Vegetative growth and productivity ofEichhornia azurea with special emphasis on leaf dynamics

The lifeform and the biological production of pure stands ofEichhornia azurea Kunth in three lakes in tropical Brazil were studied. The lifeform ofE. azurea is termed ‘semi-emergent’, because the plant has well developed trailing stems just under the water, and the aerial lamina emerges with the thick petiole. The density of shoot apices was 9.9, 17.2 and 17.1 m⁻² in Lake Dom Helvecio, Lake Jacaré and Lake Carioca, respectively. The mean daily increment of the apical shoot biomass was between 1.8 and 4.8 g m⁻² day⁻¹. The mean leaf life-span in Lake Dom Helvecio, Lake Jacaré and Lake Carioca was estimated to be 78, 49 and 64 days in the wet season and 73, 70 and 73 days in the dry season, respectively. The stem life-span was estimated to be about 28 months. Starch content in the current years' stem ranged from 24 to 118 mg g⁻¹ dry matter with fluctuations, the amplitude of which decreased with age. The differences for most of the growth parameters, such as density of shoot apices, daily increment of biomass and leaf life-span, between dry and wet season are smaller than those among the three lakes. Both the decrease in daily dry matter production and the increase in leaf life-span occurred in order from Lake Dom Helvecio to Lake Jacaré and Lake Carioca. The low productivity ofE. azurea is considered to be related to a low leaf area index, a long time interval for the emergence of new leaves, long leaf life-span and a low capacity for branching.     

Determination of Reduced, Protein-Unbound, and Total Concentrations of N-Acetyl- -cysteine and -Cysteine in Rat Plasma by Postcolumn Ligand Substitution High-Performance Liquid Chromatography

A high-performance liquid chromatographic assay was developed for the quantitative determination of the sulfur-containing amino acids N-acetyl- -cysteine (NAC) and -cysteine (Cys) in rat plasma. The thiols were separated by reverse-phase ion-pair chromatography, and the column eluent was continuously mixed with an iodoplatinate-containing solution. The substitution of sulfur of the thiol compound with iodide was quantitatively determined by measuring changes in the absorption at 500 nm. The low-molecular-weight disulfides and mixed disulfide conjugates of thiols with proteins were entirely reduced to the original reduced compounds by dithiothreitol. By reducing these two types of disulfides separately during sample pretreatment, the reduced, protein-unbound, and total thiol concentrations could also be determined. Validation testing was performed, and no problems were encountered. The limit of detection was approximately 20 pmol of thiol on the column. The present method was used to measure the plasma concentrations of NAC and Cys in the rat after a bolus intravenous administration of NAC, focusing on disulfide formation. The binding of NAC to protein through mixed disulfide formation proceeds in a time-dependent and reversible manner. Moreover, this “stable” covalent binding might limit total drug elimination, while the unbound NAC is rapidly eliminated. Consequently, the analytical method described in this study is very useful for the determination of plasma NAC and Cys, including disulfide conjugates derived from them.     

Histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow (Capricornis crispus, Artiodactyla, Mammalia) with special reference to glandular structures

The histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow ( Capricornis crispus ) was studied by light microscopic histochemical methods, particularly lectin histochemistry. The eccrine glands present exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-D-galactose and N-acetyl-neuraminic acid) especially in the cells of the secretory acini, the free surface of the collecting duct cells also showed distinct positive reactions with most of the histochemical methods. The thick epidermis of the nasolabial skin contained smaller amounts of glycoproteins. The results obtained are discussed with regard to possible functions of the glandular secretions. This substance mixture may particularly improve water retention on the skin surface, and protect against physical damage as well as microbial contamination. There seem to be no basic differences of muzzle functions between wild and domesticated bovine species.     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.     

Intra-Arterial Transplantation of Allogeneic Mesenchymal Stem Cells Mounts Neuroprotective Effects in a Transient Ischemic Stroke Model in Rats: Analyses of Therapeutic Time Window and Its Mechanisms

Objective

Intra-arterial stem cell transplantation exerts neuroprotective effects for ischemic stroke. However, the optimal therapeutic time window and mechanisms have not been completely understood. In this study, we investigated the relationship between the timing of intra-arterial transplantation of allogeneic mesenchymal stem cells (MSCs) in ischemic stroke model in rats and its efficacy in acute phase.

