Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli

The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.     

Anterograde axonal transport of endopeptidase 24.15 in rat sciatic nerves

Axonal transport of endopeptidase 24.15 (EP24.15), a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. At 48h after ligation, a significant amount of the axonal transport of EP24.15 activity was found in the proximal segment, while axonal transport of deamidase activity, a lysosomal enzyme, increased in both proximal and distal segments. Western blot analysis of EP24.15 showed that EP24.15 immunoreactivity in the proximal segment was 1.8-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in the immunoreactive EP24.15 in the proximal segment in comparison with that in the middle segment. In the distal segment, no axonal transport of EP24.15 was found in all methods examined, indicating that EP24.15 is mainly transported by an anterograde axonal flow. These observations suggest that EP24.15 may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity

The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.     

Growth and maturation of the 'autumn-fruiting type' of Sargassum horneri (Fucales, Phaeophyta) and comparisons with the 'spring-fruiting type'

The growth and maturation period of the autumn-fruiting population ot Sargassum horneri C. Agardh (Phaeophyta) was investigated in Hiroshima Bay, Seto Inland Sea. Surveyed traits were compared to those of the spring-fruiting type and ecological features of this species were discussed. The annual lifetime of the autumn-fruiting population could be divided into four phases according to the daily increase in thalius length:phase I from December to May (increase in length < 0.1 mm/ day), phase II from May to September (= 0.3–1.0 mm/ day), phase Ml (from September to December > 10 mm/day) and phase IV which was the senescence phase from December to March. Receptacle formation‘as observed in November and gamete release from November to February. Conversely, the spring-fruiting type germinated in April and exhibited swifter growth in its early stage of development than the autumn-fruiting type. Rapid increase in thalius iength in autumn was common in both fruiting types, although the spring-fruiting type continued to grow during winter. Receptacle formation of the spring-fruiting type started in February but gamete release was not observed until April and May. The difference in life-history patterns of both types of S. horneri was in the overwintering period. The autumn-fruiting type spent that season as germi-ings or as young plants exhibiting slow-paced growth, while the spring-fruiting type overvwintered as adult thal-li preparing for gamete release in spring.     

Molecular cloning and expression analysis of Fshr and Lhr in relation to Fshb and Lhb subunits during the period of temperature‐dependent sex determination in pejerrey Odontesthes bonariensis

In this study, we cloned and characterized the follicle stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) cDNAs of pejerrey Odontesthes bonariensis, a species with temperature‐dependent sex determination (TSD), and analyzed their expression in relation to Fshb and Lhb subunits during gonadogenesis at temperatures producing only females (17°C, FPT), both sexes (25°C, MixPT), and only males (29°C, MPT). The pejerrey Fshr cDNA had 3,069 bp for a mature protein of 694 amino acids (aa) and a signal peptide of 22 aa; the Lhr cDNA had 2,936 bp for a mature protein of 676 aa and a signal peptide of 25 aa. With the exception of Lhr in fish at the MPT, all genes showed significant increases and/or peaks of expression before histological differentiation of the gonads regardless of temperature. Larvae at the FPT had lower Fshb and Lhb but higher Lhr expression during the TSD period than those at the MPT; a clear pattern could not be ascertained for Fshr. At the MixPT, Fshb, Lhb, and Lhr mRNA increased in approximately half of the fish during TSD and sex differentiation and the sex ratio was 55.2% male. Based on the above results, it is suggested that animals with high Fshb and Lhb and low Lhr values represent putative males. These evidences, together with other studies, suggest that temperature may signal through the pituitary (differential expression of Fshb and Lhb) down to the gonads (differential expression of Lhr), probably affecting the regulation of steroidogenesis during the TSD process of pejerrey. Mol. Reprod. Dev. 77: 521–532, 2010. © 2010 Wiley‐Liss, Inc.     

Agrobacterium tumefaciens-mediated transformation of the vegetative dikaryotic mycelium of the cultivated mushroom Flammulina velutipes

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.     

Effect of prostaglandin E2 on PZ-peptidase and several other peptidase activities in a clonal osteoblast-like cell line derived from newborn mouse calvaria

The effects of prostaglandin E2(PGE2) on the degradation of collagen and non-collagenous peptides in clonal osteoblastic MC3T3-E1 cells were investigated by using highly sensitive assay methods for PZ-peptidase, collagenase-like peptidase (CL-peptidase), dipeptidyl-aminopeptidase (DAP), leucine aminopeptidase (LAP), and post-proline cleaving enzyme (PPCE). PGE2, at concentrations of 0.1 to 4.0 micrograms/ml, doubled the PZ-peptidase and CL-peptidase activities in the cells on 24 h culturing in a dose-dependent manner. PGE2, at a concentration of 2.0 micrograms/ml, enhanced the specific activities of PZ-peptidase, CL-peptidase, DAP, LAP, and PPCE for 75 h after the start of PGE2 stimulation. The time dependent changes in PZ-peptidase and CL-peptidase activities showed similar patterns, and 3- and 2-fold increases were seen after 48 h, respectively. The protein and DNA contents gradually increased after addition of PGE2. Since the PZ-peptidase and CL-peptidase, involved in degradation of collagen peptides, were significantly induced by PGE2 in comparison with LAP and PPCE, involved in the degradation of non-collagenous peptides, these results show that PGE2 specifically stimulates induction of collagen catabolizing enzymes in clonal osteoblasts.     

Axonal transports of tripeptidyl peptidase II in rat sciatic nerves

Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12-72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.