Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-γ, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a “forbidden” cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line and vice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cells in vivo.

Commitment to natural killer cells requires the helix–loop–helix inhibitor Id2

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2−/− mice that exhibit defective NK cell development. In the Id2−/− FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2−/− FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2−/− fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2−/− and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings1 : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [3H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m−2 s−1) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.

Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ0 and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4+CD8– cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-loxP-mediated gene inversion is used to change the specificity of the BCR on mature IgM+ IgD+ B cells in vivo to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive bcl-2 transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Plo1 Kinase Recruitment to the Spindle Pole Body and Its Role in Cell Division in Schizosaccharomyces pombe

Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.

Restrictions to Carbon Dioxide Conductance and Photosynthesis in Spinach Leaves Recovering from Salt Stress

Salt accumulation in spinach (Spinacia oleracea L.) leaves first inhibits photosynthesis by decreasing stomatal and mesophyll conductances to CO2 diffusion and then impairs ribulose-1,5-bisphosphate carboxylase/oxygenase (S. Delfine, A. Alvino, M. Zacchini, F. Loreto [1998] Aust J Plant Physiol 25: 395–402). We measured gas exchange and fluorescence in spinach recovering from salt accumulation. When a 21-d salt accumulation was reversed by 2 weeks of salt-free irrigation (rewatering), stomatal and mesophyll conductances and photosynthesis partially recovered. For the first time, to our knowledge, it is shown that a reduction of mesophyll conductance can be reversed and that this may influence photosynthesis. Photosynthesis and conductances did not recover when salt drainage was restricted and Na content in the leaves was greater than 3% of the dry matter. Incomplete recovery of photosynthesis in rewatered and control leaves may be attributed to an age-related reduction of conductances. Biochemical properties were not affected by the 21-d salt accumulation. However, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and content were reduced by a 36- to 50-d salt accumulation. Photochemical efficiency was reduced only in 50-d salt-stressed leaves because of a decrease in the fraction of open photosystem II centers. A reduction in chlorophyll content and an increase in the chlorophyll a/b ratio were observed in 43- and 50-d salt-stressed leaves. Low chlorophyll affects light absorptance but is unlikely to change light partitioning between photosystems.

Characteristics of the deep ocean carbon system during the past 150,000 years: ΣCO2 distributions, deep water flow patterns, and abrupt climate change

Studies of carbon isotopes and cadmium in bottom-dwelling foraminifera from ocean sediment cores have advanced our knowledge of ocean chemical distributions during the late Pleistocene. Last Glacial Maximum data are consistent with a persistent high-ΣCO2 state for eastern Pacific deep water. Both tracers indicate that the mid-depth North and tropical Atlantic Ocean almost always has lower ΣCO2 levels than those in the Pacific. Upper waters of the Last Glacial Maximum Atlantic are more ΣCO2-depleted and deep waters are ΣCO2-enriched compared with the waters of the present. In the northern Indian Ocean, δ13C and Cd data are consistent with upper water ΣCO2 depletion relative to the present. There is no evident proximate source of this ΣCO2-depleted water, so I suggest that ΣCO2-depleted North Atlantic intermediate/deep water turns northward around the southern tip of Africa and moves toward the equator as a western boundary current. At long periods (>15,000 years), Milankovitch cycle variability is evident in paleochemical time series. But rapid millennial-scale variability can be seen in cores from high accumulation rate series. Atlantic deep water chemical properties are seen to change in as little as a few hundred years or less. An extraordinary new 52.7-m-long core from the Bermuda Rise contains a faithful record of climate variability with century-scale resolution. Sediment composition can be linked in detail with the isotope stage 3 interstadials recorded in Greenland ice cores. This new record shows at least 12 major climate fluctuations within marine isotope stage 5 (about 70,000–130,000 years before the present).

Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.

Rational engineering of enzymes involves introducing key amino acids guided by a knowledge of protein structure to effect a desirable change in function. To date, all successful attempts to change specificity have been limited to substituting individual amino acids within a protein fold. However, the infant field of protein engineering will only reach maturity when changes in function can be generated by rationally engineering secondary structures. Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to systematically invert the coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant, which is twice as active as wild type, contains four amino acid substitutions and an alpha-helix and loop that replaces the original beta-turn. These results demonstrate that rational engineering of secondary structures to produce enzymes with novel properties is feasible.

Study of the role of retinoblastoma protein in terminal differentiation of murine erythroleukemia cells.

Hexamethylenebisacetamide-induced terminal differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells can be inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. The inhibition is shown to be correlated with prevention of dephosphorylation of retinoblastoma protein (pRB) in cells and bypass of G1 prolongation in the cell cycle. These results suggest that pRB-mediated G1 prolongation is necessary for MEL cells to commit to terminal differentiation. However, further experiments demonstrate that the simple cell cycle exit is not sufficient for commitment to terminal differentiation. Induction of dephosphorylation of pRB and subsequent G1 prolongation by forskolin does not lead MEL cells to differentiate. Additional pRB has been expressed in MEL cells by transfection with a neo-resistant plasmid containing RB cDNA under the control of a cytomegalovirus promoter. Exogenously expressed pRB is hyperphosphorylated in logarithmically growing MEL cells without any noticeable change in growth rate between the transfected cell line and the parental cell line. This result suggests that pRB in MEL cells is regulated by protein kinases and protein phosphatases and not by transcription.