Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation,
and transmembrane signaling. A mammalian sialidase located in plasma ...membranes is unique in specifically hydrolyzing gangliosides,
suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for
the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme
from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp
boxes characteristic of sialidases and sharing 19â38% sequence identity with other sialidases. Northern blot and polymerase
chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In
COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside
substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2,
both α2â3 and α2â8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed
to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of
the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase
has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.
We isolated cDNA encoding a multispecific organic anion transporter 2 (OAT2) from the mouse kidney cDNA library. Isolated mouse OAT2 (mOAT2) consisted of 1623 base pairs that encoded a 540-amino acid ...residue protein with 12 putative membrane-spanning domains, and the amino acid sequence was 87% identical to that of rat OAT2 (rOAT2). The gene coding for mOAT2, Slc22a7, is found on chromosome 17C. Northern blot analysis revealed that the mOAT2 mRNA is abundantly expressed in the male mouse kidney, whereas it was predominantly expressed in both the liver and kidney of female mice. When expressed in Xenopus laevis oocytes, mOAT2 mediated the high affinity transport of glutarate (K(m) = 15.8 +/- 3.2 microM) and prostaglandin E2 (K(m) = 5.2 +/- 0.5 nM) in a sodium-independent manner. mOAT2-expressing oocytes also mediated the uptake of alpha-ketoglutarate, glutarate, prostaglandin E2, p-aminohippuric acid, methotrexate, ochratoxin A, valproate, and allopurinol. However, we did not observe mOAT2-mediated uptake of salicylate. A wide range of structurally unrelated organic anions inhibited mOAT2-mediated glutarate uptake especially erythromycin, a potent inhibitor. These results indicate that isolated mOAT2 is a multispecific organic anion transporter having some differences in substrate specificity compared with rOAT2. In addition, we found that there exists a sex- and species-related differential gene expression of the OAT2 isoform.
Strains of Thermomyces lanuginosus could be differentiated into two groups based on their ability to produce xylanase using xylan or xylose as sole of carbon source. One group of these strains ...produced high xylanase activity either in the medium using xylan or xylose as a sole of carbon source. The xylanase production by T. lanuginosus ATCC 44008, THKU-11, and THKU-25, which were the representative members of this group, increased when xylose was added to the xylan medium. In contrast, there was another group that produced high xylanase activity only in the xylan medium. Addition of xylose to the xylan medium resulted decreasing of xylanase production in T. lanuginosus ATCC 46882, TISTR 3465 and THKU-85 that belonged to this group. The finding indicated that induction and repression mechanism of xylose on xylanase expression was different among the strains of T. lanuginosus. In addition, phylogenetic analysis obtained from random amplified polymorphic DNA (RAPD) pattern using primer UBC 241 pointed to greater diversity of high and low xylanase producing strains using xylose as a carbon source.
Sex-related differential gene expression of organic anion transporters (rOAT1, rOAT2, and rOAT3) in rat brain, liver, and kidney was investigated. There were no sex differences in the expression of ...rOAT1 mRNA. rOAT2 mRNA was abundant in the liver and weakly expressed in the kidney of male rats; however, the OAT2 gene was strongly expressed in both organs of females. The abundance of rOAT2 mRNA markedly increased in castrated male rat kidney; however, treatment of castrated male rats with testosterone led to a decrease of rOAT2 mRNA. Expression of rOAT3 mRNA in intact female rats was found in the kidney and brain, whereas in males rOAT3 mRNA was also found in the liver. rOAT3 mRNA markedly decreased in the liver of castrated male rats but increased in testosterone-treated castrated male rats. Moreover, rOAT3 mRNA increased in the hypophysectomized female rat liver, indicating that rOAT3 is an inducible isoform. The present findings suggest that sex steroids play an important role in the expression and maintenance of OAT2/3 isoforms in the rat liver and kidney. Our results provide information on the differential gene expression of OAT isoforms with sex hormone dependency.
A novel compound (2) and a known one (1) were isolated from the mushroom, Sparassis crispa. Both compounds inhibited melanin synthesis and MRSA growth.
A factorial design was performed to find the best conditions of pH and temperature for beta -xylanase activity and to maintain its activity for prolonged periods of time of pure xylanase produced by ...newly isolated Thermomyces lanuginosus THKU-49. The central composite design (CCD) used for the analysis of treatment combinations showed that a regression models of optimization of xylanase activity and xylanase stability were good agreement to experimental results with R super(2) = 0.98 and 0.99, respectively. The maximum activity of xylanase was obtained at 66 degree C and pH 6.3. The temperature and pH for maximum enzyme stability was 70 degree C and pH 7.3, respectively. Under this condition xylanase having half-life of 825 min indicated the highest thermostable xylanase.
A novel enzyme,
N-acylamino acid racemase (NAAR) which catalyzes the interconversion of the enantiomers of
N-acylamino acid, but does not act on amino acids, has been found in the actinomycetes
...Streptomyces
atratus Y-53 and
Amycolatopsis sp. TS-1-60, isolated from soil. These strains also produced
l- and
d-aminoacylases simultaneously. Furthermore, another 13 strains of actinomycetes with NAAR activity were observed from the type culture collection of the Institute for Fermentation, Osaka (IFO).
Thermostable
N-acylamino acid racemase from
Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40°C, was purified to homogeneity and characterized. The enzyme was stable at 55°C for 30
min and catalyzed the racemization of optically active
N-acylamino acids such as
N-acetyl
d- or
l-methionine,
N-acetyl-
l-valine,
N-acetyl-
l-tyrosine and
N-chloroacetyl-
l-valine. In addition, this enzyme also catalyzed the racemization of the dipeptide
l-alanyl-
l-methionine. The optically active amino acids,
N-alkyl-amino acids and ethyl ester derivatives of
N-acetyl-
d and
l-methionine, however, were not racemized. Enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co
2+, Mn
2+ and Fe
2+ and was inhibited by the addition of EDTA and PCMB.
The NAAR gene from
Amycolatopsis sp. TS-1-60, consists of an open reading frame of 1104 nucleotides, which specifies a 368-amino acid protein with a molecular weight of 39,411. No significant sequence homology was found between the DNA sequence or the deduced amino acid sequence of NAAR and those of known racemases and epimerases in data bases. However, comparison of the amino acid sequences of mandelate racemase and NAAR showed that NAAR has partial homology with the catalytic and metal ion binding sites of that enzyme. The amount of NAAR produced by an
E.
coli transformant hosting a T7 expression plasmid was 1100-fold more than that produced by
Amycolatopsis sp. TS-1-60.
Bioreactors for the production of optically active amino acids were constructed with DEAE Toyopearl-immobilized NAAR and
d- or
l-aminoacylase.
d- or
l-Methionine was continuously produced with a high yield from
N-acetyl
dl-methionine by these bioreactors.
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