CD36 or alphavbeta3 and alphavbeta5 integrins are not essential for MHC class I cross-presentation of cell-associated antigen by CD8 alpha+ murine dendritic cells

Cross-presentation of cell-associated Ag is thought to involve receptor-mediated uptake of apoptotic cells by dendritic cells (DC), and studies with human DC strongly implicate the endocytic receptor CD36 and the integrins alpha(v)beta(3) and/or alpha(v)beta(5) in this process. In the mouse, cross-presentation was recently shown to be a function of CD8alpha(+) DC. Here we report that CD36 is expressed on CD8alpha(+), but not on CD8alpha(-), DC. To address the role of CD36 in cross-presentation we compared CD36(-/-) and CD36(+/+) H-2(b) DC for their ability to stimulate naive OT-1 T cells specific for OVA plus H-2K(b) in the presence of OVA-loaded MHC-mismatched splenocytes as a source of cell-associated Ag for cross-presentation. Surprisingly, no difference was seen between CD36(-/-) and CD36(+/+) CD8alpha(+) DC in their ability to cross-present cell-associated OVA or to capture OVA-bearing cells. Furthermore, the proliferation of CFSE-labeled OT-1 cells in response to OVA cross-presentation in vivo was normal in CD36(-/-) bone marrow chimeras, also arguing against a necessary role for CD36 in cross-presentation by DC or other APC. DC doubly deficient for beta(3) and beta(5) integrins were similarly unimpaired in their ability to cross-present OVA-bearing cells in vitro. These data demonstrate that in the mouse, receptors other than CD36 or beta(3) and beta(5) integrins can support the specialized cross-presenting function of CD8alpha(+) DC.     

Tumor-induced L-selectinhigh suppressor T cells mediate potent effector T cell blockade and cause failure of otherwise curative adoptive immunotherapy

Tumor-specific effector T cells (T(E)) are naturally sensitized within the L-selectin(low) (CD62L(low)) fraction of tumor-draining lymph nodes (TDLN). Whether isolated from day 9 (D9) or day 12 (D12) TDLN, 5 million L-selectin(low) T(E) could be culture activated and adoptively transferred to achieve complete rejection of established intradermal, pulmonary, and brain tumors. Surprisingly, although 25 million unfractionated T cells from D9 TDLN were equally effective, even 100 million unfractionated T cells from D12 TDLN seldom prevented lethal intradermal tumor progression, despite a pronounced therapeutic excess of T(E). This highly reproducible treatment failure was due to cotransfer of tumor-induced, L-selectin(high) suppressor T cells (T(S)) which were also present in D12 TDLN. In contrast, D9 TDLN and normal spleens lacked L-selectin(high) T(S). Only those L-selectin(high) D12 TDLN T cells that down-regulated L-selectin during culture activation were suppressive in vivo and in vitro, and, like L-selectin(low) T(E), trafficked promptly into tumors following i.v. administration. This is the first demonstration that adoptive immunotherapy can fail as a direct result of passenger T(S) that share certain phenotypic and trafficking features of T(E), even when otherwise curative doses of T(E) have been administered. Furthermore, in contrast to recently described CD4(+)CD25(+) T(S) and plasmacytoid dendritic cell-activated T(S), tumor-induced L-selectin(high) T(S) prevent tumor rejection via blockade of sensitized, activated T(E) rather than via afferent blockade.     

Partial characterization of a tapeworm-secreted signal factor inducing sustained spike potentials in the smooth muscle of the rat small intestine

The rat tapeworm Hymenolepis diminuta alters the myoelectric activity of the small intestine. To determine if secreted factors from the tapeworm are responsible for these alterations of intestinal smooth muscle activity, tapeworm-conditioned medium (TCM) obtained from in vitro culture was infused via an indwelling cannula into the duodenum of an uninfected rat. Myoelectric recordings were analyzed for sustained spike potentials (SSP) and repetitive bursts of action potentials (RBAP), the previously characterized tapeworm modifications of the normal interdigestive myoelectric pattern. Results indicated that TCM initiated SSP, but not RBAP in the intestine of the uninfected rat. The SSP-inducing signal factor activity, present in TCM, was retained after boiling, prolonged freezing, proteinase treatment, and passage through a 10-kDa exclusion filter. The signal factor was soluble in the aqueous phase on lipid extraction. It was concluded that the SSP-inducing signal factor is a nonproteinaceous, heat-resistant, low-molecular weight, water soluble molecule.     

Snow tussocks, chaos, and the evolution of mast seeding

One hitherto intractable problem in studying mast seeding (synchronous intermittent heavy flowering by a population of perennial plants) is determining the relative roles of weather, plant reserves, and evolutionary selective pressures such as predator satiation. We parameterize a mechanistic resource-based model for mast seeding in Chionochloa pallens (Poaceae) using a long-term individually structured data set. Each plant's energy reserves were reconstructed using annual inputs (growing degree days), outputs (flowering), and a novel regression technique. This allowed the estimation of the parameters that control internal plant resource dynamics, and thereby allowed different models for masting to be tested against each other. Models based only on plant size, season degree days, and/or climatic cues (warm January temperatures) fail to reproduce the pattern of autocovariation in individual flowering and the high levels of flowering synchrony seen in the field. This shows that resource-matching or simple cue-based models cannot account for this example of mast seeding. In contrast, the resource-based model pulsed by a simple climate cue accurately describes both individual-level and population-level aspects of the data. The fitted resource-based model, in the absence of environmental forcing, has chaotic (but often statistically periodic) dynamics. Environmental forcing synchronizes individual reproduction, and the models predict highly variable seed production in close agreement with the data. An evolutionary model shows that the chaotic internal resource dynamics, as predicted by the fitted model, is selectively advantageous provided that adult mortality is low and seeds survive for more than 1 yr, both of which are true for C. pallens. Highly variable masting and chaotic dynamics appear to be advantageous in this case because they reduce seed losses to specialist seed predators, while balancing the costs of missed reproductive events.     

Flavonolignans from Hyparrhenia hirta

Leaves of Hyparrhenia hirta yielded the rare diastereoisomeric flavonolignans tricin 4'-O-(erythro-beta-guaiacylglyceryl) ether and tricin 4'-O-(threo-beta-guaiacylglyceryl) ether together with their 7-O-glucosides, which are the first flavonolignan glycosides to be isolated as natural products. A complete set of (1)H and (13)C NMR resonance assignments obtained for both flavonolignan aglycones indicates the need for revision of data published previously for these compounds and for a reassessment of their original stereochemical designation.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

Arabinoxylan biosynthesis in wheat. Characterization of arabinosyltransferase activity in Golgi membranes

Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[(14)C]-beta-L-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[(14)C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[(14)C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [(14)C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn(2+) being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [(14)C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.     

Leucine-derived cyano glucosides in barley

Barley (Hordeum vulgare) seedlings contain five cyano glucosides derived from the amino acid L-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyano glucoside content, but the relative content of the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting L-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the alpha-, as well as beta- and gamma-, carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert L-[(14)C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal cell layers after biosynthesis. A beta-glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.