Chilling Delays Circadian Pattern of Sucrose Phosphate Synthase and Nitrate Reductase Activity in Tomato1

Overnight low-temperature exposure inhibits photosynthesis in chilling-sensitive species such as tomato (Lycopersicon esculentum) and cucumber by as much as 60%. In an earlier study we showed that one intriguing effect of low temperature on chilling-sensitive plants is to stall the endogenous rhythm controlling transcription of certain nuclear-encoded genes, causing the synthesis of the corresponding transcripts and proteins to be mistimed when the plant is rewarmed. Here we show that the circadian rhythm controlling the activity of sucrose phosphate synthase (SPS) and nitrate reductase (NR), key control points of carbon and nitrogen metabolism in plant cells, is delayed in tomato by chilling treatments. Using specific protein kinase and phosphatase inhibitors, we further demonstrate that the chilling-induced delay in the circadian control of SPS and NR activity is associated with the activity of critical protein phosphatases. The sensitivity of the pattern of SPS activity to specific inhibitors of transcription and translation indicates that there is a chilling-induced delay in SPS phosphorylation status that is caused by an effect of low temperature on the expression of a gene coding for a phosphoprotein phosphatase, perhaps the SPS phosphatase. In contrast, the chilling-induced delay in NR activity does not appear to arise from effects on NR phosphorylation status, but rather from direct effects on NR expression. It is likely that the mistiming in the regulation of SPS and NR, and perhaps other key metabolic enzymes under circadian regulation, underlies the chilling sensitivity of photosynthesis in these plant species.

Nitric oxide plays a key role in adaptive control of locomotion in cat

Diverse roles in cellular functions have been ascribed to nitric oxide (NO), and its involvement in induction of long-term depression in cerebellar Purkinje cells has been demonstrated. Manipulations of NO concentration or its synthesis in cerebellar tissues therefore provide a means for investigating roles of NO in cerebellar functions at both cellular and behavioral levels. We tested adaptive control of locomotion to perturbation in cats, and found that this form of motor learning was abolished by application of either an inhibitor of NO synthase or a scavenger of NO to the cerebellar cortical locomotion area. This finding supports the view that NO in the cerebellum plays a key role in motor learning.

The “Spot 14” gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: Implications for control of tumor metabolism

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. “Spot 14” (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (THRSP) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.

Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria

Speciation involves the establishment of genetic barriers between closely related organisms. The extent of genetic recombination is a key determinant and a measure of genetic isolation. The results reported here reveal that genetic barriers can be established, eliminated, or modified by manipulating two systems which control genetic recombination, SOS and mismatch repair. The extent of genetic isolation between enterobacteria is a simple mathematical function of DNA sequence divergence. The function does not depend on hybrid DNA stability, but rather on the number of blocks of sequences identical in the two mating partners and sufficiently large to allow the initiation of recombination. Further, there is no obvious discontinuity in the function that could be used to define a level of divergence for distinguishing species.

Genetic control of abscisic acid biosynthesis in maize

Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.

Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes

Analysis of perforin-deficient mice has identified the cytolytic pathway and perforin as the preeminent effector molecule in T cell-mediated control of virus infections. In this paper, we show that mice lacking both granzyme A (gzmA) and granzyme B (gzmB), which are, beside perforin, key constituents of cytolytic vesicles, are as incapable as are perforin-deficient mice of controlling primary infections by the natural mouse pathogen ectromelia, a poxvirus. Death of gzmA×gzmB double knockout mice occurred in a dose-dependent manner, despite the expression of functionally active perforin and the absence of an intrinsic defect to generate splenic cytolytic T cells. These results establish that both gzmA and gzmB are indispensable effector molecules acting in concert with perforin in granule exocytosis-mediated host defense against natural viral pathogens.

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

RhoA GTPase and Serum Response Factor Control Selectively the Expression of MyoD without Affecting Myf5 in Mouse Myoblasts

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

Toll-related receptors and the control of antimicrobial peptide expression in Drosophila

Insects defend themselves against infectious microorganisms by synthesizing potent antimicrobial peptides. Drosophila has appeared in recent years as a favorable model to study this innate host defense. A genetic analysis of the regulation of the antifungal peptide drosomycin has demonstrated a key role for the transmembrane receptor Toll, which prompted the search for mammalian homologs. Two of these, Toll-like receptor (TLR)2 and TLR4, recently were shown to play a critical role in innate immunity against bacteria. Here we describe six additional Toll-related genes (Toll-3 to Toll-8) in Drosophila in addition to 18-wheeler. Two of these genes, Toll-3 and Toll-4, are expressed at a low level. Toll-6, -7, and -8, on the other hand, are expressed at high levels during embryogenesis and molting, suggesting that, like Toll and 18w, they perform developmental functions. Finally, Toll-5 is expressed only in larvae and adults. By using chimeric constructs, we have tested the capacity of the signaling Toll/IL-1R homology domains of these receptors to activate antimicrobial peptide promoters and found that only Toll and Toll-5 can activate the drosomycin promoter in transfected cells, thus demonstrating specificity at the level of the Toll/IL-1R homology domain. In contrast, none of these constructs activated antibacterial peptide promoters, suggesting that Toll-related receptors are not involved in the regulation of antibacterial peptide expression. This result was independently confirmed by the demonstration that a dominant-negative version of the kinase Pelle can block induction of drosomycin by the cytokine Spaetzle, but does not affect induction of the antibacterial peptide attacin by lipopolysaccharide.

Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occured at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin β1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin β1 and fibronectin in a MEK-ERK–dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Independence of metastatic ability and extravasation: metastatic ras-transformed and control fibroblasts extravasate equally well.

Escape of cancer cells from the circulation (extravasation) is thought to be a major rate-limiting step in metastasis, with few cells being able to extravasate. Furthermore, highly metastatic cells are believed to extravasate more readily than poorly metastatic cells. We assessed in vivo the extravasation ability of highly metastatic ras-transformed NIH 3T3 cells (PAP2) versus control nontumorigenic nontransformed NIH 3T3 cells and primary mouse embryo fibroblasts. Fluorescently labeled cells were injected intravenously into chicken embryo chorioallantoic membrane and analyzed by intravital videomicroscopy. The chorioallantoic membrane is an appropriate model for studying extravasation, since, at the embryonic stage used, the microvasculature exhibits a continuous basement membrane and adult permeability properties. The kinetics of extravasation were assessed by determining whether individual cells (n = 1481) were intravascular, extravascular, or in the process of extravasation, at 3, 6, and 24 h after injection. Contrary to expectations, our results showed that all three cell types extravasated with the same kinetics. By 24 h after injection > 89% of observed cells had completed extravasation from the capillary plexus. After extravasation, individual fibroblasts of all cell types demonstrated preferential migration within the mesenchymal layer toward arterioles, not to venules or lymphatics. Thus in this model and for these cells, extravasation is independent of metastatic ability. This suggests that the ability to extravasate in vivo is not necessarily predictive of subsequent metastasis formation, and that postextravasation events may be key determinants in metastasis.

Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule.

The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.