Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, syn-thesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation. Accepted: 19 August 1997     

Co-expression of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase and catalase in <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce α-ketoglutaric acid by whole-cell biocatalyst

Objectives

To improve the production of α-ketoglutaric acid (α-KG) from l-glutamate by whole-cell biocatalysis.

Results

A novel and highly active l-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg^?1 at pH 6.0, 35 ^oC. The apparent K_m and V_max values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg^?1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H₂O₂, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l^?1 with a conversion rate from l-glutamate of 98.5% after 12 h.

Conclusions

An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.

     

Methane emission from paddy soils as affected by wheat straw returning mode

To study influence of wheat straw returning mode on CH₄ emission from paddy soils, a field experiment was conducted at Jurong, Jiangsu Province, China in 2006. Five treatments, no wheat straw applied (CK), wheat straw evenly incorporated into the topsoil (WI), wheat straw buried in ditches (WD), wheat straw strip-mulched onto the field surface (WM) and wheat straw burned in-situ (WB), were laid out in a randomized block design. Results showed that seasonal CH₄ emissions ranged from 6.9 to 28.1 g CH₄ m^?2. Wheat straw incorporation significantly increased CH₄ emission. No significant difference was observed between Treatments WI and WD in total CH₄ emission. Compared with Treatment WI, Treatment WM reduced total CH₄ emission by 32% and Treatment WB by 42%. Treatment WM was about 27% higher than Treatment CK in grain yield. Based on the findings, the best management of wheat straw prior to rice cultivation is strip-mulching wheat straw onto the field surface, which largely reduced CH₄ emission from rice fields with no decrease in rice yield.     

Long-Term Selenium-Deficient Diet Induces Liver Damage by Altering Hepatocyte Ultrastructure and MMP1/3 and TIMP1/3 Expression in Growing Rats

The effects of selenium (Se)-deficient diet on the liver were evaluated by using growing rats which were fed with normal and Se-deficient diets, respectively, for 109 days. The results showed that rats fed with Se-deficient diet led to a decrease in Se concentration in the liver, particularly among male rats from the low-Se group. This causes alterations to the ultrastructure of hepatocytes with condensed chromatin and swelling mitochondria observed after low Se intake. Meanwhile, pathological changes and increased fibrosis in hepatic periportal were detected by hematoxylin and eosin and Masson’s trichrome staining in low-Se group. Furthermore, through immunohistochemistry (IHC) staining, higher expressions of metalloproteinases (MMP1/3) and their tissue inhibitors of metalloproteinases (TIMP1/3) were observed in the hepatic periportal of rats from the low-Se group. However, higher expressions of MMP1/3 and lower expressions of TIMP1/3 were detected in hepatic central vein and hepatic sinusoid. In addition, upregulated expressions of MMP1/3 and downregulated expressions of TIMP1/3 at the messenger RNA (mRNA) and protein levels also appeared to be relevant to low Se intake. In conclusion, Se-deficient diet could cause low Se concentration in the liver, alterations of hepatocyte ultrastructure, differential expressions of MMP1/3 and TIMP1/3 as well as fibrosis in the liver hepatic periportal.     

Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A

An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-beta-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 angstroms resolution showed that AaManA adopted a (beta/alpha)8-barrel fold. Two catalytic residues were identified: Glu151 at the C terminus of beta-stand beta4 and Glu231 at the C terminus of beta7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr95 and Cys150. We propose a model of substrate binding in AaManA in which Glu282 interacts with the axial OH-C(2) in-2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.     

How do we get a perfect complement of digits?

A crucial issue in limb development is how a correct set of precisely shaped digits forms in the digital plate. This process relies on patterning across the anterior-posterior axis of the limb bud, which is under the control of Sonic hedgehog emanating from the zone of polarizing activity. Recently, Sonic hedgehog function in the limb bud has been shown to have a dual character controlling both growth and patterning of the digital field. This finding has prompted the proposal of new models of how these two functions are achieved, and this will be discussed in this review.     

Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Moench

Summary Immature embryos of 20 sorghum genotypes were cultured on MS 5 medium containing MS mineral salts supplemented with 2,4-D, zeatin, glycine, niacinamide, Ca-pantothenate, L-asparagine, and vitamins. For regeneration, calli were transferred onto the same medium with the exception that IAA was substituted for 2,4-D. In general, immature embryos obtained 9–12 days after pollination resulted in the best redifferentiation. Ability of calli to regenerate varied among genotypes; cultivars C401-1 and C625 had the highest redifferentiation frequencies. Ability to redifferentiate was heritable and acted as a dominant trait. At least two gene pairs were involved. Regenerated R₀ plants were planted in a greenhouse and their selfed (R₁ and R₂) progenies were planted in the field and examined for morphological and cytological variations. The majority of the phenotypic variations noted in R₀ were not transmitted to later generations. However, variants for plant height, degree of fertility, and midrib color persisted in R₁ and R₂ generations. A variation in tallness was attributable to one dominant mutant gene. Short stature and male sterility variants appeared to be consequences of recessive mutant genes controlling those traits. Minor variations in peroxidase banding patterns were found among R₀ plants.This study was supported by a research grant from Kansas Sorghum Commission and by a Research Fellowship to the senior author from the Ministry of Agriculture, Animal Husbandry, and Fisheries, China. Contribution 86-456-J from the Kansas Agricultural Experiment Station     

Allelopathy of diterpenoids on three species of soil ciliates

Allelopathy of diterpenoids extracted from plants of the genus Robdosia on three common species of soil ciliates, Colpoda inflata, Colpoda cucullus and Euplotes muscicola, was studied by acute toxicity test, sub-lethal effect test and morphological observation. Acute toxicity test showed that there was remarkable toxicity of the diterpenoids on the individuals of the three soil ciliate species, and there was close correlation between toxicity and concentration of the diterpenoids. 12 h-LC₅₀ values of the diterpenoids on the individuals of C. inflata, C. cucullus and E. muscicola were 161.40 mg L^?1, 94.80 mg L^?1 and 83.70 mg L^?1 respectively, and 24 h-LC₅₀ values were 114.90 mg L^?1, 92.30 mg L^?1and 65.80 mg L^?1 separately. Sub-lethal effect test of soil ciliates suggested that there existed significant inhibition of the diterpenoids on population growth of the three ciliates with dose-dependant relationships, population density and growth rate of the test group was obviously lower than that of the control group. Morphological observation indicated that diterpenoids affected the body shapes of the three ciliates and made them shorter and thicker, and the higher the concentration of diterpenoids, the greater the affection. The results are of great significance for understanding the functions of ciliates and their relationships to other organisms, and for the application of allelopathy in biological pest control in the soil ecosystems.