Methods

Adult male Wistar rats weighing 200 to 250g received right middle cerebral artery occlusion (MCAO) for 90 minutes. MSCs (1×10⁶cells/ 1ml PBS) were intra-arterially injected at either 1, 6, 24, or 48 hours (1, 6, 24, 48h group) after MCAO. PBS (1ml) was intra-arterially injected to control rats at 1 hour after MCAO. Behavioral test was performed immediately after reperfusion, and at 3, 7 days after MCAO using the Modified Neurological Severity Score (mNSS). Rats were euthanized at 7 days after MCAO for evaluation of infarct volumes and the migration of MSCs. In order to explore potential mechanisms of action, the upregulation of neurotrophic factor and chemotactic cytokine (bFGF, SDF-1α) induced by cell transplantation was examined in another cohort of rats that received intra-arterial transplantation at 24 hours after recanalization then euthanized at 7 days after MCAO for protein assays.

Results

Behavioral test at 3 and 7 days after transplantation revealed that stroke rats in 24h group displayed the most robust significant improvements in mNSS compared to stroke rats in all other groups (p’s<0.05). Similarly, the infarct volumes of stroke rats in 24h group were much significantly decreased compared to those in all other groups (p’s<0.05). These observed behavioral and histological effects were accompanied by MSC survival and migration, with the highest number of integrated MSCs detected in the 24h group. Moreover, bFGF and SDF-1α levels of the infarcted cortex were highly elevated in the 24h group compared to control group (p’s<0.05).

Conclusions

These results suggest that intra-arterial allogeneic transplantation of MSCs provides post-stroke functional recovery and reduction of infarct volumes in ischemic stroke model of rats. The upregulation of bFGF and SDF-1α likely played a key mechanistic role in enabling MSC to afford functional effects in stroke. MSC transplantation at 24 hours after recanalization appears to be the optimal timing for ischemic stroke model, which should guide the design of clinical trials of cell transplantation for stroke patients.     

Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.     

Carbonic anhydrase activators: the first activation study of the human secretory isoform VI with amino acids and amines

The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4 x 10(5)s(-1) and k(cat)/K(M) of 4.9 x 10(7)M(-1)s(-1) (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction, of the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of amino acids and amines were shown to act as CA VI activators, with variable efficacies. l-His, l-Trp, and dopamine showed weak CA VI activating effects (K(A)s in the range of 21-42 microM), whereas d-His, d-Phe, l-DOPA, l-Trp, serotonin, and some pyridyl-alkylamines were better activators, with K(A)s in the range of 13-19 microM. The best CA VI activators were l-Phe, d-DOPA, l-Tyr, 4-amino-l-Phe, and histamine, with K(A)s in the range of 1.23-9.31 microM. All these activators enhance k(cat), having no effect on K(M), participating thus in the rate determining step in the catalytic cycle, the proton transfer reactions between the enzyme active site and the environment.     

Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1

DNA methyltransferase 1 (Dnmt1) is an enzyme that recognizes and methylates hemimethylated CpG after DNA replication to maintain methylation patterns. Although the N-terminal region of Dnmt1 is known to interact with various proteins, such as methyl-CpG-binding protein 2 (MeCP2), the associations of protein kinases with this region have not been reported. In the present study, we found that a 110-kDa protein kinase in mouse brain could bind to the N-terminal domain of Dnmt1. This 110-kDa kinase was identified as cyclin-dependent kinase-like 5 (CDKL5) by LC-MS/MS analysis. CDKL5 and Dnmt1 were found to colocalize in nuclei and appeared to interact with each other. Catalytically active CDKL5, CDKL5(1-352), phosphorylated the N-terminal region of Dnmt1 in the presence of DNA. Considering that defects in the MeCP2 or CDKL5 genes cause Rett syndrome, we propose that the interaction between Dnmt1 and CDKL5 may contribute to the pathogenic processes of Rett syndrome.