Effect of vitamin E on reperfusion injuries during reconstructive vascular operations on lower limbs.

INTRODUCTION: The challenge against reperfusion injury and tissue oxidative stress, especially in vascular surgical interventions has an essential importance to reach the optimal clinical result. Numerous experimental attempts have proved the positive antioxidant effect of vitamin E in both chronic and acute phase models. In our study we monitored the effect of continuous preoperative treatment with vitamin E, on oxidative stress and tissue inflammation reactions developed after reconstructive operations. PATIENTS AND METHODS: 32 patients have been involved in a randomized, prospective study, all suffering from AFS occlusion proved by angiography, and all undergone supragenual reconstruction. Duration of ischemia and amount of tissues under vascular clamping were almost the same in all patients. In the group treated with E-vitamin, we administered 1 x 200 mg of vitamin E p/o from the preoperative day till the 7th post operative day. Patients of the second group did not receive vitamin E. MATERIALS AND METHODS: Peripheral blood samples were collected immediately before operation and at the end of the second reperfusion hour (early reperfusion period). Late reperfusion period has been monitored by analyzing blood samples taken at 24th hour and 7th day next to the operative ischemia. Among oxidative stress parameters, direct measurement of reactive oxygen intermediator (ROI) and determination of antioxidant state (GSH, Total-SH group, SOD) have been performed. Malondialdehyde was chosen as marker for lipidperoxidation. Inflammation reactions were monitored up on expression of adhesion molecules (CD11a and CD18). We also controlled the oscillation of myeloperoxidase (MPO) activity. RESULTS: Our study has proved that preoperative (from the preoperative day till the 7th post operative day) administration of 200 mg vitamin E could reduce the level of oxidative stress developed after ischemic-reperfusion insult (lipidproxidation, antioxidant enzymes). According to our results, the prooxidant-antioxidant imbalance also diminished in the group with E-vitamin treatment. We proved that elective administration of vitamin E could decrease the WBC activity (MPO activity, free radicals production, expression of adhesion molecules) and its consequential local inflammation process, during early reperfusion.

Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid.

Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.

Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos.

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of alpha-eleostearic (18:3Delta(9cis, 11trans,13trans)) and alpha-parinaric (18:4Delta(9cis,11trans, 13trans,15cis)) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of alpha-eleostearic and alpha-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Delta(12)-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of alpha-eleostearic and alpha-parinaric acids, neither of which is present in untransformed soybean embryos. alpha-Eleostearic and alpha-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Delta(12)-oleic acid desaturase-type enzymes that we have termed "conjugase."

A 62-kD sucrose binding protein is expressed and localized in tissues actively engaged in sucrose transport.

Sucrose transport from the apoplasm, across the plasma membrane, and into the symplast is critical for growth and development in most plant species. Phloem loading, the process of transporting sucrose against a concentration gradient into the phloem, is an essential first step in long-distance transport of sucrose and carbon partitioning. We report here that a soybean 62-kD sucrose binding protein is associated with the plasma membrane of several cell types engaged in sucrose transport, including the mesophyll cells of young sink leaves, the companion cells of mature phloem, and the cells of the developing cotyledons. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely parallel the rate of sucrose uptake in the cotyledon. Molecular cloning and sequence analysis of a full-length cDNA for this 62-kD sucrose binding protein indicated that the protein is not an invertase, contains a 29-amino acid leader peptide that is absent from the mature protein, and is not an integral membrane protein. We conclude that the 62-kD sucrose binding protein is involved in sucrose transport, but is not performing this function independently.

Substance use in young adults with schizophrenic disorders.

Use of nonprescribed mood altering substances is pervasive and problematic in young adults with serious mental illnesses in community care. Fifty-eight percent of young adult clients with clearly defined schizophrenia or schizophrenia-related disorders participating in a long-term community treatment study were rated by staff or themselves as using alcohol, cannabis, or other street drugs several times a week or more. We interviewed in depth a random sample of these "significant users" to obtain their perspective on their frequencies, patterns, histories, contributing factors to, and effects of substance use and their related treatment experiences. Results revealed these clients' substance use to be of long duration and deeply entrenched, with current use often involving multiple substances including both street drugs and substances of "everyday life" (e.g., caffeine, nicotine). Clients reported compelling reasons for use including anxiety reduction, relief of boredom, and a means for social contact. Staff and clients clearly view substance use quite differently, with the latter focusing at least as much on consequences of symptom relief as symptom exacerbation. Treatment implications are discussed.

Identification of membrane protein associated with sucrose transport into cells of developing soybean cotyledons.

The photolyzable sucrose derivative 6'-deoxy-6'-(4-azido-2-hydroxy)-benzamidosucrose (6'-HABS), competitively inhibited the influx of [(14)C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of (125)I-labeled 6'-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl alpha-d-thioglucopyranoside. Glucose, raffinose, or phenyl alpha-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6'-HABS was attached to 6-deoxy-6-aminoglucose and (125)I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter.

Substrate recognition by a sucrose transporting protein.

Protoplasts derived from developing soybean cotyledons were used to study substrate recognition by a sucrose transporting protein in plant membranes. When used as alternate substrate inhibitors of [14C] sucrose influx, five different fructosyl-substituted sucrose derivatives, phenyl-alpha-D-glucopyranoside, and phenyl-alpha-D-thioglucopyranoside proved to bind effectively to the sucrose carrier-active site. These results are interpreted to indicate that a large portion of substrate recognition by this carrier may arise from the interaction of a relatively hydrophobic portion of the sucrose molecule and a hydrophobic region of the carrier protein binding site. Binding of phenyl-alpha-D-thioglucopyranosides in which various substitutions were made for the glucosyl hydroxyls shows that the glucosyl hydroxyls at positions 3, 4, and 6 are involved in substrate recognition by the carrier protein.