Biochemical differences in the skin of two blueberries (Vaccinium corymbosum) varieties with contrasting firmness: Implication of ions, metabolites and cell wall related proteins in two developmental stages.

The pursuit of firmer and better-quality blueberries is a continuous task that aims at a more profitable production. To this end it is essential to understand the biological processes linked to fruit firmness, which may diverge among tissues. By contrasting varieties with opposing firmness, we were able to elucidate events that, taking place at immature stage, lay the foundation to produce a firmer ripe fruit. A deep analysis of blueberry skin was carried out, involving diverse comparative approaches including proteomics and metabolomics coupled to immunolocalization assays. In'O'Neal' (low firmness) enhanced levels of aquaporins, expansins and pectin esterases at the green stage were found to be critical in distinguishing it from 'Emerald' (high firmness). The latter featured higher levels of ABA, low methyl esterified pectins in tricellular junctions and high levels of catechin at this stage. Meanwhile, in 'Emerald' 's ripe fruit epicarp, several mechanisms of cell wall reinforcement such as calcium and probably boron bridges, appear to be more prominent than in 'O'Neal'. This study highlights the importance of cell wall reorganization and structure, abundance of specific metabolites, water status, and hormonal signalling in connection to fruit firmness. These findings result particularly valuable in order to improve the fertilization procedures or in the search of molecular markers related with firmness.

Proteomic and metabolomic approaches unveil relevant biochemical changes in carbohydrate and cell wall metabolisms of two blueberry (Vaccinium corymbosum) varieties with different quality attributes.

Quality maintenance in rapidly decaying fruit such as blueberries (Vaccinium corymbosum) is of essential importance to guarantee the economic success of the crop. Fruit quality is a multifaceted subject that encompasses flavor, aroma, visual and physical issues as main factors. In this paper we report an ample characterization of different biochemical and physical aspects in two varieties (O'Neal and Emerald) of blueberries that differ in firmness, aspect, flavor and harvesting times, at two different phenological stages (fruit set vs. ripe), with the intention of unveiling how the metabolic signature of each contributes to their contrasting quality. To this effect a metabolomic, ionomic and proteomic approach was selected. The results presented here show marked differences in several variables at the two stages and between varieties. Emerald is an early variety with a large, good taste and firm fruit, while O'Neal is soft, medium sized and very sweet. Proteomic data comparison between both cultivars showed that, at fruit set, processes related with the response to inorganic compounds and small molecule metabolisms are relevant in both varieties. However, solute accumulation (mainly amino acids and organic acids), enzymes related with C: N balance, water transport and cell wall recycling are enhanced in Emerald. In ripe fruit, Emerald showed an enrichment of proteins associated with TCA, nitrogen, small molecules and cell wall in muro recycling processes, while mannitol and fatty acid metabolism were enhanced in the soft variety. The measured variation in metabolite levels gave strong support to the precedent results. This study suggests that at fruit set, a composite scenario of active metabolic recycling of the cell wall, improved C: N balance and solute accumulation give place to a more efficient carbon and water resource management. During the ripe stage, an increased and efficient in muro and metabolic recycling of the cell wall, added to enhanced inositol and secondary metabolism may be responsible for a best turgor conservation in Emerald. These findings may yield clues for improvements in fertilization practices, as well as to assist the guided development of new varieties based on biochemical quality.

Plant cytosolic pyruvate kinase: a kinetic study.

The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP. Mg2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc.

Isolation and sequence of an active-site peptide from maize leaf phosphoenolpyruvate carboxylase inactivated by pyridoxal 5'-phosphate.

An active-site peptide from maize (Zea mays L.) phosphoenolpyruvate carboxylase has been isolated, sequenced and identified in the primary structure following chemical modification/inactivation of the enzyme by pyridoxal 5'-phosphate and reduction with sodium borohydride. The amino acid sequence of the purified dodecapeptide is Val-Gly-Tyr-Ser-Asp-Ser-Gly-L*ys-Asp-Ala-Gly-Arg, which corresponds exactly to residues 599-610 in the deduced primary sequence of the maize-leaf enzyme. Comparative analysis of the deduced amino acid sequences of the enzyme from Escherichia coli, Anacystis nidulans and C3, C4 and Crassulacean acid metabolism plants indicates that they all contain this specific lysyl group, as well as a high degree of sequence homology flanking this species-invariant residue. This observation suggests a critical role for Lys-606 during catalysis by maize phosphoenolpyruvate carboxylase. This represents the first identification of a specific, species-invariant active-site residue in the enzyme.

IGF-I mRNA and signaling in the diabetic retina.

