Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.     

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

Quantitative trait loci influencing protein and starch concentration in the Illinois Long Term Selection maize strains

A study was initiated to determine the number, chromosomal location, and magnitude of effect of QTL (quantitative trait loci or locus depending on context) controlling protein and starch concentration in the maize (Zea mays L.) kernel. Restriction fragment length polymorphism (RFLP) analysis was performed on 100 F₃ families derived from a cross of two strains, Illinois High Protein (IHP), X Illinois Low Protein (ILP), which had been divergently selected for protein concentration for 76 generations as part of the Illinois Long Term Selection Experiment. These families were analyzed for kernel protein and starch in replicated field trials during 1990 and 1991. A series of 90 genomic and cDNA clones distributed throughout the maize genome were chosen for their ability to detect RFLP between IHP and ILP. These clones were hybridized with DNA extracted from the 100 F₃ families, revealing 100 polymorphic loci. Single factor analysis of variance revealed significant QTL associations of many loci with both protein and starch concentration (P < 0.05 level). Twenty-two loci distributed on 10 chromosome arms were significantly associated with protein concentration, 19 loci on 9 chromosome arms were significantly associated with starch concentration. Sixteen of these loci were significant for both protein and starch concentration. Clusters of 3 or more significant loci were detected on chromosome arms 3L, 5S, and 7L for protein concentration, suggesting the presence of QTL with large effects at these locations. A QTL with large additive effects on protein and starch concentration was detected on chromosome arm 3L. RFLP alleles at this QTL were found to be linked with RFLP alleles at the Shrunken-2 (Sh2) locus, a structural gene encoding the major subunit of the starch synthetic enzyme ADP-glucose pyrophosphorylase. A multiple linear regression model consisting of 6 significant RFLP loci on different chromosomes explained over 64 % of the total variation for kernel protein concentration. Similar results were detected for starch concentration. Thus, several chromosomal regions with large effects may be responsible for a significant portion of the changes in kernel protein and starch concentration in the Illinois Long Term Selection Experiment.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

The SKS of the KMSKS signature of class I aminoacyl-tRNA synthetases corresponds to the GKT/S sequence characteristic of the ATP-binding site of many proteins

On the basis of protein modification studies and primary structure comparison, we propose that the SKS sequence within the KMSKS signature of the class 1 aminoacyl-tRNA synthetases corresponds to the GKT(or S) sequence considered as a signature of the nucleotide triphosphate-binding site of many proteins.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

Pythium aphanidermatum root rot of pawpaw (Carica papaya L.) in Nigeria

Root rot of pawpaw (Carica papaya L.) reported in Nigeria is caused byPythium aphanidermatum which was consistently isolated from diseased plant parts and highly pothogenic. Out of 16 different media tested, it grew best on corn-meal-agar (CMA) and CMA supplemented with cellulose and sucrose. The highest number of oospores/ml was on CMA with average diameter of 19.9±0.1 µm. The symptom is characterized by dark brown rot of roots, absence of secondary roots and disintegration of internal tissue of the main root. These cause the progressive decline of the aerial parts of the tree untill it dies.     

Epidermal calcium-binding protein: a marker of early differentiation of basal layer keratinocytes of rats

Epidermal calcium-binding protein (ECaBP) is present in the cells of the basal layer of the epidermis and other stratified epithelia. Since the basal layer compartment contains at least two types of cells: slow-cycling, poorly-differentiated, and actively proliferating, more differentiated cells, it was of interest to determine whether they both contained ECaBP. Basal and nearly suprabasal layer keratinocytes from newborn rat epidermis were fractionated into three fractions on the basis of cell size, using low-gravity sedimentation. The cell differentiation in each subgroup was estimated by cell size, morphology, cell cycle stage, RNA/DNA content, and the presence of specific keratins. The presence of ECaBP in these fractions was detected by immunocytochemistry and immunoblotting. Double staining with ECaBP antibodies and propidium iodide followed by flow cytometry was used to correlate ECaBP production and the stage of cell cycle. The relative cell size, measured by the light scattering was used to study the relationship between cell size and ECaBP production. The results show that small keratinocytes with low DNA and RNA content (G₀ cells) do not express ECaBP. ECaBP was found only in intermediate size basal keratinocytes with higher DNA and RNA contents, corresponding to actively proliferating S phase cells. Large keratinocytes, which express suprabasal keratin and have low DNA and high RNA content, cease to express ECaBP. ECaBP may, therefore, be a useful marker for assessing the movement of cells from poorly differentiated reserve compartment towards proliferation and further differentiation in both physiological and pathological situations.