Tissular distributions and kinetics of two renal 99mTc-radiopharmaceuticals in rat.

A digital radioimager (RI), conventional radioautography (RA), and tracks microradioautography (MRA) were used to assess the biodistributions and kinetics of 99mTc-dimercaptosuccinic (99mTc-DMSA) and 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) in rat at both macroscopic and microscopic levels. Three groups of male Wistar rats were studied. Using gamma-counting, kidney, liver, spleen and blood kinetics of both tracers were assessed in the three groups. Using RA and RI, renal slices were analyzed in group 1 the animals being sacrified from 2 to 60 min after injection of 99mTc-MAG3, and in group 2 the animals being sacrificed from 0.5 to 24 hr after injection of 99mTc-DMSA. Using MRA, renal slices were analyzed for 99mTc-DMSA (group 3). RA films and RI images displayed the variation with time of the cortical and medullary uptakes of the tracers. No regional heterogeneity within the different structures could be seen neither with RA films nor with MRA. The remaining activity in the blood 24 hr after injection of 99mTc-DMSA was evaluated. The tissular distributions of both tracer being homogenous, mean values of cortical uptake seems to be acceptable for dosimetric studies. Our results incite to use of 99mTc-MAG3 instead of 99mTc-DMSA when both tracers may be indicated.     

Study on the ophthalmic diseases in ICR mice and BALB/c mice.

In pharmaceutical companies and research institutes, many toxicity tests are performed with laboratory animals. This study was performed to produce reference data for eye toxicity tests and to investigate the ophthalmic diseases of 408 ICR mice and 119 BALB/c mice, which are commonly used as subjects in toxicity tests. The experimental animals without clinical disorders were selected regardless of sex. The ophthalmic diseases were examined by using special ophthalmic instruments: direct ophthalmoscope, indirect ophthalmoscope, slit-lamp biomicroscope and focal illuminator. The most prevalent ocular variation within normal limits was hyaloid vessel remnant (ICR mice, 28.2%; BALB/c mice, 31.9%) and the incidence gradually decreased with age. The ocular diseases found in ICR mice were retinal degeneration (9.8%), corneal scar (4.2%), focal cataract (2.2%), anisocoria (1.2%), corneal ulcer (0.2%) and uveitis (0.2%). In BALB/c mice, corneal scar (9.2%), focal cataract (1.7%) and corneal ulcer (0.8%) were the ocular diseases found.     

Production of 3,6-anhydro-D-galactose from κ-carrageenan using acid catalysts

This study describes acid-catalyzed production of 3,6-anhydro-D-galactose (D-AnG) from κ-carrageenan, a sulfated polysaccharide with an alternating backbone consisting of D-AnG and D-galactose (D-Gal). We analyzed four hydrolysis products (D-AnG, 5-hydroxymethylfurfural (HMF), levulinic acid (LA), and D-Gal) and reducing sugar contents during acid hydrolysis. Acid screening was carried out using seven acid catalysts which have different acidity. The catalysts showing high D-AnG production and high selectivity were chosen for subsequent experiments. We selected four acid catalysts (HCOOH, CH₃COOH, HNO₃, and HCl), and studied the effects of catalyst acidity, hydrolysis temperature T, and reaction time t on the production of D-AnG and other hydrolysis products. The optimal condition for maximum production of D-AnG by κ-carrageenan hydrolysis was T = 100°C and t = 30 min using 0.2 M HCl. Under this condition, 2.81 g/L D-AnG (33.5% of theoretical maximum) could be obtained from 2% (w/v) κ-carrageenan. In general, the maximum values of D-AnG, D-Gal, and the sum of two by-products (HMF and LA) increased with the acidity of catalysts. However, HNO3 was an exception in that the maximum production levels of HMF and LA were unusually low compared with other acid catalysts. D-AnG was successfully purified from acid hydrolysates using silica gel chromatography and the product was nearly 100% pure. This effective D-AnG production could facilitate future studies on the conversion of D-AnG to biofuels and biochemicals.     

Purification of properoxinectin, a myeloperoxidase homologue and its activation to a cell adhesion molecule

Peroxidases are important mediators of innate immune reactions throughout the animal kingdom. In many arthropods a myeloperoxidase homologue, peroxinectin, is known to function as a cell adhesion factor and an opsonin. Here, we report in the freshwater crayfish Pacifastacus leniusculus the isolation of properoxinectin, inactive in cell adhesion, and we also show that properoxinectin is produced in the mature blood cells whereas the hematopoietic tissue contains very little of this protein. Both properoxinectin and peroxinectin are catalytically active as peroxidases, at least when using low molecular weight substrates. The extracellular processing of properoxinectin into an active cell adhesion protein was found to involve proteolytic steps shared with the prophenoloxidase activating system to yield catalytically active phenoloxidase. Thus, the regulation of activities by two ancient metalloproteins, both potentially producing highly toxic substances aimed at pathogens, is carried out by limited proteolysis. The proteolytic processing is triggered in the presence of microbial compounds such as beta-glucans or lipopolysaccharide after the release of properoxinectin and prophenoloxidase activating serine proteinases from the blood cells.     

