of blood. We verify our stabilized finite element numerical method with the benchmark lid-driven cavity flow problem. Numerical simulations show that the Newtonian model gives similar velocity profiles in the 2-dimensional cavity given different height and width dimensions, given the same Reynolds number. Conversely, the SDF model gave dissimilar velocity profiles, differing from the Newtonian velocity profiles by up to 25% in velocity magnitudes. This difference can affect estimation in platelet distribution within blood vessels or magnetic nanoparticle delivery. Wall shear stress (WSS) is an important quantity involved in vascular remodeling through integrin and adhesion molecule mechanotransduction. The SDF model gave a 7.3-fold greater WSS than the Newtonian model at the top of the 3-dimensional cavity. The SDF model gave a 37.7-fold greater WSS than the Newtonian model at artery walls located immediately after bifurcations in the idealized femoral artery tree. The pressure drop across arteries reveals arterial sections highly resistive to flow which correlates with stenosis formation. Numerical simulations give the pressure drop across the idealized femoral artery tree with the SDF model which is approximately 2.3-fold higher than with the Newtonian model. In atherosclerotic lesion models, the SDF model gives over 1 Pa higher WSS than the Newtonian model, a difference correlated with over twice as many adherent monocytes to endothelial cells from the Newtonian model compared to the SDF model.