IGF-I promotes the survival of multiple cell types by activating the IGF-I receptor (IGF-IR), which signals downstream to a serine/threonine kinase termed Akt. Because in diabetes vascular and neural cells of the retina undergo accelerated apoptosis, we examined IGF-I synthesis and signaling in the human and rat diabetic retina. In retinas obtained postmortem from six donors aged 64 +/- 8 years with a diabetes duration of 7 +/- 5 years, IGF-I mRNA levels were threefold lower than in the retinas of six age-matched nondiabetic donors (P = 0.005). In the retinas of rats with 2 months' duration of streptozotocin-induced diabetes, IGF-I mRNA levels were similar to those of control rats, but after 5 months of diabetes they failed to increase to the levels recorded in age-matched controls (P < 0.02). Retinal IGF-I expression was not altered by hypophysectomy, proving to be growth-hormone independent. IGF-IR levels were modestly increased in the human diabetic retinas (P = 0.02 vs. nondiabetic retinas) and were unchanged in the diabetic rats. Phosphorylation of the IGF-IR could be measured only in the rat retina, and was not decreased in the diabetic rats (94 +/- 18% of control values). In the same diabetic rats, phosphorylation of Akt was 123 +/- 21% of control values. There was not yet evidence of increased apoptosis of retinal microvascular cells after 5 months of streptozotocin-induced diabetes. Hence, in the retina of diabetic rats, as in the retina of diabetic human donors, IGF-I mRNA levels are substantially lower than in age-matched nondiabetic controls, whereas IGF-IR activation and signaling are not affected, at least for some time. This finding suggests that in the diabetic retina, the activation of the IGF-IR is modulated by influences that compensate for, or are compensated by, decreased IGF-I synthesis.

Increased expression of tissue plasminogen activator and its inhibitor and reduced fibrinolytic potential of human endothelial cells cultured in elevated glucose.

In diabetic patients, elevated plasma levels of t-PA and PAI-1 accompany impaired fibrinolysis. To identify mechanisms for these abnormalities, we examined whether vascular endothelial cells exposed to high glucose upregulate t-PA and PAI-1 production and whether ambient PA activity is decreased concomitantly. In 17 cultures of human umbilical vein endothelial cells grown to confluency in 30 mM glucose, the t-PA antigen released to the medium in 24 h was (median) 52 ng/10(6) cells (range 10-384) and the PAI-1 antigen was 872 ng/10(6) cells (range 217-2074)--both greater (P less than 0.02) than the amounts released by paired control cultures grown in 5 mM glucose--29 ng/10(6) cells (range 7.5-216) and 461 ng/10(6) cells (range 230-3215), respectively. In the presence of high glucose, the steady-state levels of t-PA and PAI-1 mRNAs were increased correspondingly (median 142 and 183% of control, respectively, P less than 0.05); high glucose per se and hypertonicity contributed to the upregulation in additive fashion. The PA activity of conditioned medium from cultures exposed to high glucose was 0.4 IU/ml (range 0.2-0.6), which was significantly lower (P less than 0.02) than the PA activity of control medium (0.5 IU/ml, range 0.2-0.9). No difference was observed when comparing the PA activities of acidified conditioned media, expected to be depleted of inhibitors. Thus, high glucose coordinately upregulates endothelial t-PA and PAI-1 expression through effects exerted at the pretranslational level and enhanced by even mild degrees of hypertonicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol.

Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo.

Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant, Pineapple.

Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.

Fibroblast Na+-Li+ countertransport rate is elevated in essential hypertension.

OBJECTIVES: Elevated erythrocyte Na+- Li+ countertransport (SLC) rates are commonly found in essential hypertension. We have recently shown that human skin fibroblasts functionally express a phloretin-sensitive Na+-H+ exchange (NHE) which may also be similar to erythrocyte SLC because of amiloride-insensitivity. DESIGN AND METHODS: We investigated whether elevations in fibroblast SLC parallel the known elevations in erythrocyte SLC and in cell NHE that characterize essential hypertension. RESULTS: Higher fibroblast SLC rates were found among hypertensive patients (n = 23, median 48.8 nmol Li+/ mg(protein) per min) than in 19 normotensive individuals of similar age and sex (median 14.8 nmol Li+/mg(protein) per min, P= 0.0002). As expected, erythrocyte SLC was elevated in patients with hypertension (median 411 versus 329 micromol/l(cell) per h, P= 0.0273), but was not quantitatively related to fibroblast SLC. Finally, fibroblast NHE exchange activity was higher in essential hypertension (median Vmax 14.2 versus 7.6 mmol H+/l(cell) per min, P= 0.002), but was unrelated to fibroblast SLC. CONCLUSIONS: These findings extend to human skin fibroblasts the notion of abnormal Li+ transport in essential hypertension, and appear to be in accordance with the hypothesis that fibroblast SLC may be independent of NHE. However, molecular studies will be required to understand whether distinct exchangers and/or regulation mechanisms underlie these dysregulations.

