Novel use of the wild species Cephalaria joppensis for silage preparation and its nutritive value for feeding lactating dairy cows.

This study presents a novel method for use of the wild plant species Cephalaria joppensis (CJ) as agricultural forage for ruminants. Domesticated CJ tends to have higher crop mass yield per hectare than a commercial wheat variety (W) but is similar in in vitro dry matter (DM) digestibility. This study was composed of 3 experiments. Experiment 1 aimed to measure effects of ensiling CJ versus W in packed polyethylene-wrapped bales. Three types of ensiled bales were produced for each plant: 1) direct-cut CJ versus W packed solely; 2) direct-cut CJ versus W mixed as sole roughage source together with dietary ingredient and packed in bales to create CJ total mixed ration (CJ-TMR) or W-TMR; 3) CJ silage versus W silage mixed as one-third of dietary roughage source together with two-thirds sorghum (S) silage and additional dietary ingredients and packed in bales to create CJ-S-TMR or W-S-TMR. Data showed that packing and wrapping created anaerobic conditions within the 4 types of TMR bales while reducing pH (4.12 to 4.37). Dry matter loss during ensilage was higher for the 2 types of TMR containing W compared with CJ. Ensilage decreased soluble nitrate content as well as yeast and mold contamination, and the 4 types of TMR bales were characterized by a long outdoor shelf life (3 mo) and high stability under aerobic exposure. Experiment 2 aimed to measure the intake and digestibility by sheep of the 4 types of packed TMR after 90 d of ensiling. Data demonstrated higher voluntary intake of the CJ-TMR compared with the other TMR types. The CJ-TMR was characterized by higher digestibility of DM, crude protein, and neutral detergent fiber components compared with the CJ-S-TMR. Experiment 3 examined intake, digestibility, and milk production by 21 pairs of lactating cows individually fed CJ-S-TMR versus W-S-TMR. Similar intake (21.6 to 22.0 kg/d) and digestibility of DM and crude protein were observed in cows fed the 2 TMR types (68 to 69% and 66 to 68%, respectively). However, neutral detergent fiber and cellulose digestibility were slightly higher in the cows fed W-S-TMR and this was reflected in a small increase in their milk and energy-corrected milk yield (36.5 and 31.4 kg/cow per day, respectively) compared with cows fed CJ-S-TMR (35.5 and 30.4 kg/cow per day, respectively). Results demonstrate that direct-cut CJ used as is, or CJ silage can be included and ensiled in TMR bales for feeding productive ruminants as a substitute for wheat silage.

Does side matter? Effect of single lung transplant laterality on outcomes.

BACKGROUND: Previous works have suggested that recipients of left single lung transplant (SLT) have a worse outcome than those receiving right SLT. We evaluated the effect of SLT laterality on outcomes. METHODS: We performed a retrospective study of SLT recipients followed up at our center. One hundred and nineteen patients were reviewed (53 left SLT, 66 right SLT). We extracted data on lung function, exercise capacity, relative graft perfusion, airway complications, acute rejection episodes, infections and mortality. RESULTS: There was no significant difference between right and left lung recipients with regard to baseline demographic and physiological characteristics. Lung function, exercise capacity and relative graft perfusion improved in both groups following transplantation. We observed a higher graft perfusion in right-sided grafts compared to left ( P = 0.048). There was no significant difference between the two groups in physiological outcomes, rejection or infection episodes, the presence of chronic rejection or mortality. We observed a statistically higher need for bronchial stent insertion during early follow-up amongst the left lung recipients ( P = 0.022). CONCLUSIONS: Both right and left lungs are equally suitable for transplantation. The left-sided bronchial anastomosis may be more vulnerable to complications.

Discovering new ordered phases of block copolymers

We propose a new and general method for discovering novel ordered phases of block copolymer melts. The method involves minimizing a free energy functional in an arbitrary unit cell with respect to the composition profile and the dimensions of the unit cell, without any prior assumption of the microphase symmetry. Varying the initial conditions allows to search for different stable and metastable structures. Application of this method to ABC star and linear triblock copolymers using an approximate free energy reveals new morphologies not yet observed in experiment.

P-glycoprotein-overexpressing multidrug-resistant cells are resistant to infection by enveloped viruses that enter via the plasma membrane.

