Time to chill: effects of simulated global change on leaf ice nucleation temperatures of subarctic vegetation

We investigated the effects of long-term (7-yr) in situ CO(2) enrichment (600 μmol/mol) and increased exposure to UV-B radiation, the latter an important component of global change at high latitudes, on the ice nucleation temperatures of leaves of several evergreen and deciduous woody ericaceous shrubs in the subarctic (68° N). Three (Vaccinium uliginosum, V. vitis-idaea, and Empetrum hermaphroditum) of the four species of shrubs studied showed significantly higher ice nucleation temperatures throughout the 1999 growing season in response to CO(2) enrichment and increased exposure to UV-B radiation relative to the controls. The same species also showed a strong interactive effect when both treatments were applied together. In all cases, leaves cooled to below their ice nucleation temperatures failed to survive the damage resulting from intracellular ice formation. Our results strongly suggest that future global change on a decadal time scale (atmospheric CO(2) increases and polar stratospheric O(3) destruction) will lead to increased foliage damage of subarctic vegetation by severe late spring or early autumnal frosting events. Indeed, in support of our experimental findings, there is now some evidence that increases in atmospheric CO(2) concentration over the past three to four decades may already have acted in this manner on high-elevation arboreal plants in the Swedish Scandes. The implications for vegetation modeling in a future "greenhouse" world and palaeoclimate estimates from high-latitude plant fossils dating to the high-CO(2) environment of the Mesozoic are discussed.     

A novel family of retroviral vectors for the rapid production of complex stable cell lines

The production of stable cell lines is an important technique in cell biology, and it is often the rate-limiting step in studies involving the characterization of the function of novel genes or gene mutations. To facilitate this process, a novel family of retroviral vectors, the pE vector family, has been generated. The retroviral sequences in the pE vectors have been taken from the Moloney murine leukemia virus (MMLV) vector pMFG, which has been shown to express cDNA inserts more consistently and at higher levels than earlier generations of MMLV vectors. These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA. The pE vectors have an episomal design to allow long-term production of high-titer virus without the need for subcloning the producer line. Using a strategy of combinatorial infection followed by combinatorial drug selection, we demonstrate that the pE vectors can be used to generate stable, polyclonal cell lines expressing at least three novel cDNAs in less than 2 weeks. The use of these vectors will thus dramatically accelerate the production of complex stable cell lines.     

APAP, a sequence-pattern recognition approach identifies substance P as a potential apoptotic peptide

We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro-apoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro-apoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve pro-apoptotic peptides for chemotherapy.     

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.     

Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.     

Immunoendocrine interactions during chronic cysticercosis determine male mouse feminization: role of IL-6

Taenia crassiceps cysticercosis results in an impressive feminization in male mice during chronic infection, characterized by increased serum estradiol levels 100 times their normal values, while those of testosterone and dihydrotestosterone are decreased by 85 and 95% respectively. Concomitantly, the levels of follicle-stimulating hormone and IL-6 are increased 70 and 90 times their normal values in the infected male mice. Since a specific Th1/Th2 shift of the immune response has been previously reported during the chronic infection, and this shift may be associated with the feminization process, we proposed that this shift is induced by immunoendocrine interactions during the disease, and this gives way to a change in the initial resistance to the infection in the male mice, which become as susceptible as female mice. To confirm this hypothesis, we depleted immune system activity in two different ways: total body irradiation and neonatal thymectomy. Our results show that when immune system activity is depleted using either strategy, the male mice do not feminize, and the levels of follicle-stimulating hormone and IL-6 are inhibited. Depletion of IL-6 using IL-6(-/-) knockout mice does not produce the feminization process stated above, while restitution of the IL-6(-/-) knockout, irradiated, and thymectomized mice with murine recombinant IL-6 restores the feminization process. Expression of the IL-6 gene was found only in the testes and spleen of infected animals. Our results illustrate the importance of immunoendocrine interactions during a parasitic disease and show a possible new mechanism of parasite establishment in an initially resistant host.     

Characterization of the active sites in decay-accelerating factor

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.