Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H₂O₂). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-¹³C₂,6,6-²H₂]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H₂O₂, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H₂O₂ caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H₂O₂ exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H₂O₂-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H₂O₂ detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.     

chs-4, a class IV chitin synthase gene fromNeurospora crassa

InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a reverse genetics approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 ^RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.A. Beth Din and C. A. Specht contributed equally to this work     

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

State mutability and ethnic civil society: the Palestinian Arab minority in Israel

This article identifies the phenomenon of ethnic civil society activism as mobilization that seeks to empower an ethnic community and challenges the institutional order. The case of the Palestinian Arab minority in Israel is discussed and is used to reveal the conditions under which disaffected minorities pursue the path of ethnic civil society. The study finds that an analytical framework that stresses the mutability of state structures and changes in broader state-society relations provides a better explanation than existing theories of ethnic conflict. State-society characteristics conducive to this type of mobilization are a well-institutionalized state that can prevent deviation from the state's foundational rules and a counterbalanced dispersion of authority that limits regime capacity to control society.