A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Biological Activities of an Abscisic Acid Analog in Barley, Cress, and Rice

Biological activities of an abscisic acid (ABA) analog, RCA-7a[l-(3-carboxyl-5-methylphenyl)-l-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene],carrying a phenyl group in the side chain are reported. ( +)-RCA-7a was approximately 3-fold less active than ( + )-ABAin the inhibition of      

Protease inhibitors prevent the development of human rotavirus-induced diarrhea in suckling mice.

Oral inoculation of human rotavirus MO strain (serotype 3) into 5-day-old BALB/c mice caused gastroenteritis characterized by diarrhea (90% on the average, on day 2). Using this animal model, preventive effect of antiviral agents on the development of rotavirus-induced diarrhea was examined. The infectivity of human rotavirus was enhanced by treatment with protease in vitro. A cysteine protease inhibitor, E-64-c, was given orally at 12 hr and 24 hr after MO infection. Oral administration of 0.3 mg of E-64-c decreased the diarrhea ratio to 17.5% on day 2 and to 10% on day 3. Oral administration of 0.15 mg of cysteine protease inhibitor, ovocystatin, completely prevented the diarrhea on day 2. Serine protease inhibitor, aprotinin (0.15 mg x 2), also prevented the diarrhea on day 2 to 14.3%. These protease inhibitors were nontoxic in vitro and to suckling mice. The histopathological changes in the small intestine due to infection recovered 2 days after MO infection in mice treated with E-64-c and ovocystatin. These results suggest that protease inhibitors are protective agents for human rotavirus infection by inhibiting proteases required for viral replication.     

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/ c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (106 cells) and left flank (2 × 105 cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibitition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 × 107 cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.     

Needle area measurement by the cut method and estimation of specific leaf area inCryptomeria japonica

Needle surface area inCryptomeria japonica was measured using a newly proposed cut method. Sample needles of various lengths were taken from foliage shoots belonging to various height layers of nine trees growing at three sites. Needles were cut into small pieces with a hand-made cutter made of razor blades and washers. By measuring the circumference and thickness of each piece, its lateral area was calculated and summed to give the total surface area of the needle. For estimating the surface area of a needle (s), two linear parameters of needle size termedy _n* and /were proposed:y _n* was the distance between the needle tip and the uppermost point of attachment of the needle to the shoot, whilel was the distance between the needle tip and the lowermost point of attachment. The power-form relationship betweens andl was superior to thes-y _n* relationship, since the former did not differ significantly among shoots of different diameter. Based on thes-l relationship, the total surface area of a shoot was estimated from thel-census of the shoot. Specific leaf area of a shoot (SLA), defined as half of the shoot surface area divided by the dry weight of the shoot, decreased from 90 to 3 [cm²g(dry wt)⁻¹] with the diameter of the woody tissue of the foliage shoot.     

Identification of cellular factors required for the budding of koala retrovirus

Koala retrovirus (KoRV) is a unique gammaretrovirus that is currently endogenizing into its host and considered to be associated with leukemia, lymphoma and immunosuppression in koalas (Phascolactos cinereus). In this study, it was demonstrated that WWP2 or WWP2‐like E3 ubiquitin ligases possessing the WW domain closely related to WWP2 and Vps4A/B are involved in KoRV budding. These data suggest that KoRV Gag recruits the cellular endosomal sorting complex required for transport machinery through interaction of the PPPY L ‐domain with the WW domain(s) of WWP2 and that progeny virions are released from cells by utilizing the multivesicular body sorting pathway.     

Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO₂ concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidase-labeled antibody method under the light microscope. The distribution of Asp-hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mM cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.