AtPLC2, a gene encoding phosphoinositide-specific phospholipase C,is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized.The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian -type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.     

The pathway of ammonia assimilation in the silkworm,Bombyx mori

Ammonia can easily be assimilated into amino acids and used for silk-protein synthesis in the silkworm, Bombyx mori. To determine the metabolic pathway of ammonia assimilation, silkworm larvae were injected with methionine sulfoximine (MS), a specific inhibitor of glutamine synthetase (GS). Activity of GS in the fat body 2h after treatment with 400&mgr;g MS decreased to less than 10% of the control activity, whereas MS had no effect on the activity of glutamate dehydrogenase (GDH), another enzyme which could possibly be responsible for ammonia assimilation. Glutamine concentration in the hemolymph rapidly decreased after MS treatment, while the ammonia level in the hemolymph sharply increased. Glutamine concentration in the hemolymph 4h after injection decreased with increasing doses of MS, whereas ammonia concentration increased in proportion to the MS dose. MS strongly blocked the incorporation of (15)N label into silk-protein in larvae injected with (15)N ammonia acetate, while it slightly inhibited the incorporation of (15)N-amide glutamine into silk-protein. These results suggest that ammonia is mainly assimilated into glutamine via the action of GS and then converted into other amino acids for silk-protein synthesis and that GDH does not play a major role in ammonia assimilation in B. mori.     

Anti-inflammatory activity of superoxide dismutase conjugated with sodium hyaluronate

Superoxide dismutase (SOD) from bovine erythrocytes was conjugated with sodium hyaluronate (HA) with a mean molecular weight of 106 to have greater anti-inflammatory activity in vivo. Amino groups of SOD were coupled with carboxyl groups in the hyaluronate molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The HA-SOD conjugate was composed of 1.5 mol of SOD molecule per 1 mol of hyaluronate on the average, and retained 70% of the activity of unmodified SOD. The conjugate was essentially non-immunogenic in mice, and exhibited much higher anti-inflammatory activities than HA or SOD in models of inflammatory diseases such as ischemic oedema of the foot-pad in mice, carrageenin-induced pleurisy and adjuvant arthritis in rats. This revised version was published online in November 2006 with corrections to the Cover Date.     

Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature

1. 1. Myosin and its subfragment-1 (Sl) from carp acclimated to 10°C showed higher actin-activated Mg²⁺-ATPase activity and lower thermostability than their counterparts from carp acclimated to 30°C. Accordingly, filament velocity for the 10°C-acclimated carp myosin was higher at any measuring temperatures from 3 to 23°C than that for the 30°C-acclimated carp myosin.

2. 2. Three types of cDNA clones encoding myosin heavy chains were isolated from thermally acclimated carp. The 10 and 30°C types were predominating in carp acclimated to 10 and 30°C, respectively, whereas the intermediate type was found as a minor component in the 10°C-acclimated carp with an intermediate feature in both DNA nucleotide and deduced amino acid sequences between those of the 10 and 30°C types.

3. 3. The three types of myosin rod all showed a typical coiled-coil structure of -helices. DSC scans demonstrated that myosin rod prepared from carp acclimated to 10°C had a lower thermostability than that from carp acclimated to 30°C, showing that low thermostability in cold-acclimated carp myosin prevails over the entire molecule.

4. 4. cDNA clones encoding myosin alkali light chains were isolated from thermally acclimated carp. Northern blot analysis showed that the ratios of LC3/LC1 mRNAs were significantly higher (3.92) in the 30°C- than 10°C-acclimated (3.10) carp.

     

Structural determination of a directly mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2',4'-tetranitrobiphenyl in Salmonella typhimurium TA98.

In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2',4'-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO2 and Al2O3 column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2',4'-tetranitrobiphenyl with S9 mix at 37 degrees C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4'-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4'-diamino-2',4-dinitrobiphenyl by its chemical and physico-chemical properties.     

Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus

A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.     

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.     

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.