The multidrug resistance gene product P-glycoprotein confers drug resistance to tumor cells by acting as a transporter that blocks the entry into the cell of a great variety of drugs and hydrophobic peptides. In this study we find that in drug-resistant cells, the insertion of the influenza virus fusion protein (hemagglutinin-2) into the plasma membrane is blocked and that the fusion of the viral envelope with the plasma membrane of these cells is impaired. Multidrug-resistant cells display significant resistance to infection by envelope viruses that invade cells by fusion with the plasma membrane, but not to infection by pH-dependent viruses that penetrate cells by fusion with endocytic vesicles. These observations suggest that multidrug resistance phenomena may protect cells from infection by a large group of disease-causing viruses that includes human immunodeficiency virus, herpes simplex virus, and some cancer-inducing retroviruses.

Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids.

We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [125I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [125I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed.

Detection of nearest neighbors to specific fluorescently tagged ligands in rod outer segment and lymphocyte plasma membranes by photosensitization of 5-iodonaphthyl 1-azide.

Lima bean agglutinin-fluorescein 5-isothiocyanate conjugate (FluNCS-lima bean lectin) interacts with specific receptor molecules on membranes both from the rod outer segment (ROS) of the frog retina and from S49 mouse lymphoma cells. When [125I]-5-iodonaphthyl 1-azide (125I-INA), which freely and randomly partitions into the lipid bilayer, is added to membranes and the suspension is irradiated at 480 nm, the FluNCS-conjugated lectin photosensitizes the [125I]INA but only at discrete sites. This results in the selective labeling of specific proteins: an 88-kDa protein on ROS membranes and a 56-kDa protein on S49 plasma membranes. Labeling is dependent upon the interaction of the FluNCS-lectin with glycosylated receptor sites, since N-acetylgalactosamine, but not methyl alpha-mannoside, blocked labeling of the 56-kDa protein on S49 membranes. In contrast, a random labeling pattern of membrane proteins was observed upon irradiation at 480 nm using other fluorescein conjugates, such as FluNCS-bovine serum albumin (FluNCS-BSA) or FluNCS-soybean trypsin inhibitor (FluNCS-STI), which interact with cell membranes in a nonselective manner, or with N-(fluorescein-5-thiocarbamoyl)-n-undecyclamine (FluNCS-NHC11), which is freely miscible in the membrane lipid. Random labeling was also obtained by direct photoexcitation of [125I]INA at 314 nm, with no distinct labeling of the 88- and 56-kDa proteins in the respective membranes. These results suggest that protein ligands can be used to guide sensitizers to discrete receptor sites and lead to their selective labeling by photosensitized activation of [125I]INA [Raviv, Y., Salomon, Y., Gitler, C., & Bercovici, T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6103-6107].(ABSTRACT TRUNCATED AT 250 WORDS)

Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.

The apolar azide of 5-iodonaphthalene-1-azide (Ina) partitions into the lipid bilayer of biological membranes. Upon photolysis at 314 nm, it is rapidly converted into the reactive nitrene, which efficiently attaches covalently to lipid-embedded domains of proteins and, to a lesser extent, to membrane phospholipids. Above 370 nm, Ina absorption is negligible and photolysis at these wavelengths does not occur. However, on addition of the photosensitizing molecule 3-aminopyrene, trifluoperazine, or 8-anilinonaphthalene-1-sulfonate, followed by irradiation at 380 nm, efficient conversion of Ina to reactive species was observed, as measured by [125I]Ina-labeling of membrane proteins and inactivation of the hormonal response of adenylate cyclase. Irradiation at 480 nm in the presence of a fluorescein derivative of n-undecylamine also resulted in a pattern of [125I]Ina-labeled membrane proteins and hormone uncoupling indistinguishable from that obtained following direct photolysis at 314 nm. Photosensitization of the azide molecules is confined to the vicinity of the photosensitizer chromophore. This allowed selective labeling of chromophore-bearing proteins in solution or in membranes. Bovine serum albumin-fluorescein conjugate, in the presence of nonderivatized soluble proteins, was exclusively labeled by [125I]Ina when irradiated at 480 nm, but random labeling occurred on photolysis at 314 nm. Likewise, rhodopsin in rod outer segment membranes from frog retina was exclusively labeled by [125I]Ina upon photosensitization at 380 nm. Random labeling again occurred on direct irradiation at 314 nm. The results suggest that selective labeling in complex biological systems may be achieved by photosensitized activation of azides.

Reversible penetration of alpha-glutathione S-transferase into biological membranes revealed by photosensitized labelling in situ.

