A nonchromatographic assay for sorbitol in chemically complex growth media containing sorbose

Summary Microbiologists who lack gas-chromatographic equipment cannot easily study the microbial conversion of sorbitol to sorbose, because there is no other proven method for quantitatively measuring sorbitol in complex growth media. The purpose of this study was to determine if the polyol assay suggested by Mäkinen in 1980 could be used to measure sorbitol depletion in chemically complex bacteriological-culture media containing sorbose. After we pretreated the culture medium to remove interfering phosphate complexes and slightly modified Mäkinen's assay, we were able to detect 0.1 mg/ml differences in sorbitol concentrations that varied between 0.9 and 1.5 mg of sorbitol/ml. The presence of sorbose in the complex medium did not affect the assay. Growing cultures ofGluconobacter oxydans were used to test this assay, because these bacteria reportedly oxidize sorbitol to sorbose and quantitatively release the sorbose into the growth medium. When samples of the culture medium removed during growth were centrifuged to remove cells and precipitated to remove interfering substances then tested with the mofidied Mäkinen assay, these cultures showed a sorbitol depletion rate of 4.9 (±0.1) mg/ml h^–1. The Fehling's assay on the same cultures showed a sorbose accumulation rate of 5.12 (±0.01) mg/ml h^–1. We concluded that the modified Mäkinen phosphate-interference assay can be satisfactorily used to quantitatively screen cultures for their ability to oxidize sorbitol.     

Mitochondrial control of cell death induced by hyperosmotic stress

HeLa and HCT116 cells respond differentially to sorbitol, an osmolyte able to induce hypertonic stress. In these models, sorbitol promoted the phenotypic manifestations of early apoptosis followed by complete loss of viability in a time-, dose-, and cell type-specific fashion, by eliciting distinct yet partially overlapping molecular pathways. In HCT116 but not in HeLa cells, sorbitol caused the mitochondrial release of the caspase-independent death effector AIF, whereas in both cell lines cytochrome c was retained in mitochondria. Despite cytochrome c retention, HeLa cells exhibited the progressive activation of caspase-3, presumably due to the prior activation of caspase-8. Accordingly, caspase inhibition prevented sorbitol-induced killing in HeLa, but only partially in HCT116 cells. Both the knock-out of Bax in HCT116 cells and the knock-down of Bax in A549 cells by RNA interference reduced the AIF release and/or the mitochondrial alterations. While the knock-down of Bcl-2/Bcl-X_L sensitized to sorbitol-induced killing, overexpression of a Bcl-2 variant that specifically localizes to mitochondria (but not of the wild-type nor of a endoplasmic reticulum-targeted form) strongly inhibited sorbitol effects. Thus, hyperosmotic stress kills cells by triggering different molecular pathways, which converge at mitochondria where pro- and anti-apoptotic members of the Bcl-2 family exert their control. A. Criollo and L. Galluzzi contributed equally to this work.     

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

Astrocytes constitute a major class of glial cells in the CNS, and play crucial roles in physiological functioning, performance and maintenance of the CNS, as well as promotion of neuronal migration and maturation. Astrocytes have also been directly and indirectly implicated in the pathophysiology of various trauma occurrences, development of neurodegenerative diseases and nerve regeneration. To further understand mechanisms by which astrocytes elicit these effects, the first critical step in the study of astrocytes is the preparation of purified astrocytes cultures. Here we describe a simple and convenient procedure for producing rat primary astrocyte cultures of high purity, viability and proliferation. For astrocyte culture, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, seeding density and composition of the culture medium. Using immunofluorescent antibodies against GFAP and OX-42 in combination of Hoechst 33342 fluorescent staining, we found that the purity of the astrocyte cultures was >99%. Astrocytes had high viability as measured by 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, flow cytometric analysis was used to measure and observe variations in the cell cycle after 1–2 passages and proliferation of astrocytes was detected with a high percentage of cells stand in S+G₂/M phase. Therefore, the method described here is ideal for experiments, which require highly pure astrocyte cultures.     