Vascular wall von Willebrand factor in human diabetic retinopathy.

PURPOSE: To reconstruct the role played by vascular endothelium in the elevation of circulating von Willebrand factor (vWf) in diabetic patients with microangiopathy and, specifically, to determine whether storage and synthesis of vWf is altered in diabetic retinal vessels. METHODS: Trypsin digests were prepared from retinas obtained post mortem from 11 patients (age 62 +/- 9 years, mean +/- SD) with 9 +/- 5 years of diabetes and 12 nondiabetic control subjects matched for age and sex. Trypsin digests were inspected for the presence of lesions of diabetic retinopathy; vWf protein was localized by indirect immunofluorescence; and vWf mRNA levels were studied by in situ hybridization. RESULTS: vWf immunofluorescence was present in vessels of all sizes. The granular fluorescence was localized to the endothelial cell cytoplasm. Pattern and intensity of staining in diabetic microvessels and large vessels were similar to those observed in the vessels of nondiabetic subjects. The amount of vWf mRNA detected by in situ hybridization in retinal endothelial cells was similar in diabetic (0.92 +/- 0.32 grains/cell) and control (0.91 +/- 0.42 grains/cell) microvessels. Likewise, no differences were observed in vWf mRNA levels in the large vessels of diabetic (0.073 +/- 0.034% grain area) and control (0.069 +/- 0.018 grain area) subjects. CONCLUSIONS: These observations are compatible with the occurrence in diabetes of the slow release of endothelial vWf through the pathway of vWf secretion not linked to synthesis, ie, the regulated pathway.

Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia.

PURPOSE: To examine the relationship between early retinal capillary cell apoptosis and late histologic lesions of diabetic retinopathy and to compare the effects of aminoguanidine (AMG) on the retinopathies caused by diabetes and galactose feeding. METHODS: Rats with alloxan-induced diabetes and rats fed a 30% galactose diet (known to induce diabetic-like retinopathy) were assigned randomly to receive diet with (2.5 g/kg diet) or without AMG. After 6 to 8 months of diabetes or galactosemia, retinal trypsin digests were prepared, and capillary cell apoptosis was quantitated using the Tdt-mediated dUTP nick-end labeling (TUNEL) reaction in association with morphologic evidence of nuclear fragmentation. At 18 months duration, pericyte ghosts and acellular capillaries were quantitated in the isolated vasculature. Several advanced glycation end products (AGEs) were measured at 4 months of study and at 18 months of study by established methods to assess biochemical effects of AMG. RESULTS: As expected, both diabetic and galactosemic rats showed increased frequency of TUNEL-positive capillary cells at 6 to 8 months and vascular lesions characteristic of retinopathy at 18 months. AMG inhibited both the early apoptosis and late histopathology in the diabetic rats, but neither of these abnormalities in the galactosemic rats. In contrast to its preventative effect on retinopathy in the diabetic rats, AMG showed no inhibitory effect on levels of hemoglobin AGE, or tail collagen pentosidine, fluorescence, and thermal breaking time. Diabetes of 4 months' duration did not cause a detectable increase in retinal levels of several AGEs. CONCLUSIONS: The frequency of early apoptosis in retinal microvascular cells predicted the development of the histologic lesions of retinopathy in diabetes as well as in galactosemia. The beneficial effect of AMG on retinal lesions in diabetes is exerted on pathways that are either not operative or are less important in galactosemia and that may not relate to the accumulation of AGEs.

Low Ca(2+) pump activity in diabetic nephropathy.

