Seed Storage Glutelin α-2 Subunit Lacking Mutants in Rice

Nine rice Oryza sativa L.） mutant lines lacking the seed storage glutelin α-2 subunit were obtained from the progenies of fertilized egg cells treated with N-methy-N-nitrosourea (MNU). The mutants could be classified into three types: the α-1 subunit increased type (α-1H/α-2L), decreased the β-2 subunit decreased type (β-2L/α-2L) and the α-3 subunit increased type (α-3H/α-2L) according to their SDS-PAGE profiles. Two-dimensional electrophoresis analysis revealed that all of the mutants lacked a polypeptide of pI 6.71/α-2, while new polypeptides of pI 6.50/α-1 and pI 6.90/α-3 formed in α-1H/α-2L and α-3H/α-2L mutants respectively. Although the β-2L/α-2L mutants did not form new polypeptide, their pI 8.74/β-2 polypeptide was also decreased, suggesting that the two polypeptides decreased in β-2L/α-2L mutants might derive from the same glutelin precursor. These mutant lines are very useful in studying genetic characterisation,the mechanism of genetic regulation on biosynthesis, gene function and proteomics of rice seed storage glutelin.     

Induction of Bcl-xL Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-κB in Apoptosis-Resistant T-Cell Transfectants with Tax

Human T-cell leukemia virus type 1 (HTLV-1) Tax is thought to play a pivotal role in immortalization of T cells. We have recently shown that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by interleukin-2 (IL-2) deprivation and converted its growth from being IL-2 dependent to being IL-2 independent. In this study, we demonstrate that constitutive expression of bcl-xl but not bcl-2, bcl-xs, bak, bad, or bax was associated with apoptosis resistance after IL-2 deprivation in CTLL-2 cells that expressed Tax. Transient-transfection assays showed that bcl-x promoter was transactivated by wild-type Tax. Similar effects were observed in mutant Tax retaining transactivating ability through NF-kappaB. Deletion or substitution of a putative NF-kappaB binding site identified in the bcl-x promoter significantly decreased Tax-induced transactivation. This NF-kappaB-like element was able to form a complex with NF-kappaB family proteins in vitro. Furthermore, Tax-induced transactivation of the bcl-x promoter was also diminished by the mutant IkappaBalpha, which specifically inhibits NF-kappaB activity. Our findings suggest that constitutive expression of Bcl-x(L) induced by Tax through the NF-kappaB pathway contributes to the inhibition of apoptosis in CTLL-2 cells after IL-2 deprivation.     

Potentiation of recombinant L-type Ca channel currents by alpha1-adrenoceptors coexpressed in baby hamster kidney (BHK) cells.

In cardiac myocytes, the effect of alpha1-adrenergic stimulation on L-type Ca current remains to be clarified. We examined this issue by the transient coexpression of alpha1-adrenoceptors on BHKC12 cells, where recombinant Ca channels composed of cardiac alpha1 subunit and skeletal beta, gamma, alpha2/delta subunits were stably expressed. After transfection of plasmid DNA encoding bovine alpha1C-adrenoceptors, bath-applied phenylephrine potentiated the cloned Ca channel current during perforated-patch whole-cell recording by 26+/-6% in 6 out of 12 cells. The potentiation was elicited also by methoxamine, and was blocked by prazosin. Phenylephrine also increased the channel open probability during cell-attached single channel recording in 7 out of 15 cells. The ratio of successful modulation of Ca channels was in accordance with the ratio of successful expression of alpha1-adrenoceptors, as estimated by beta-galactosidase staining. These results suggest that the stimulation of alpha1C-adrenoceptors is linked to potentiation of cardiac L-type Ca current. BHK cells provide a valuable expression system to study the modulation of Ca channels evoked by a receptor stimulation.     

Occurrence of Cello-Oligosaccharides in the Apoplast of Auxin-Treated Pea Stems

Treatment of pea (Pisum sativum L.) hypocotyl segments with indole-3-butyric acid, which promotes segment elongation, increased the solubilization of both xyloglucan and cello-oligosaccharides in the apoplast of auxin-treated pea stems. The cello-oligosaccharides were isolated from the apoplastic solution with a charcoal/Celite column and were identified as cellobiose, cellotriose, and cellotetraose after subsequent thin-layer chromatography and paper electrophoresis. Cello-oligosaccharides in the apoplastic fraction were monitored using cellobiose dehydrogenase. Both xyloglucan and cello-oligosaccharides appeared to be formed concurrently within 30 min after treatment with the auxin, and the cello-oligosaccharides increased with stem elongation even after 2 h. The total activity of cellulase did not increase for up to 4 h.     