Multiple independent kinase cascades are targeted by hyperosmotic stress but only one activates stress kinase p38

In this report, we analyse the effects of osmotic shock on signal transduction in CHO cells. We demonstrate that at least three different kinase cascades are switched on upon osmotic shock, namely PKA, AMPK, and MLTK. Whereas PKA from cells treated with forskolin activated stress kinase p38, PKA from cells treated with sorbitol did not activate p38, although the enzyme is activated in both cases as analysed in vitro using a specific peptide target. Further, osmolar shock activated AMPK but treatment of the cells with the AMPK activator 5-amino-4-imidazolecarboxamide (AICAr) did not result in p38 activation, strongly suggesting that AMPK is not involved in stress kinase activation. Transfection of CHO cells with dominant negative recombinants of MLTKalpha resulted in inhibition of sorbitol-mediated p38 activation, indicating that the mixed-lineage kinase is involved in the activation of p38 by sorbitol. Finally, in CHO cells overexpressing wild-type MLTKalpha, no activation of AMPK of PKA could be demonstrated, indicating that the activated kinase cascades are not involved in a cross-talk process.     

Is sorbital dehydrogenase gene expression affected by streptozotocin-diabetes in the rat?

The polyol pathway, which comprises the enzymes aldose reductase and sorbitol dehydrogenase, is recognised to play a major role in the pathogenesis of diabetic complications. Although there has been extensive research on aldose reductase, the role of sorbitol dehydrogenase has been overlooked. This study examined the response of sorbitol dehydrogenase gene expression to streptozotocin-diabetes (STZ-diabetes) in the rat and whether these changes were reversed by insulin. STZ-diabetes increased testicular sorbitol dehydrogenase gene expression in a manner that was not reversible by insulin but had no effect on gene expression in kidney and brain. A secondary question was the relationship between sorbitol dehydrogenase and aldose reductase gene expression in STZ-diabetes. STZ-diabetes increased renal dose reductase gene expression in a manner that was not reversible by insulin but had no effect on gene expression in the brain, testes and muscle. Thus, STZ-diabetes causes changes in sorbitol dehydrogenase gene expression which do not parallel those in aldose reductase, implying that expression of the two genes is not regulated via a common mechanism. Furthermore, changes in sorbitol dehydrogenase and aldose reductase gene expression cannot be fully explained on the basis of the osmoregulatory hypothesis, suggesting that regulation is mediated via mechanisms that are multifactorial and tissue-specific.     

2-C-methyl-D-erythritol is a major carbohydrate in petals of Phlox subulata possibly involved in flower development

2-C-methyl-D-erythritol, a soluble carbohydrate that is not ubiquitously found in higher plants, was detected in the ethanol extract from Phlox subulata petals and isolated using HPLC. The isolated compound was identified by 1H-NMR, 13C-NMR and Cl-MS spectra. 2-C-methyl-D-erythritol was a major soluble carbohydrate in petals, leaves and stems. In petals, the concentration of 2-C-methyl-D-erythritol markedly increased during flower development and opening and was similar in concentration to glucose, a ubiquitous metabolic sugar. This suggests that 2-C-methyl-D-erythritol may contribute to flower opening in association with glucose in the P. subulata.     

Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus

The capacity to accumulate winter polyols (mainly ribitol and sorbitol) during cold-acclimation in Pyrrhocoris apterus is restricted only to the adults that have previously entered diapause. The enzymatic complement involved in polyol biosynthesis was found to differ in a complex manner between diapause and non-diapause adults. Nearly 100% of glycogen phosphorylase (GPase) was present in its active form in non-diapause adults irrespective of their acclimation status. In contrast, less than 40% of GPase was present in its active form in diapause adults prior to cold-acclimation and the inactive form was rapidly activated upon transition from 5 to 0 degrees C, concomitantly with the start of rapid polyol accumulation. The flow of carbon released by activation of glycogen degradation might be routed to the pentose cycle because the activity of glucose-6-P dehydrogenase (G(6)P-DH) was significantly higher and it increased with cold-acclimation in diapause adults while it was relatively low and it decreased with cold-acclimation in non-diapause adults. Reducing equivalents in the form of NADPH, which were generated in the pentose cycle, might require re-oxidation. Such re-oxidation might be achieved during reduction of sugars to polyols. The activity of NADP(H)-dependent aldose reductase (AR) was about 20-fold higher in diapause than in non-diapause adults. Similarly, the activity of NAD(H)-dependent polyol dehydrogenase (PDH) was higher in diapause adults. In addition, we found a very high activity of an unusual enzyme, NADP(H)-dependent ketose reductase (KR), exclusively in diapause adults. KR might be involved in reduction of fructose to sorbitol. Although its affinity for fructose as a substrate was low (K(M)=0.64M), its activity was about 10-fold higher than that of PDH with fructose. Moreover, the activity of KR significantly increased with cold-acclimation while that of PDH remained unchanged. Different electrophoretic mobilities in PAGE gel suggested that KR and PDH are two different enzymes with specific requirement for NADP(H) or NAD(H), respectively, as co-factors.     

Chemotaxonomic markers in Digitalideae (Plantaginaceae)

In a chemosystematic investigation of Digitalideae (Plantaginaceae), the water-soluble part of extracts of two species of Digitalis, two species of Isoplexis, as well as Erinus alpinus and Lafuentea rotundifolia were studied with regard to their content of main carbohydrates, iridoids and caffeoyl phenylethanoid glycosides (CPGs). Digitalis and Isoplexis contained sorbitol, cornoside and a number of other phenylethanoid glycosides including the new tyrosol beta-D-mannopyranoside, sceptroside but were found to lack iridoid glucosides. Erinus contained mainly glucose, the new 8,9-double bond iridoid, erinoside, and a number of known iridoid glucosides including two esters of 6-rhamnopyranosylcatalpol, as well as the CPG poliumoside. Finally, Lafuentea was characterized by the presence of glucose, aucubin and cryptamygin B but apparently lacked CPGs. The chemosystematic significance of the isolated compounds is discussed.     

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO<Subscript>2</Subscript> assimilation in apple leaves

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, Greensleeves apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15–30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. ¹⁴CO₂ pulse-chase labeling at ambient CO₂ demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO₂ assimilation in response to photon flux density or intercellular CO₂ concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO₂ assimilation in source leaves was sustained at both ambient CO₂ and saturating CO₂.     

Diverse associations in the ternary systems of β-cyclodextrin, simple carbohydrates and phenyl derivatives of inorganic oxoacids

Complex formation reactions of phenylboronic, phenylphosphonic, phenylarsonic and 4-aminophenyl arsonic acids with β-cyclodextrin (cycloheptaamylose, β-CD) and some simple carbohydrates (mannitol, sorbitol, glucose) have been studied using spectrophotometric, potentiometric methods and solubility measurements, supplemented with HPLC and IR analyses of the solid samples. Equilibrium constants have been determined at ionic strength of 0.2 M (NaCl) and 25 °C. β-CD forms the most stable complexes with the neutral, undissociated forms of the acids, the stability constants are as follows: phenylboronic acid: 320 ± 36, phenylphosphonic acid: 108 ± 25, phenylarsonic acid: 97 ± 4 and 4-aminophenyl arsonic acid: 107 ± 10. The stability constants for the β–CD-complexes of the ionic forms are much lower. Ternary complexes of low stability could be detected in the case of phenylphosphonic acid and sorbitol with the undissociated form and with glucose and the dianion. In more concentrated solutions phenylboronic acid forms insoluble complexes with mannitol, sorbitol and β-CD. The solid phases obtained in the ternary systems are predominantly mixtures of ester type 3:1 complexes with the carbohydrate and 1:1 inclusion complex with the β-CD. No significant interaction has been found with glucose. The phenomena can be explained by the differences in the structures of the components and by the changes in the H-bonding network of β-CD on the complex formation.