Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5′-UTR, a coding sequence of 1515 bp and a 104-bp 3′-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (55 kDa) and the tetrameric protein (230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.     

Thermodynamically controlled synthesis of amide bonds catalyzed by highly organic solvent-resistant penicillin acylase derivatives

A study of various direct condensations between different amines, having very high pK values, and unmodified acyl donors has been performed. This has been possible by the use of a very stable PGA derivative. First, it has been found that the higher the cosolvent concentration, the higher the pK of the acyl donor and thus the higher the yield. Therefore, these high concentrations of cosolvents seem to be a requisite for certain enzymatic condensations. Using ethanolamine and 2-hydroxy-2-phenylethyl-amine as nucleophiles and phenyl acetic acid as the acyl donor, the increase in the diglyme concentration from 50 to 90% (v/v) permitted improvement of not only the yield (reaching values higher than 99% in both cases) but also the reaction rates (by 360- or 3-fold, respectively). However, even when using PGA preparations stabilized by multipoint covalent attachment, it was not possible to obtain these results by inactivation of the enzyme derivative. Thus, in the protection of the octylamine with phenylacetic acid in 90% diglyme, the enzymatic activity was more than 20-fold higher using the hydrophilized derivative than the glyoxyl PGA, which allowed us to obtain a yield higher than 99%. Thus, the use of hydrophilized derivatives that are very stable even in the presence of high concentrations of organic solvents opens new opportunities in the use of PGA in organic chemistry.     

Effects of reducing agents on glutathione metabolism and the function of carotid body chemoreceptor cells

Two current hypotheses of O2 sensing in the carotid body (CB) chemoreceptors suggest participation of oxygen reactive (ROS) species, but they are mechanistically opposed. One postulates that hypoxia decreases ROS levels; the other that hypoxia increases them. Yet, both propose that the ensuing alteration in the cellular redox environment is the key signal triggering hypoxic chemoreception. Since the glutathione redox pair is the main cellular buffer for ROS and the main determinant of the general redox environment of the cells, a way to test whether ROS participate in chemoreception is to determine glutathione levels and to correlate them with the activity of CB chemoreceptor cells. We found that hypoxia does not alter the glutathione reduction potential but that it activates chemoreceptor cell neurosecretion. Incubation of tissues with reduced glutathione increases the glutathione-reducing potential but does not activate chemoreceptor cells in normoxia nor does it modify hypoxic activation. Like reduced glutathione, N-acetylcysteine promoted a general reducing environment in the cells without alteration of chemoreceptor cell activity. N-(mercaptopropionyl)-glycine, like the two previous agents, increases the reduction potential of glutathione. In contrast, the compound activated chemoreceptor cells in normoxia, promoting a dose- and Ca(2+)-dependent neurosecretion and a potentiation of the hypoxic responses. The existence of multiple relationships between glutathione reduction potential in the cells and their activity indicates that the general cellular redox environment is not a factor determining chemoreceptor cell activation. It cannot be excluded that the local redox environments of restricted microdomain(s) in the cells with specific regulating mechanisms are important signals for chemoreceptor cell activity.     

Genome-wide patterns of carbon and nitrogen regulation of gene expression validate the combined carbon and nitrogen (CN)-signaling hypothesis in plants

Background  

Carbon and nitrogen are two signals that influence plant growth and development. It is known that carbon- and nitrogen-signaling pathways influence one another to affect gene expression, but little is known about which genes are regulated by interactions between carbon and nitrogen signaling or the mechanisms by which the different pathways interact.     

Combined treatment with vessel dilator and kaliuretic hormone in persons with congestive heart failure

Vessel dilator and kaliuretic hormone, two cardiovascular peptide hormones, enhance urine flow 2- to 13-fold and 4-fold, respectively, in persons with class III New York Heart Association congestive heart failure (CHF). The natriuresis and diuresis secondary to vessel dilator and kaliuretic hormone are not blunted as are atrial natriuretic peptide and brain natriuretic peptide effects in persons with CHF compared with healthy individuals. The present investigation determined if the two peptide hormones that do not have blunted effects in persons with CHF may have added beneficial effects when given simultaneously to individuals with class III CHF. Together with each at 100 ng/kg of body weight per minute, vessel dilator and kaliuretic hormone increased urine flow rate 3.5-fold (P < 0.05) compared with their 60-min baseline and control CHF subjects' urine flow rates. Combined, they enhanced the excretion rate of sodium a maximum of 3.6-fold (P < 0.05) with 2.5- and 2-fold enhancement 2 and 3 hrs after infusion. These data indicate that vessel dilator and kaliuretic hormone have diuretic and natriuretic effects when used in combination, but these effects are not additive over their individual effects in persons with CHF.     

Expression of lung uncoupling protein-2 mRNA is modulated developmentally and by caloric intake

Lung expresses a high concentration of uncoupling protein-2 (UCP-2) mRNA, but neither its pulmonary regulation nor function is known. We measured lung UCP-2 mRNA expression in two animal models: in neonatal rats when both the metabolic rate, as measured by oxygen consumption, and levels of serum free fatty acids (FFAs) increase and in adult mice during decreased food intake, when levels of serum FFAs increase but the metabolic rate decreases. In rat lung, the concentration of UCP-2 mRNA was low and unchanged during late gestation, increased approximately twofold within 6 hrs after birth, and, compared with late gestation, remained approximately threefold higher from day 1 to adulthood. The early postnatal rise in the lung UCP-2 mRNA concentration was partially blocked by an antithyroid drug and was increased by treatment with triiodothyronine. Unlike lung, heart UCP-2 mRNA levels were lower during adulthood than at day 15. In adult mice, lung UCP-2 mRNA concentrations increased approximately fivefold within 12 hrs of 67% calorie restriction (CR), remained elevated during 2 weeks of CR, fell to control levels within 24 hrs of refeeding (CR-RF), and positively correlated with serum FFA concentrations. Heart UCP-2 expression during CR and CR-RF was similar to that of lung; liver UCP-2 mRNA levels were slightly lower during CR and returned to control levels during CR-RF. These data suggest that the regulation of UCP-2 is at least partly tissue-specific and that, in the adult mouse, lung UCP-2 is regulated not by oxygen consumption but by FFAs. Moreover, lung UCP-2 mRNA levels in mice fed ad libitum was increased by the intraperitoneal administration of Intralipid, a 20% fat emulsion. On the basis of these data in adult mice, together with the findings of others that levels of FFAs increase by 2 hrs after birth, we propose lung UCP-2 is regulated by FFA.     

Dendritic cells from chronic lymphocytic leukemia patients are normal regardless of Ig V gene mutation status

Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor alpha, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal- DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.