Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h^−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h^−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h^−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.     

Separation and quantification of serum alkaline phosphatase isozymes in the rat by affinity electrophoresis

The purpose of this study was to examine the possibility of separation and quantification of serum alkaline phosphatase (ALP) isozymes in rats by wheatgerm lectin affinity electrophoresis. Cellulose acetate electrophoresis of the liver and bone ALPs without lectin results in overlapping bands, but in the presence of lectin, the mobility of the band of bone enzyme was retarded and well separated from the liver enzyme band. With this affinity electrophoretic method, we determined the serum ALP isozymes in fed and fasting rats grouped by age. As a result, the absolute activity of bone isozyme showed a downward trend with age in the fed and fasting rats. The serum ALP activity was steadily higher in fed rats than in fasting rats, and the increase was due to intestinal ALP isozyme. There was low activity bordering complete absence in liver isozyme under both nutritional conditions. The affinity electrophoretic method provided a rapid, reproducible, and relatively simple technique for further clinical characterization of ALP isozyme in the rat serum.     

Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100-108) and NLS2 in the ligand-binding domain (residues 111-320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220-258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317-358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.     

Genetic diversity in the Japanese rosy bitterling, Rhodeus ocellatus kurumeus (Cyprinidae)

Comparison of meristic characters (pored lateral line scales, vertebrae, and fin rays), and PCR-RFLP analysis in the D-loop and ND1 regions of mitochondrial DNA were performed to estimate the genetic diversity in local populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus. In terms of meristic characters, the Fukuoka population was the largest in both range and variance of the number of pored lateral line scales and vertebrae (abdominal and caudal), and Osaka was the second, whereas the Kagawa population showed the smallest range and variance in these characters. In PCR-RFLP analysis, 11 haplotypes (3 in Fukuoka, 2 in Okayama, 2 in Kagawa, and 4 in Osaka) were observed, and nucleotide sequence divergence (NSD) was approximately two times larger in ND1 (mean, 0.61%) than in D-loop (mean, 0.31%). In the neighbor-joining (NJ) tree, based upon the NSD value in ND1, haplotypes were arranged into four clades, which corresponded to the locality of each haplotype. The Fukuoka population was conspicuously apart from the other populations (mean, 0.90% in NSD), but the remaining three showed a similar genetic distance with each other (mean, 0.48%–0.52% in NSD). In haplotype diversity of mtDNA, half the stations in Osaka and all in Kagawa were monomorphic. Especially, two haplotypes endemic to Kagawa were randomly distributed, irrespective of drainages. Rhodeus o. kurumeus in Fukuoka inhabits small rivers and creeks (open water systems), while that in Kagawa and Osaka lives in small ponds (closed water systems). Taking the information of morphology, mtDNA, and habitat into consideration, the low genetic diversity in Kagawa and Osaka populations of R. o. kurumeus is thought to be mainly the result of the isolation of their habitat. Received: January 14, 2001 / Revised: June 14, 2001 / Accepted: July 1, 2001     

Marine Hydroquinone Zonarol Prevents Inflammation and Apoptosis in Dextran Sulfate Sodium-Induced Mice Ulcerative Colitis

Background and Aim

We previously identified an anti-inflammatory compound, zonarol, a hydroquinone isolated from the brown algae Dictyopteris undulata as a marine natural product. To ascertain the in vivo functions of zonarol, we examined the pharmacological effects of zonarol administration on dextran sulfate sodium (DSS)-induced inflammation in a mouse model of ulcerative colitis (UC). Our goal is to establish a safe and effective cure for inflammatory bowel disease (IBD) using zonarol.

Methods and Results

We subjected Slc:ICR mice to the administration of 2% DSS in drinking water for 14 days. At the same time, 5-aminosalicylic acid (5-ASA) at a dose of 50 mg/kg (positive control) and zonarol at doses of 10 and 20 mg/kg, were given orally once a day. DSS-treated animals developed symptoms similar to those of human UC, such as severe bloody diarrhea, which were evaluated by the disease activity index (DAI). Treatment with 20 mg/kg of zonarol, as well as 5-ASA, significantly suppressed the DAI score, and also led to a reduced colonic ulcer length and/or mucosal inflammatory infiltration by various immune cells, especially macrophages. Zonarol treatment significantly reduced the expression of pro-inflammatory signaling molecules, and prevented the apoptosis of intestinal epithelial cells. Finally, zonarol protected against in vitro lipopolysaccharide (LPS)-induced activation in the RAW264.7 mouse macrophage cell line.

Conclusions

This is the first report that a marine bioproduct protects against experimental UC via the inhibition of both inflammation and apoptosis, very similar to the standard-of-care sulfasalazine, a well-known prodrug that releases 5-ASA. We believe that the oral administration of zonarol might offer a better treatment for human IBDs than 5-ASA, or may be useful as an alternative/additive therapeutic strategy against UC, without any evidence of side effects.     

Modification of chemical properties of cell walls by silicon and its role in regulation of the cell wall extensibility in oat leaves

Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.     

Association of mast cell-derived VEGF and proteases in Dengue shock syndrome

Background

Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls.

Methodology/Principal Findings

The study was performed at Children''s Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9.

Conclusions

As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.     

Green and Red Brazilian Propolis: Antimicrobial Potential and Anti-Virulence against ATCC and Clinically Isolated Multidrug-Resistant Bacteria

Brazilian green and red propolis stand out as commercial products for different medical applications. In this article, we report the antimicrobial activities of the hydroalcoholic extracts of green (EGP) and red (ERP) propolis, as well as guttiferone E plus xanthochymol (8) and oblongifolin B (9) from red propolis, against multidrug-resistant bacteria (MDRB). We undertook the minimal inhibitory (MIC) and bactericidal (MBC) concentrations, inhibition of biofilm formation (MICB₅₀), catalase, coagulase, DNase, lipase, and hemolysin assays, along with molecular docking simulations. ERP was more effective by displaying MIC and MBC values <100 μg mL⁻¹. Compounds 8 and 9 displayed the lowest MIC values (0.98 to 31.25 μg mL⁻¹) against all tested Gram-positive MDRB. They also inhibited the biofilm formation of S. aureus (ATCC 43300 and clinical isolate) and S. epidermidis (ATCC 14990 and clinical isolate), with MICB₅₀ values between 1.56 and 6.25 μg mL⁻¹. The molecular docking results indicated that 8 and 9 might interact with the catalase's amino acids. Compounds 8 and 9 have great antimicrobial potential.