Effect of supplementation of Agaricus mushroom meal extracts on enzyme activities in peripheral leukocytes of calves.

To investigate the effect of Agaricus mushroom meal on the energy metabolism in animal tissues; plasma glucose, triglyceride, cholesterol and immunoreactive insulin (IRI) concentrations and activities of enzymes related to energy metabolism in plasma and peripheral leukocytes were measured in Japanese Black WagyuxHolstein F1 calves supplemented with Agaricus blazei Murill (A. blazei) extract in milk-replacer at the dose of 60g/head/day for 4 weeks. Activities of malate dehydrogenase and aspartate aminotransferase in cytosol and glutamate dehydrogenase in mitochondria, and the malate dehydrogenase/lactate dehydrogenase ratio in cytosol in peripheral leukocytes of calves with A. blazei were significantly higher than those in control calves without A. blazei. It was concluded that supplementation of Agaricus mushroom meal extract was effective in activation of enzymes related to energy metabolism in peripheral leukocytes of calves.

A cardiotonic steroid bufalin-induced differentiation of THP-1 cells. Involvement of Na+, K(+)-ATPase inhibition in the early changes in proto-oncogene expression.

Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1 beta (IL-1 beta). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto- oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ medium. These findings indicate that an increased intracellular Na+ concentration resulting from the Na+, K(+)-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.

Purification and characterization of the Clostridium josui porphobilinogen deaminase encoded by the hemC gene from a recombinant Escherichia coli.

The porphobilinogen deaminase encoded by the Clostridium josui hemC gene was purified from a recombinant Escherichia coli strain and its properties were characterized. The optimal temperature and pH of the purified enzyme were 65 degrees C and 7.0, respectively. This enzyme was quite thermostable: it retained 86% of the original activity after incubation at 70 degrees C for 1 h. The Km and Vmax values of the enzyme were 65 microM and 3.3 micromol/h/mg for porphobilinogen, respectively.

Inhibition of insulin-induced chondrogenic differentiation of embryonic carcinoma cells by a teratogenic compound YM9429.

A teratogenic compound cis-1-[4-(p-menthane 8-yloxy)phenyl]piperidine (YM9429) induces cleft palate selectively in rat fetuses. The effect of YM9429 on chondrogenic differentiation was investigated using mouse embryonic carcinoma ATDC5 cells that produce chondrocyte specific extracellular matrix upon insulin stimulation. YM9429 at concentrations that showed no growth-inhibitory effect on logarithmic proliferating cells suppressed insulin-mediated increases in Alcian blue staining and expression of type II collagen almost completely. Under the identical conditions, insulin-stimulated cell growth was only partially blocked by the compound. The early response genes such as c-fos and c-jun were induced by insulin even in the presence of YM9429. On the other hand, YM9429 inhibited accumulation of cAMP during the differentiation process. These results indicate that YM9429 selectively inhibits in vitro chondrogenic differentiation of ATDC5 cells.

Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui.

The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes.

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product.

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.

Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome.

The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of C. josui CipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticum CipC, but C. josui CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of the C. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.