Elevated cell Na(+)-H(+) exchange (NHE) activity characterizes diabetic nephropathy (DN), but the mechanisms of this abnormality are unclear. Recent evidence suggests that NHE and the Ca(2+) pump share similar regulatory pathways, but whether abnormalities in Ca(2+) metabolism characterize DN is not known. We investigated Ca(2+) efflux rates, NHE activity, cytosolic Ca(2+) ([Ca(2+)](i)) concentrations, and intracellular pH (pH(i)) in human skin fibroblasts from 20 patients with type 1 (insulin-dependent) diabetes and nephropathy; 20 patients with diabetes with normoalbuminuria matched for age, sex, and duration of diabetes; and 10 individuals without diabetes. Ca(2+) pump-mediated Ca(2+) efflux was significantly lower in patients with nephropathy than in patients with normoalbuminuria and individuals without diabetes (0.074 +/- 0.01 versus 0.115 +/- 0.01 versus 0.131 +/- 0.02 nmol.mg(protein)(-1).min(-1); analysis of variance [ANOVA], P = 0.015). Elevated maximal velocity of the Na(+)-H(+) exchanger was confirmed in fibroblasts from patients with nephropathy (14.4 +/- 1.2 versus 7.1 +/- 0.7 versus 8.0 +/- 1.2 mmol H(+).l cell(-1).min(-1); ANOVA, P < 0.0001). A reverse correlation between Ca(2+) pump activity and NHE rates could be shown. Adjustment for glycated hemoglobin and plasma lipid levels did not affect these findings. Finally, [Ca(2+)](i) concentrations and pH(i) were normal in all patients. Low Ca(2+) pump activity is a concomitant event of elevated NHE rates in DN; the molecular dysfunction(s) underlying these abnormalities remains to be established.

[Evaluation in experimental animals of a mathematical model of foreseeing variations in values of stable glycosylated hemoglobin]. / Valutazione sull'animale da esperimento di un modello matematico per la previsione delle variazioni dei valori di emoglobina glicosilata stabile.

Assay of glycosylated hemoglobin provides reliable information on metabolic control in diabetes mellitus over a period of about 90 days. This is why it is currently used as a parameter of blood glucose control in diabetic patients. However, at present little is known about the kinetics of stable glycosylated hemoglobin variations as a result of circadian changes in blood glucose level. The authors describe a mathematical model which allows to foresee glycosylated hemoglobin variability as a result of alterations of blood glucose equilibrium.

[A multicenter study of the biometric parameters of Tuscan newborns]. / Indagine policentrica sui parametri biometrici neonatali toscani.

We combined data concerning weight, length, chest's perimeter and head's circumference of newborns of 16 Tuscan hospitals on 1985, with sample of 4,215 boys and 4,119 girls, from 33.th to 42.th gestational week. These data have been statistically elaborated to obtain values of the four considered parameters and the respective standard deviation. For the distribution of the 16 hospital in the region, we think that these data are representatives of Tuscan newborn's reality. We have moreover considered whether data of Tuscan newborns, with parents proceeding from different regions, were dissimilar from those with autochthonous parents. The statistical comparison of the two reality, with "T-Student's test', is resulted not significant. We calculated then values of the four parameters with their standard deviation, for each hospital participating to the research, excluding groups with to small numerousness, i.e. 33rd, 34th, 35th and 42nd gestational week. Besides, we valued if data of each Hospital were statistically different from those related to the whole Tuscan sample. The results of this testing, performed with "T-Student's test", are illustrated in the schedules. The differences seem to be referred to local conditions, that will be object of new researches. At present, we can only emphasize that the chest's perimeter is the parameter with the larger variability, so that we will exclude this size from the next research.

[Scoliosis: ten years' experience of screening]. / La scoliosi: esperienza di dieci anni di dépistage.

Scoliosis is a permanent lateral deflection of backbone, associated to a vertebra's rotation and twisting on their vertical axle, and it is one of more common diseases of pediatric age. It exist a great difference between the structural conformations, that are true pathologies, and the functional ones or paramorphisms, without bony alterations. Since about 80% of real scoliosis is idiopathic. The pediatrician's task is of precociously identify the appearance of a scoliotic bend and differentiate the structural and evolutive shapes from simple scoliotic posture. Therefore for ten years, in the preventive medicine programme of school age, we affected a scoliosis research in the secondary school using as clinical method the "bending test" that appraises three essential parameters: size's triangle, gibbus and limbs' asymmetry. Before examination an anamnestic form is compiled and the sexual maturity's degree is appraised. The subjects with suspect of scoliosis are asked to a subsequential control performed in our department's ambulatory service by a doctor of Scoliosis's Center of Pozzolatico (Florence), in order to decide whether to take radiographs and to define, if necessary, treatment. We verified 4453 pupils and of these the 8.9% has been asked to the specialist's control. Of these 106, namely the 2.8% of total pattern, have been X-rayed and 46, namely the 0.9% of total pattern, have been orthopaedically treated because affected by evolutive scoliosis. If we consider the last quinquennium's statistics, we remark that treatment's incidence is lower to 0.4% conforming to literature's data. We never observed false negatives whereas false positive have been only 0.8% of total pattern.(ABSTRACT TRUNCATED AT 250 WORDS)