Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.     

Single unit activity in the guinea-pig cochlear nucleus during sleep and wakefulness.

The effects of waking and sleep on the response properties of auditory units in the ventral cochlear nucleus (CN) were explored by using extracellular recordings in chronic guinea-pigs. Significant increases and decreases in firing rate were detected in two neuronal groups, a) the "sound-responding" and b) the "spontaneous" (units that do not show responses to any acoustic stimuli controlled by the experimenter). The "spontaneous" may be considered as belonging to the auditory system because the corresponding units showed a suppression of their discharge when the receptor was destroyed. The auditory CN units were characterized by their PSTH in response to tones at their characteristic frequency and also by the changes in firing rate and probability of discharge evaluated during periods of waking, slow wave and paradoxical sleep. The CNS performs functions dependent on sensory inputs during wakefulness and sleep phases. By studying the auditory input at the level of the ventral CN with constant sound stimuli, it was shown that, in addition to the firing rate shifts, some units presented changes in the temporal probability of discharge, implying central actions on the corresponding neurons. The mean latency of the responses, however, did not show significant changes throughout the sleep-waking cycle. The auditory efferent pathways are postulated to modulate the auditory input at CN level during different animal states. The probability of firing and the changes in the temporal pattern, as shown by the PSTH, are thus dependent on both the auditory input and the functional brain state related to the sleep-waking cycle.     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica , comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.     

Cell Wall Solubilization in Pedicel Abscission of Begonia Flower Buds

Effects of metabolic inhibitors and growth regulators on the course of abscission and on the activities of cell wall solubilizing enzymes were studied in pedicel explants of Begonia flower buds. Actinomycin D, chloramphenicol and 2,4-dinitrophenol slightly retarded abscission, whereas cycloheximide exerted a strong inhibition if applied until 10.5 h after explant excision. Indoleacetic acid retarded and ethylene promoted abscission and cell wall solubilization. However, the activities of cell wall solubilizing enzymes did not correspond with the course of abscission. No polygalacturonase and pectic acid and pectin transeliminases could be detected in the abscission zone during abscission, whereas a low pectin methylesterase activity did not change. Endo- and exocellulase activities did not increase until about 10 h after the onset of abscission, indicating that they are the result rather than the cause of abscission.     

Hormonal Regulation of Pedicel Abscission in Begonia Flower Buds

In order to analyse the hormonal regulation of flower bud shedding in Begonia, levels of indoleacetic acid (IAA), abscisic acid (ABA) and ethylene were determined in buds and pedicels. The translocation and metabolism of ¹⁴C-labeled IAA in pedicel segments were also studied. In a monoecious Begonia fuchsioides hybrid, abscising male flower buds contain about 1% of the IAA present in non-abscising female flowers. In a male Begonia davisii hybrid, the seasonal variation in bud drop coincides with changes in the IAA content of the buds, while also the release of IAA from the bud to the pedicel is hampered. Abscission zones of these pedicels always contain abscission promoting ethylene concentrations. The tissue is prevented from responding with abscission by IAA from the flower buds. The buds also contain ABA but without influencing abscission considerably. Pretreatment with ethylene or ABA does not affect IAA transport in pedicel segments. The rate of this transport is 4–6 mm × h^–1:; the capacity increases with the transverse area. In young segments, IAA is decarboxylated and also otherwise metabolized.     

In Type 1 Diabetes a Subset of Anti-Coxsackievirus B4 Antibodies Recognize Autoantigens and Induce Apoptosis of Pancreatic Beta Cells

Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients’sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients’ sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.