Cytochemical localization of 5′-nucleotidase in the enteric ganglia and in smooth muscle cells of the guinea-pig

Brain Cell Biology - Cytochemical techniques were used to study the localization of 5′-nucleotidase in the enteric ganglia and in smooth muscle cells of the guinea-pig ileum, iris and vas...     

Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.     

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.     

The novel adipocytokine visfatin exerts direct cardioprotective effects

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 x 10(-6) micromol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1+/-4.1% in control hearts to 27.3+/-4.0% (n>or= 6/group, P<0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8+/-5.5% and 45.9+/-8.4%, respectively, versus 27.3+/-4.0% with visfatin; n>or= 6/group, P<0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2+/-4.6% in control to 49.2+/-3.7%(n>200 cells/group, P<0.05), an effect that was abrogated by wortmannin and U0126 (68.1+/-5.2% and 59.7+/-6.2%, respectively; n>200 cells/group, P>0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2+/-4 sec. in control to 120+/-7 sec. (n>20 cells/group, P<0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.     

Sophisticated selectionism as a general theory of knowledge

Human knowledge is a phenomenon whose roots extend from the cultural, through the neural and the biological and finally all the way down into the Precambrian “primordial soup.” The present paper reports an attempt at understanding this Greater System of Knowledge (GSK) as a hierarchical nested set of selection processes acting concurrently on several different scales of time and space. To this end, a general selection theory extending mainly from the work of Hull and Campbell is introduced. The perhaps most drastic change from previous similar theories is that replication is revealed as a composite function consisting of what is referred to as memory and synthesis. This move is argued to drastically improve the fit between theory and human-related knowledge systems. The introduced theory is then used to interpret the subsystems of the GSK and their interrelations. This is done to the end of demonstrating some of the new perspectives offered by this view.

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.     

Red-light- and gibberellic-acid-enhanced α-galactosidase activity in germinating lettuce seeds,cv. Grand Rapids

Dry lettuce (Lactuca sativa L.) seeds (achenes) contain -galactosidase (EC 3.2.122) at a level which is maintained in the imbibed dormant state in darkness. Both red light (R) and gibberellic acid promote an increase in enzyme activity several hours prior to the completion of germination. Germination and enzyme activity are not essentially linked, however, for the latter can increase while the former is inhibited. -Galactosidase activity increases within the cotyledons and the endosperm following R stimulation, but the axis is essential to perceive the stimulus and to promote and maintain the increase in enzyme activity. A diffusible factor (or factors) is produced by and-or released from irradiated axes, and it migrates to the cotyledons (and possibly endosperm) to promote the increase in -galactosidase activity. Gibberellic acid, particularly in the presence of benzyladenine, can replace the requirement for irradiated axes.Abbreviations GA₃ gibberellic acid - R red light     

Genomic data reject the hypothesis of a prosimian primate clade

The phylogenetic position of tarsiers within the primates has been a controversial subject for over a century. Despite numerous morphological and molecular studies, there has been weak support for grouping tarsiers with either strepsirrhine primates in a prosimian clade or with anthropoids in a haplorrhine clade. Here, we take advantage of the recently released whole genome assembly of the Philippine tarsier, Tarsius syrichta, in order to infer the phylogenetic relationship of Tarsius within the order Primates. We also present estimates of divergence times within the primates. Using a 1.26 million base pair multiple sequence alignment derived from 1078 orthologous genes, we provide overwhelming statistical support for the presence of a haplorrhine clade. We also present divergence date estimates using local relaxed molecular clock methods. The estimated time of the most recent common ancestor of extant Primates ranged from 64.9 Ma to 72.6 Ma, and haplorrhines were estimated to have a most recent common ancestor between 58.9 Ma and 68.6 Ma. Examination of rates of nucleotide substitution in the three major extant primate clades show that anthropoids have a slower substitution rate than either strepsirrhines or tarsiers. Our results provide the framework on which primate morphological, reproductive, and genomic features can be reconstructed in the broader context of mammalian phylogeny.