Fluorescent lipid analogue 3,3'-dioctadecyloxacarbocyanine incorporated into biological membranes was used to induce photoactivation of a hydrophobic probe 5-[125I]iodonaphthyl-1-azide (125INA) by energy transfer and to thereby confine subsequent radiolabelling of proteins to the lipid bilayer. This approach was applied in bovine chromaffin cells to discover cytosolic proteins that reversibly penetrate into membrane domains. alpha-Glutathione S-transferase (alpha-GST) was identified as the only labelled protein in bovine chromaffin-cell cytosol, indicating that it inserts reversibly into the membrane lipid bilayer. The selectivity of the labelling towards the lipid bilayer is demonstrated by showing that influenza virus haemagglutinin becomes labelled by 125INA only after the insertion of this protein into the target membrane. The molar 125INA:protein ratio was used as a quantitative criterion for evaluation of the penetration of proteins into the membrane lipid bilayer. This ratio was calculated for four integral membrane proteins and four soluble proteins that interact with biological membranes. The values for four integral membrane proteins (erythrocyte anion transporter, multidrug transporter gp-170, dopamine transporter and fusion-competent influenza virus haemagglutinin) were 1, 8, 2 and 2, respectively, whereas for soluble proteins (annexin VII, protein kinase C, BSA and influenza virus haemagglutinin) the values were 0.002, 0, 0.002 and 0.02, respectively. The molar ratio for alpha-GST was found to be 1, compatible with the values obtained for integral membrane proteins.

Selective photoinduced uncoupling of the response of adenylate cyclase to gonadotropins by 5-iodonaphthyl 1-azide.

5-Iodonaphthyl 1-azide (INA) has been previously shown to selectively label, on photolysis, only those proteins in contact with the membrane lipids. Low concentrations (less than 10 microM) of INA added to rat ovarian plasma membranes induced, on photoactivation, a selective and complete loss of the response of the adenylate cyclase to stimulation by human chorionic gonadotropin (hCG) or luteinizing hormone (LH). In contrast, this treatment affected neither hCG binding to the receptor nor the stimulation of the enzyme by NaF. That the uncoupling of the receptor from the enzyme by INA occurred within the lipid bilayer can be derived from the finding that the prior presence neither of saturating concentrations of hCG nor of the aqueous nitrene-scavenger glutathione (GSH) prevented this effect. Photolysis at higher concentrations of INA (0.1-1 mM) led to the inhibition of the adenylate cyclase stimulated by fluoride. This effect was totally prevented by glutathione. A similar behavior was obtained with a water-soluble analogue of INA, namely, 5-diazonionapthyl 1-azide (DAN). On photoactivation with 30 microM DAN, the NaF-stimulated adenylate cyclase was inhibited, but this effect was completely prevented by added GSH. At low concentrations where its effects are restricted to the lipid core, INA may represent a useful tool to define receptor coupling with the adenylate cyclase. The capacity of INA at low concentrations to uncouple the hormone receptor from the adenylate cyclase is not restricted to the LH/hCG receptor. Other hormone receptors tested behaved similarly. Therefore, the reported findings appear to represent a general phenomenon.

Detection of influenza hemagglutinin interaction with biological membranes by photosensitized activation of [125I]iodonaphthylazide.

Fusion of influenza virus with cells is triggered by a pH-dependent conformational change in the viral envelope protein, hemagglutinin, which results in exposure of the fusion peptide and its insertion into the target membrane. We have investigated the association of hemagglutinin with erythrocyte membranes by photosensitized labeling with [125I]iodonaphthylazide. This technique relies on the collisional energy transfer from a photosensitizing chromophore to [125I]iodonaphthylazide, which selectively labels proteins in the vicinity of the chromophore. Incubation of influenza virus with erythrocyte membranes containing chromophore and [125I]iodonaphthylazide results in labeling of hemagglutinin under fusogenic conditions (pH 5 and 37 degrees C). We also examined photosensitized labeling of hemagglutinin upon incubation of the X31 strain of influenza virus with labeled erythrocyte membranes in a pre-fusion state (pH 5 and 4 degrees C). There was little hemagglutinin labeling under these conditions, although incubation of bromelain-cleaved hemagglutinin, which lacks the transmembrane region, resulted in rapid labeling. Hemagglutinin was also labeled by [125I]iodonaphthylazide photosensitized by a fluorescent substrate transported through the erythrocyte band 3 sialoglycoprotein. Hemagglutinin labeling decreased after an initial rapid rise, suggesting that the fusion site is close to the sialoglycoprotein and that [125I]iodonaphthylazide photosensitized labeling may be used to assay protein movement during fusion.

Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells.

A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Relaxation dynamics of semiflexible polymers.

We study the relaxation dynamics of a semiflexible chain by introducing a time-dependent tension. The chain has one of its ends attached to a large bead, and the other end is fixed. We focus on the initial relaxation of the chain that is initially strongly stretched. Using a tension that is self-consistently determined, we obtain the evolution of the end-to-end distance with no free parameters. Our results are in good agreement with single molecule experiments on double stranded DNA.