Protection of photosynthetic O2 evolution against heat inactivation: the role of chloride,pH and coupling status

Heat inactivation of photosynthetic O₂ evolution was studied in isolated thylakoids from spinach (Spinacia oleracea) and mangrove (Avicennia marina) leaves. Different temperatures, salt, pH and uncoupler effects were investigated. From these results and others in the literature it was concluced that chloride loss from the membrane and, more specifically, the oxygen-evolving complex of photosystem II, may be the cause of inhibition of oxygen evolution during heat inactivation.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - Tricine N-2-hydroxy-1, 1-bis (hydroxymethyl) ethyl glycine - EDTA ethylenediaminetetraacetic acid - FeCN K-ferricyanide     

Brain Metabolism and Intracellular pH During Ischaemia: Effects of Systemic Glucose and Bicarbonate Administration Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy In Vivo in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), beta-nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of arterio-cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue PCO2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by 1H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis

Summary The inheritance of both the mitochondrial DNA (mtDNA) and the nuclear-encoded extrachromosomal ribosomal DNA (rDNA) has been studied in the myxomycete, Didymium iridis, by DNA-DNA hybridization of labeled probes to total DNA at various stage of the life cycle. Both the mtDNA and rDNA populations rapidly become homogeneous in individuals, but there is a qualitative difference in the patterns of inheritance of these two molecules. One parental rDNA type was preferentially inherited in all crosses; selective replication of this molecule is tentatively proposed as the mechanism of inheritance. In contrast, either parental mtDNA type could be inherited. Since the inherited population of parental mtDNA molecules are not partitioned into cells in this coenocytic organism, no known mechanism of inheritance can explain the rapid and apparently random loss of one parental mtDNA type in individuals.     

Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

Transformation of plant mitochondria with mitochondrial DNA plasmids via protoplast fusion

Summary Brassica napus cybrid plants which contain novel nucleus-mitochondria-chloroplast combinations have been constructed, via protoplast fusion. Such fusions resulted in mitochondrial DNA plasmids being lost (at a frequency of 12.5%) or, more surprisingly, being transferred from mitochondria of one protoplast population to mitochondria of the other population (at a frequency of 6.1%). Mitochondria containing their new DNA complement became the dominant organelle population in regenerated plants and were faithfully maternally inherited through successive sexnal generations. No concomitant alterations in mitochondrial chromosome organization or nuclear chromosome number occurred. Protoplast fusion can, therefore, cure plant mitochondria of extrachromosomal DNA and, more importantly, be used to transform plant mitochondria with naturally occurring mitochondrial plasmids. The potential for mitochondrial transformation with recombinant vectors is discussed.     

The sugar beet mitochondrial genome: A complex organisation generated by homologous recombination

Summary The complete physical map of the mitochondrial genome of the Owen cytoplasm of sugar beet has been determined from overlapping cosmid clones. The genome is 386 kb in size and has a multicircular organisation generated by homologous recombination across repeated DNA elements. The location of the rRNA genes and several polypeptide genes has been determined. In addition the mitochondrial genome was found to contain a sequence of chloroplast DNA including part of the 16 S rRNA gene.     

Gating properties of channels formed by colicin Ia in planar lipid bilayer membranes

Summary Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (>+10 mV) are applied to thecis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant () of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric aroundV _o (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction inn, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be itcis orrans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change inn. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open,F _(v). For a given voltage,F _(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino-acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Cytoplasmic free calcium in Riccia fluitans L. and Zea mays L.: Interaction of Ca2+ and pH?

In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca²⁺ and pH have been measured with double-barrelled microelectrodes. Free Ca²⁺ activities of 109–187 nM (Riccia rhizoids), 94–160 nM (Riccia thalli), 145–231 nM (Zea root hairs), 84–143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca²⁺ was in the lower millimolar range (2.3±0.8 mM). A change in external Ca²⁺ from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca²⁺ which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca²⁺ activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca²⁺ regulation, and since it is energy-dependent, may involve a Ca²⁺-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca²⁺, while alkalinization of pH_c decreased the Ca²⁺ activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca²⁺ are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.Symbols and abbreviations _m, _m membrane potential difference, changes thereof - PVC polyvinylchloride