Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fernAdiantum as analyzed by microbeam irradiation

When prothalli ofAdiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389–398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m^–2) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 m·min^–1 between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement.Abbreviations B blue light - FR far-red light - IR infrared light - R red light     

Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, diphydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.     

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.     

Chemiosmotic coupling of ion transport in the yeast vacuole: Its role in acidification inside organelles

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H⁺-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl^– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl^– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl^– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK _m for Cl^– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Phytochrome-Mediated Photoorientation of Chloroplasts in Protonemal Cells of the Fern Adiantum Can be Induced by Brief Irradiation with Red Light

Measuring the ratio of the number of photooriented chloroplaststo the total number of chloroplasts, we found that photoorientationof chloroplasts in protonemata of the fern Adiantum capillus-veneriscould be induced by brief irradiation with polarized red light.After irradiation with red light (R) of 3 or 10 min, orientationalmovement was detected as early as 10 min after the irradiation;it continued during the subsequent dark period for 30–60min, after which chloroplasts gradually dispersed again. WhenR-treated protonemata were irradiated briefly with a second10-min pulse of R, 60 min after the onset of the first irradiation,the orientational response of chloroplasts was again observed.Typical red/far-red photoreversibility was apparent in the response,indicating the involvement of phytochrome. By contrast, irradiationwith polarized blue light for 10 min was ineffective, whileirradiation with blue light (B) at the same fluence for a longerperiod of time clearly induced the photoorientation of chloroplasts.It is likely that longterm irradiation is necessary for theresponse mediated by a blue-light receptor. When protonemata were irradiated with far-red light (FR) immediatelyafter R or after a subsequent dark period of 10 min, the magnitudeof the orientational response was smaller and chloroplasts dispersedmore quickly than those exposed to R alone. When FR was appliedat 50 min, when the response to R had reached the maximum level,chloroplasts again dispersed rapidly to their dark positions.These results indicate that P_FR not only induces the photoorientationmovement of chloroplasts but also fixes the chloroplasts atthe sites to which they have moved as a result of photoorientation. (Received June 2, 1993; Accepted January 11, 1994)     

Optimal conditions of fixation for immunohistochemical staining of proliferating cell nuclear antigen in tumour cells and its cell cycle related immunohistochemical expression

Abstract. Comparison of the results of immunohistochemical expression, such as proliferating cell nuclear antigen (PCNA) in archival material of tumours, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain stable and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fNation, processing and staining of samples using animal-implanted MBT-2 cells derived from chemical-induced mouse bladder carcinoma and PC10, a monoclonal antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temperature before fixation for less than 12 h, fixation in formaldehyde was less than 48 h, and paraffin-embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PCNA positive rates obtained under these conditions was compared with the bromodeoxyuridine (BrdUrd) labelling indices. Although the average PCNA positive rate was significantly higher than the BrdUrd labelling index (P?0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In order to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Occurrence of PCNA positive G₁ cells was expected at a half-life of the PCNA-protein of 20 h and a tumour cell doubling time of about 24h. Thus, the percentage of PCNA positive nuclei in a conventionally paraffin-embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of the biological malignant potential of tumours.