Aluminium causes nutrient imbalance and structural changes in the needles of Scots pine without inducing clear root injuries

The effects of aluminium chloride (AICI3) treatments (50 and 150 mg/l) on 3-year-old Scots pine (Pinus sylvestris L.) seedlings were studied in a sand culture during 2 growing periods in an open field experiment. Even by the end of the first growing period, a decline was observed in the concentrations of Ca, Mg and P within the needles, and of Ca and Mg in the roots. After the second growing period, increased N and K concentrations were observed in the needles of Al-treated seedlings. Both the needles and roots of Al-treated seedlings showed, after the second growing period, a decline in growth and increased concentrations of AI as the amount of AICI3 in the nutrient solution increased. Al-induced changes in needle structure were found to be symptomatic of a nutrient imbalance, particularly of Mg and P. Al-stress did not result in any observable changes in root anatomy or in the number of mycorrhizas. Scots pine proved to be rather resistant to Al-stress, indicating that direct Al-injuries are not likely in the field, though Al-stress may be a contributing factor in the formation of nutrient imbalances.     

Different states of sarcoplasmic reticulum membrane in the presence of acetyl phosphate and adenosine triphosphate.

In the presence of acetyl phosphate, approximately 0.8 extra sulphydryl groups/10⁵ g protein of sarcoplasmic reticulum membrane vesicles are exposed to reaction with $\text{N}$-ethylmaleimide, whereas in the presence of ATP approximately 0.6 groups/10⁵ g protein are protected. Dithiobis (nitrobenzoic acid) reacts with the membrane sulphydryl groups more slowly in the presence of ATP than in the presence of acetyl phosphate or in the absence of substrate. Sarcoplasmic reticulum membrane is degraded by trypsin at a faster rate than normal when acetyl phosphate is present as seen from changes in electrophoretic patterns, ATPase activity and Ca²⁺ uptake capacity, and at a slower rate when ATP is present as seen from the last two properties. These differences in reactivity are interpreted as being due to differences in membrane conformations induced by the two substrates.     

Competitive ability of maize pollen. Intergametophytic effects

Summary Intergametophytic influences on pollen competitive ability were studied by means of the pollen mixture technique: mixtures of pollen from different genetic sources, marked for the presence of the normal or the mutant allele of the opaque-2 gene, were used to pollinate o2o2 female plants. The variation of relative frequency of one of the two kernel types from apex to base of the resulting ear, made it possible to measure the competitive ability by regression coefficients.The effect of the pollen marker on the character was estimated by analyzing the progeny of heterozygous o2/+ plants self-pollinated through four generations. The o2 allele-bearing pollen revealed a slower germination rate than the dominant counterpart; no differences between the two pollen types were observed with regard to tube growth rate.The occurrence of intergametophytic interactions was confirmed: the competitive ability of a line varied according to the genotype of the competing pollen. Thus, this component has to be taken into account in considering male gametophyte fertilization ability.     

Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide- and Catecholamine-Biosynthetic Enzymes

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.     

Glycosylation and membrane insertion of newly synthesized rat dopamine beta-hydroxylase in a cell-free system without signal cleavage.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) is present in both membrane-bound and soluble forms in neurosecretory vesicles. This study was designed to investigate the differences between membrane-bound and soluble DBH and how they may arise from translation of a single mRNA. Antisera to a peptide corresponding to the carboxyl terminus of rat DBH was found to specifically immunoprecipitate the 77- and 73-kDa subunits of newly synthesized DBH in rat brain. Thus, both soluble and membrane-bound forms contain the same carboxyl terminus. To investigate differences at the amino terminus, full-length rat DBH mRNA, translated in a cell-free system, produced a 66-kDa peptide. An additional higher molecular mass product was synthesized upon co-translational addition of microsomal membranes. This product was glycosylated since it bound to concanavalin A-Sepharose and reverted to the 66-kDa polypeptide after treatment with endoglycosidase H. This glycosylated product was resistant to protease digestion and fractionated with microsomal membranes on sucrose gradients, indicating that it is incorporated into the microsomal membranes. Amino-terminal sequencing of the glycosylated translation product indicated that the amino-terminal "signal" sequence was not cleaved. The results indicate that in the cell-free system newly synthesized DBH undergoes glycosylation and incorporation into microsomal membranes without cleavage of the NH2-terminal signal sequence.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.