SAFB2, a new scaffold attachment factor homolog and estrogen receptor corepressor

We have characterized previously the nuclear matrix protein/scaffold attachment factor (SAFB) as an estrogen receptor corepressor and as a potential tumor suppressor gene in breast cancer. A search of the human genome for other potential SAFB family members revealed that KIAA00138 (now designated as SAFB2) has high homology to SAFB (now designated as SAFB1). SAFB1 and SAFB2 are mapped adjacent to each other on chromosome 19p13.3 and are arranged in a bidirectional divergent configuration (head to head), being separated by a short (<500 bp) GC-rich intergenic region that can function as a bidirectional promoter. SAFB1 and SAFB2 share common functions but also have unique properties. As shown previously for SAFB1, SAFB2 functions as an estrogen receptor corepressor, and its overexpression results in inhibition of proliferation. SAFB1 and SAFB2 interact directly through a C-terminal domain, resulting in additive repression activity. They are coexpressed in a number of tissues, but unlike SAFB1, which is exclusively nuclear, SAFB2 is found in the cytoplasm as well as the nucleus. Consistent with its cytoplasmic localization, we detected an interaction between SAFB2 and vinexin, a protein involved in linking signaling to the cytoskeleton. Our findings suggest that evolutionary duplication of the SAFB gene has allowed it to retain crucial functions, but also to gain novel functions in the cytoplasm and/or nucleus.     

Deletion of N-terminal residues 23-88 from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-induced Neurodegeneration

Accumulating evidence has suggested that prion protein (PrP) is neuroprotective and that a PrP-like protein/Doppel (PrPLP/Dpl) is neurotoxic. A line of PrP-deficient mice, Ngsk Prnp0/0, ectopically expressing PrPLP/Dpl in neurons, exhibits late-onset ataxia because of Purkinje cell death that is prevented by a transgene encoding wild-type mouse PrP. To elucidate the mechanisms of neurodegeneration in these mice, we introduced five types of PrP transgene, namely one heterologous hamster, two mouse/hamster chimeric genes, and two mutants, each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K), into Ngsk Prnp0/0 mice. Only MHM2.del23-88 failed to rescue the mice from the Purkinje cell death. The transgenic mice, MHM2.del23-88/Ngsk Prnp0/0, expressed several times more PrP than did wild-type (Prnp+/+) mice and PrPLP/Dpl at an equivalent level to Ngsk Prnp0/0 mice. Little difference was observed in the pathology and onset of ataxia between Ngsk Prnp0/0 and MHM2.del23-88/Ngsk Prnp0/0. No detergent-insoluble PrPLP/Dpl was detectable in the central nervous system of Ngsk Prnp0/0 mice even after the onset of ataxia. Our findings provide evidence that the N-terminal residues 23-88 of PrP containing the unique octapeptide-repeat region is crucial for preventing Purkinje cell death in Prnp0/0 mice expressing PrPLP/Dpl in the neuron.     

Characterization of pNI10 plasmid in <Emphasis Type="Italic">Pseudomonas</Emphasis>, and the construction of an improved <Emphasis Type="Italic">Escherichia</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis> shuttle vector,pNUK73

The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment encodes a putative origin of replication ( ori) and one putative replication-controlling protein (Rep). An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb). We show that expression in pseudomonads of the bromoperoxidase gene ( bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E. coli.     

A traditional Japanese-style salt field is a niche for haloarchaeal strains that can survive in 0.5% salt solution

Background

Most of the haloarchaeal strains have been isolated from hypersaline environments such as solar evaporation ponds, salt lakes, or salt deposits, and they, with some exceptions, lyse or lose viability in very low-salt concentrations. There are no salty environments suitable for the growth of haloarchaea in Japan. Although Natrialba asiatica and Haloarcula japonica were isolated many years ago, the question, "Are haloarchaea really thriving in natural environments of Japan?" has remained unanswered.

Results

Ten strains were isolated from a traditional Japanese-style salt field at Nie, Noto Peninsula, Japan by plating out the soil samples directly on agar plates containing 30% (w/v) salts and 0.5% yeast extract. They were most closely related to strains of three genera, Haladaptatus, Halococcus, and Halogeometricum. Survival rates in 3% and 0.5% SW (Salt Water, solutions containing salts in approximately the same proportions as found in seawater) solutions at 37°C differed considerably depending on the strains. Two strains belonging to Halogeometricum as well as the type strain Hgm. borinquense died and lysed immediately after suspension. Five strains that belonged to Halococcus and a strain that may be a member of Halogeometricum survived for 1–2 days in 0.5% SW solution. Two strains most closely related to Haladaptatus possessed extraordinary strong tolerance to low salt conditions. About 20 to 34% of the cells remained viable in 0.5% SW after 9 days incubation.

Conclusion

In this study we have demonstrated that haloarchaea are really thriving in the soil of Japanese-style salt field. The haloarchaeal cells, particularly the fragile strains are suggested to survive in the micropores of smaller size silt fraction, one of the components of soil. The inside of the silt particles is filled with concentrated salt solution and kept intact even upon suspension in rainwater. Possible origins of the haloarchaea isolated in this study are discussed.     

Peptide synthesis of aspartame precursor using organic-solvent-stable PST-01 protease in monophasic aqueous-organic solvent systems

The PST-01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water-soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Cbz-Asp-Phe-OMe) synthesis from N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and L-phenylalanine methyl ester (Phe-OMe) in the presence of water-soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz-Asp-Phe-OMe were attained by the PST-01 protease when 30 mM Cbz-Asp and 500 mM Phe-OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 degrees C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz-Asp-Phe-OMe using the PST-01 protease attained 83% in the presence of 50% (v/v) DMSO.     

Stabilities and conformational transitions of various proteases in the presence of an organic solvent

The half-life of the activity of the PST-01 protease that was secreted by organic solvent-tolerant Pseudomonas aeruginosa PST-01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST-01 protease, alpha-chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST-01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.     

Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.     

Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.