Human CD4+ effector memory T cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of IL-12 to proliferate, produce IFN-gamma, and eradicate tumor cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-gamma. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45(+) leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-alpha. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4(+) effector memory T cell. We conclude that quiescent CD4(+) effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-gamma, leading to tumor cell eradication.     

Protein kinase C-theta is an early survival factor required for differentiation of effector CD8+ T cells

CD8(+) T cells are crucial for host defense against invading pathogens and malignancies. However, relatively little is known about intracellular signaling events that control the genetic program of their activation and differentiation. Using CD8(+) T cells from TCR-transgenic mice crossed to protein kinase C-theta (PKCtheta)-deficient mice, we report that PKCtheta is not required for Ag-induced CD8(+) T cell proliferation, but is important for T cell survival and differentiation into functional, cytokine-producing CTLs. Ag-stimulated PKCtheta(-/-) T cells underwent accelerated apoptosis associated with deregulated expression of Bcl-2 family proteins and displayed reduced activation of ERKs and JNKs. Some defects in the function of PKCtheta(-/-) T cells (poor survival and reduced Bcl-2 and Bcl-x(L) expression, CTL activity, and IFN-gamma expression) were partially or fully restored by coculture with wild-type T cells or by addition of exogenous IL-2, whereas others (increased Bim(EL) expression and TNF-alpha production) were not. These findings indicate that PKCtheta, although not essential for initial Ag-induced proliferation, nevertheless plays an important role in promoting and extending T cell survival, thereby enabling the complete genetic program of effector CD8(+) differentiation. The requirement for PKCtheta in different types of T cell-dependent responses may, therefore, depend on the overall strength of signaling by the TCR and costimulatory receptors and may reflect, in addition to its previously established role in activation, an important, hitherto unappreciated, role in T cell survival.     

STRUCTURALLY PRESERVED FOSSIL PLANTS FROM ANTARCTICA: II. A PERMIAN MOSS FROM THE TRANSANTARCTIC MOUNTAINS

An anatomically preserved moss from the late Permian of Antarctica is described. The fossils include delicate, unistratose leaves and axes with numerous rhizoids preserved in a silicified peat. Leaves exhibit a multi-layered midrib of thick-walled cells, and a lamina of rhomboidal cells that become more elongate toward the margin. The axes consist entirely of parenchyma with a central column of slightly smaller diam cells. Rhizoids are attached to the outer layer of the cortex and surround all the axes. These Permian fossils represent the oldest anatomically preserved bryophytes yet described from Gondwanaland and compare most closely with modern members of the Bryidae.     

Conserved cysteine 126 in triosephosphate isomerase is required not for enzymatic activity but for proper folding and stability

In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in the folding of wtTIM, as indicated by spectroscopic studies. C126S and C126A have K(M) and k(cat) values that are concomitantly reduced by only 4-fold and 1.5-fold, respectively, compared to those of wtTIM; in either case, however, the catalytic efficiency (k(cat)/K(M)) of the enzyme is barely affected. The affinity of mutated TIMs for the competitive inhibitor 2-phosphoglycolate augmented also slightly. In contrast, greater susceptibility to thermal denaturation resulted from mutation of Cys126, especially when it was changed to Ser. By using values of the rate constants for unfolding and refolding, we estimated that, at 25 degrees C, C126A and C126S are less stable than wtTIM by about 5.0 and 9.0 kcal mol(-)(1), respectively. Moreover, either of these mutations slows down the folding rate by a factor of 10 and decreases the recovery of the active enzyme after thermal unfolding. Thus, Cys126 is required for proper stability and efficient folding of TIM rather than for enzymatic catalysis.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.     

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.     

Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae

Aging is believed to be a nonadaptive process that escapes the force of natural selection. Here, we challenge this dogma by showing that yeast laboratory strains and strains isolated from grapes undergo an age- and pH-dependent death with features of mammalian programmed cell death (apoptosis). After 90-99% of the population dies, a small mutant subpopulation uses the nutrients released by dead cells to grow. This adaptive regrowth is inversely correlated with protection against superoxide toxicity and life span and is associated with elevated age-dependent release of nutrients and increased mutation frequency. Computational simulations confirm that premature aging together with a relatively high mutation frequency can result in a major advantage in adaptation to changing environments. These results suggest that under conditions that model natural environments, yeast organisms undergo an altruistic and premature aging and death program, mediated in part by superoxide. The role of similar pathways in the regulation of longevity in organisms ranging from yeast to mice raises the possibility that mammals may also undergo programmed aging.     

Cataphylls of the Middle Triassic cycad Antarcticycas schopfii and new insights into cycad evolution

Cataphylls associated with the Middle Triassic stem genus Antarcticycas are described, and their impact on understanding cycad evolution is discussed. The cataphylls of Antarcticycas are triangular in outline and flattened adaxially with lateral flanges. The outer surfaces are covered with a ramentum of filamentous hairs, the epidermis is a single cell layer thick, and the ground tissue is parenchymatous with mucilage canals and sclereids. Vascular bundles form a distinct inverted omega-shaped pattern characteristic of the Cycadales observed in petioles of extant species. The structures in Antarcticycas are interpreted as cataphylls based on overall morphology, presence of straight vascular strands in the cortex of the associated stem, and lack of fascicular cambia in the vascular bundles. Because much of the overall diversity of Cycadales is represented by fossils, integrating fossil taxa into explicit phylogenetic hypotheses is important for understanding cycad evolution. Therefore, character and minimum age mapping were performed on a phylogeny of extant and fossil taxa including Antarcticycas. The results suggest that major extant lineages of Cycadales had diverged by the Permian to Triassic and that certain synapomorphies for Cycadales had evolved by the Permian. Evidence of insect feeding on Antarcticycas suggests that associations between cycads and insects are ancient.     

Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. eIF5A-1 mRNA (1.3 kb) and protein (18 kDa) are constitutively expressed in human cells. In contrast, expression of eIF5A-2 mRNA (0.7-5.6 kb) and eIF5A-2 protein (20 kDa) varies widely. Whereas eIF5A-2 mRNA was demonstrable in most cells, eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high overexpression of eIF5A-2 mRNA. Multiple forms of eIF5A-2 mRNA (5.6, 3.8, 1.6 and 0.7 kb) were identified as the products of one gene with various lengths of 3'-UTR, resulting from the use of different polyadenylation (AAUAAA) signals. The eIF5A-1 and eIF5A-2 precursor proteins were modified comparably in UACC-1598 cells and both were similarly stable. When eIF5A-1 and eIF5A-2 coding sequences were expressed from mammalian vectors in 293T cells, eIF5A-2 precursor was synthesized at a level comparable to that of eIF5A-1 precursor, indicating that the elements causing inefficient translation of eIF5A-2 mRNA reside outside of the open reading frame. On sucrose gradient separation of cytoplasmic RNA, only a small portion of total eIF5A-2 mRNA was associated with the polysomal fraction, compared with a much larger portion of eIF5A-1 mRNA in the polysomes. These findings suggest that the failure to detect eIF5A-2 protein even in eIF5A-2 mRNA positive cells is, at least in part, due to inefficient translation.