Breakdown of self-incompatibility in a natural population of Petunia axillaris (Solanaceae) in Uruguay containing both self-incompatible and self-compatible plants

 Many members of the Solanaceae display a type of gametophytic self-incompatibility which is controlled by a single multiallelic locus, called the S-locus. From our previous survey of more than 100 natural populations of Petunia axillaris (a solanaceous species) in Uruguay, we had found that the majority of the populations of subspecies axillaris were comprised of virtually all self-incompatible individuals. The rest were ”mixed populations” which contained mostly self-incompatible and some self-compatible individuals. In this study, we examined the self-incompatibility behavior and determined the S-genotypes of 33 plants raised from seeds obtained from one such mixed population, designated U1. We found that 30 of the 33 plants (designated U1–1 through U1–33) were self-incompatible and a total of 18 different S-alleles were represented. To determine the S-genotypes of the three self-compatible plants (U1–2, U1–16, and U1–22) and the possible causes for the breakdown of their self-incompatibility, we carried out reciprocal crosses between each of them and each of the 18 S-homozygotes (S ₁ S ₁ through S ₁₈ S ₁₈ ) obtained from bud-selfed progeny of 14 of the 30 self-incompatible plants. For U1–2 and U1–16, we also carried out additional crosses with U1–25 (with S ₁ S ₁₃ genotype) and an S ₁₃ S ₁₅ plant (obtained from a cross between an S ₁₃ -homozygote and an S ₁₅ -homozygote), respectively. Based on all the pollination results and analysis of the production of S-RNases, products of S-alleles in the pistil, we determined the S-genotypes of U1–2, U1–16, and U1–22, and propose that the breakdown of self-incompatibility in these three plants is caused by suppression of the production of S₁₃-RNase from the S ₁₃ -allele they all carry. We have termed this phenomenon ”stylar-part suppression of an S-allele” or SPS. Received: 25 September 1998 / Revision accepted: 22 December 1998     

Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus sp.

Enzymatic decomposition of gelatin layers on X-ray films and repeated utilization of enzyme for potential industrialization were investigated using thermostable alkaline protease from the alkaliphilic Bacillus sp. B21-2. The decomposition of gelatin layers at 50 °C with the mutant enzyme (Ala187 was replaced by Pro) was higher than those of the wild-type and other mutant enzymes. In the repeated experiment for every 60 min (20 U ml^–1, 50 °C), the mutant enzyme could be satisfactorily used five times while three times for the wild-type enzyme.     

Design and semisynthesis of spermine-sensitive Ribonuclease S'.

Spermine-sensitive stabilization of semisynthetic Ribonuclease S' was successfully carried out by sequence specific incorporation of a poly-anion domain into alpha-helix region of S-peptide.     

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52 000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.     

Dissociation of CDK2 from Cyclin A in Response to the Topoisomerase II Inhibitor Etoposide in v-src-Transformed but Not Normal NIH 3T3 Cells

Our previous work has demonstrated that treatment of NIH 3T3 cells with etoposide (VP16), an inhibitor of DNA topoisomerase II and widely used anticancer agent, results in G2/M-phase arrest, whereas treatment of cells transformed by v-src, v-ras, or v-raf results in an S-phase blockage. The present studies describe the mechanistic aspects of this selective S-phase arrest in the v-src-transformed cells. The S-phase arrest in these cells was found to be coupled with depletion of cyclin A-dependent kinase activity. This decrease could not be explained by changes in the overall level of cyclin A, CDK2, p27, or p21 proteins. Rather, it was associated with a time-dependent reduction of CDK2 protein complexed with cyclin A following VP16 treatment. It was further shown that the decrease of cyclin A-associated CDK2 was linked to an increase of CDK2 protein in cyclin E immunocomplexes, which suggests that CDK2 might become redistributed following treatment with VP16. Thus, oncogenic transformation by v-src can trigger separation of CDK2 protein from cyclin A in response to VP16. This might contribute to the depletion of cyclin A-dependent kinase activity and the selective S-phase arrest by VP16 in v-src-transformed cells.