Age-dependent changes of the ion content and the circadian leaf movement period in thePhaseolus pulvinus

The circadian movement of the lamina of primary leaves ofPhaseolus coccineus L. depends on circadian changes of the K⁺, Cl^- and (depending on the Cl^- availability) malate content in the swelling and shrinking motor cells of the laminar pulvinus. After sowing in soil, the laminar pulvinus develops within about 26 days. When the leaves emerge from the soil (about 6 days after sowing) and the pulvinus starts with the diurnal movement (about 9 days after sowing) the pulvinar dimensions are about half of those of the mature pulvinus. The anatomical structure, however, is basically the same as in the developed pulvinus. In soil-grown plants, the K⁺, Cl^- and malate content as well as the period length of the circadian leaf movement rhythm change in the developing pulvinus. In the embryo of the dry seed, the Cl^- content is low (0.03 mmol g^-1 DW), the K⁺ content, however, 22-fold higher than the Cl^- content. When the leaves emerge from the soil, the pulvinar K⁺ and Cl^- content is the same as in the whole embryo of the dry seed. In the developing pulvinus the K⁺ content increases by a factor of 2 and the Cl^- content by a factor of 41 in the mature pulvinus. The pulvinar malate content increases between the 6th and 10th day from about 40 to 180Μmol g^-1 DW, then decreases until the 17th day and remains thereafter on a low level (around 80 Μmol g^-1 DW). These results indicate that the Cl^- availability increases in the developing pulvinus with age. It explains furthermore why in young leaves malate was found as counterion to K⁺ in the osmotic leaf movement motor, in older ones, however, Cl^-. The circadian leaf movement starts 9 days after sowing. The period length decreases during the development of the pulvinus from 31.3 to 28.6 h in leaves of intact soil-grown plants. In leaves which were cut from the plants and immersed with their petioles in distilled water, the age dependent decrease of the period length is also found. However, the period lengths are shorter by more than 1 h than in the leaves of intact plants. The increasing Cl^- availability in the developing pulvinus does not seem to be the cause for the age dependent shortening of the period length, because the period length in 22 days old Cl^- deprived pulvini is the same as in 22 days old pulvini with a high Cl^- content.     

Down‐regulation of a chitin synthase a gene by RNA interference enhances pathogenicity of Beauveria bassiana ANU1 against Spodoptera exigua (HÜBNER)

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, classes A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS‐A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while class B CHS (CHS‐B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS‐A gene from the beet armyworm, Spodoptera exigua (SeCHS‐A). The SeCHS‐A contains an open reading frame of 4,698 nucleotides, encoding a protein of 1,565 amino acids with a predicted molecular mass of approximately 177.8 kDa. The SeCHS‐A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by quantitative real‐time‐PCR analysis. Expression of SeCHS‐A gene was suppressed by feeding double‐stranded RNA (dsCHS‐A, 400 ng/larva) in the third instar larvae of S. exigua. Suppression of the SeCHS‐A gene expression significantly increased 35% of mortality on pupation of S. exigua. Also, the third instar larvae fed with dsCHS‐A significantly increased susceptibility to entomopathogenic fungi, Beauveria bassiana ANU1 at 3 days after treatment. These results suggest that the SeCHS‐A gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. bassiana.     

Identification and characterization of the bacterial <Emphasis Type="SmallCaps">d</Emphasis>-gluconate dehydratase in <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis>

Achromobacter xylosoxidans is known to utilize d-glucose via the modified Entner-Doudoroff pathway. Although d-gluconate dehydratase produced from this bacterium was purified and partially characterized previously, a gene that encodes this enzyme has not yet been identified. To obtain protein information on bacterial d-gluconate dehydratase, we partially purified d-gluconate dehydratase in A. xylosoxidans and investigated its biochemical properties. Two degenerate primers were designed based on the N-terminal amino acid sequence of the partially purified d-gluconate dehydratase. Through PCR performed using degenerate primers, a 1,782-bp DNA sequence encoding the A. xylosoxidans d-gluconate dehydratase (gnaD) was obtained. The deduced amino acid sequence of A. xylosoxidans gnaD showed strong similarity with that of proteins belonging to the dihydroxy-acid dehydratase/phosphogluconate dehydratase family (COG0129). This is in contrast to the archaeal d-gluconate dehydratase, which belongs to the enolase superfamily (COG4948). The phylogenetic tree showed that A. xylosoxidans d-gluconate dehydratase is closer to the 6-phosphogluconate dehydratase than the dihydroxy-acid dehydratase. Interestingly, a clade containing A. xylosoxidans enzyme was clustered with proteins annotated as a second and a third dihydroxy-acid dehydratase in the genomes of Clostridium acetobutylicum (Cac_ilvD2) and Streptomyces ceolicolor (Sco_ilvD2, Sco_ilvD3), indicating that the function of these enzymes is the dehydration of d-gluconate.     

The dynamics of surface acoustic wave‐driven scaffold cell seeding

Flow visualization using fluorescent microparticles and cell viability investigations are carried out to examine the mechanisms by which cells are seeded into scaffolds driven by surface acoustic waves. The former consists of observing both the external flow prior to the entry of the suspension into the scaffold and the internal flow within the scaffold pores. The latter involves micro‐CT (computed tomography) scans of the particle distributions within the seeded scaffolds and visual and quantitative methods to examine the morphology and proliferation ability of the irradiated cells. The results of these investigations elucidate the mechanisms by which particles are seeded, and hence provide valuable information that form the basis for optimizing this recently discovered method for rapid, efficient, and uniform scaffold cell seeding. Yeast cells are observed to maintain their size and morphology as well as their proliferation ability over 14 days after they are irradiated. The mammalian primary osteoblast cells tested also show little difference in their viability when exposed to the surface acoustic wave irradiation compared to a control set. Together, these provide initial feasibility results that demonstrate the surface acoustic wave technology as a viable seeding method without risk of denaturing the cells. Biotechnol. Bioeng. 2009;103: 387–401. © 2009 Wiley Periodicals, Inc.