[Molecular biological study on enzymes involved in cyclooxygenase pathway: structure of their genes and proteins].
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Amino Acid Sequence↗
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[Structural biology of the enzymes in PCB degradation pathway].
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Biodegradation, Environmental↗
[Molecular mechanism and biological function of the cytokinin signal transduction pathway].
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Arabidopsis Proteins↗
[Biological characteristics and pathological significance of alternative pathway].
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Animals↗
Gastrin, CCK, signaling, and cancer.
Gastrin, produced by G cells in the gastric antrum, has been identified as the circulating hormone responsible for stimulation of acid secretion from the parietal cell. Gastrin also acts as a potent cell-growth factor that has been implicated in a variety of normal and abnormal biological processes including maintenance of the gastric mucosa, proliferation of enterochromaffin-like cells, and neoplastic transformation. Here, we review the models used to study the effects of gastrin on cell proliferation in vivo and in vitro with respect to mechanisms by which this hormone might influence normal and cancerous cell growth. Specifically, human and animal models of hypergastrinemia and hypogastrinemia have been described in vivo, and several cells that express cholecystokinin (CCK)B/gastrin receptors have been used for analysis of intracellular signaling pathways initiated by biologically active amidated gastrins. The binding of gastrin or CCK to their common cognate receptor triggers the activation of multiple signal transduction pathways that relay the mitogenic signal to the nucleus and promote cell proliferation. A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades, including mitogen-activated protein kinase, is an important early response to these signaling peptides. Gastrin and CCK also induce rapid Rho-dependent actin remodeling and coordinate tyrosine phosphorylation of cellular proteins including the non-receptor tyrosine kinases p125fak and Src and the adaptor proteins p130cas and paxillin. This article reviews recent advances in defining the role of gastrin and CCK in the control of cell proliferation in normal and cancer cells and in dissecting the signal transduction pathways that mediate the proliferative responses induced by these hormonal GI peptides in a variety of normal and cancer cell model systems.
Animals↗
Calcium-independent haemolysis via the lectin pathway of complement activation in the guinea-pig and other species*.
We previously reported that complement-dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan-binding lectin (MBL) via the lectin pathway in man occurs in Mg-EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E-M, but once the E-M-MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E-M-MBL in guinea-pig, rat, dog and pig sera, and was further investigated in the guinea-pig, in which titres were much higher ( approximately 14-fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4- or C2-deficient guinea-pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea-pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G-75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea-pig serum to mediate haemolysis via the lectin pathway. Further, the C3-convertase formed via the lectin pathway (E-M-MBL-C4,2) lysed in C2-deficient guinea-pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea-pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea-pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology.
Animals↗
Blocking the PI3K/PKB pathway in tumor cells.
A substantial number of experimental and epidemiological studies support an important role for the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway in the biology of human cancers. Components of this signaling cascade have been found to be deregulated in a wide range of solid tumors and hematologic malignancies, and extensive anti-cancer therapeutic programs are now devoted to the identification of agents that specifically block this molecular pathway. This article focuses on the current knowledge of the alterations of the PI3K/PKB pathway in cancer cells and ongoing drug discovery efforts to therapeutically target it. Particular emphasis is placed on medicinal chemistry activities to identify and develop compounds able to modulate the kinase activity of its main molecular components.
Cell Line, Tumor↗
PTHrP: novel roles in skeletal biology.
Parathyroid hormone-related peptide (PTHrP) was discovered as the main mediator of humoral hypercalcemia associated with malignancy but is now known to be expressed by a variety of normal fetal and adult tissues. The amino-terminal region of PTHrP reveals limited but significant homology with parathyroid hormone (PTH), resulting in the interaction of either peptide with a common seven-transmembrane spanning G-protein linked receptor termed the PTH/PTHrP receptor. Targeted inactivation of PTHrP and its receptor in mice has established a critical role for this signaling pathway in chondrocyte biology and endochondral bone formation. Animals homozygous for the targeted alleles demonstrate marked skeletal deformities arising from impaired chondrocyte proliferation, premature differentiation and accelerated apoptosis. The complex processes resulting in normal endochondral bone development involve additional factors such as the hedgehog signaling pathway with which PTHrP interacts. PTHrP, like PTH, also binds to receptors on cells of the osteoblast lineage resulting in enhanced bone formation and also, indirectly, augmented osteoclastic bone resorption. The marked premature osteoporosis observed in mice heterozygous for the disrupted Pthrp allele also points to a crucial role for the protein in the maintenance of the adult skeleton. Further studies into this process are likely to reveal new facets of the pathogenesis underlying a variety of metabolic bone diseases and potentially point to new directions for therapeutic interventions.
Amino Acid Sequence↗
Pathway and ontology analysis: emerging approaches connecting transcriptome data and clinical endpoints.
The increasing use of gene expression profiling offers great promise in clinical research into disease biology and its treatment. Along with the ability to measure changing expression levels in thousands of genes at once, comes the challenge of analyzing and interpreting the vast sets of data generated. Analysis tools are evolving rapidly to meet such challenges. The next step is to interpret observed changes in terms of the biological properties or relationships underlying them. One powerful approach is to make associations between the genes that are under investigation and well-known biochemical or signaling pathways, and further to assess the significance of such associations. Similarly, genes can be mapped to standardized biological categories via an ontology resource. We discuss these approaches and several web-based resources and tools designed to facilitate such analyses. This information can be used to facilitate understanding and to help design more focused experiments for validating the relevance and importance of these biological pathways and processes in human disease and therapeutics.
Animals↗
Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects.
The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.
Androstadienes↗
Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b.
Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.
Complement Activation↗
Rac1/Cdc42 and RhoA GTPases antagonistically regulate chondrocyte proliferation, hypertrophy, and apoptosis.
UNLABELLED: The intracellular signaling pathways controlling chondrocyte physiology are largely unknown. Here we show that the small GTPases, Rac1 and Cdc42, accelerate the rate of chondrocyte differentiation and apoptosis, thereby antagonizing the activity of RhoA. These results identify Rac1 and Cdc42 pathways as novel regulators of cartilage development. INTRODUCTION: Proliferation, hypertrophic differentiation, and ultimate apoptosis of chondrocytes regulate endochondral bone growth and development, but the intracellular signaling pathways controlling chondrocyte biology are incompletely understood. In this study, we investigated the role of the small GTPases Rac1 and Cdc42 in chondrocytes. MATERIALS AND METHODS: Rac1 and Cdc42 expression during chondrogenic differentiation was assessed by RT-PCR and Western blotting. Effects of Rac1 and Cdc42 on parameters of chondrocyte biology were studied using transient transfections into primary mouse chondrocytes and stable transfections of the chondrogenic cell line ATDC5. Luciferase assays, RT-PCR, cell proliferation, alkaline phosphatases assays, staining procedures, TUNEL assays, and caspase activity assays were performed to study the chondrocyte response to overexpression of Rac1 and Cdc42 proteins. Activation of the p38 pathway was analyzed using Western blotting with phospho-specific antibodies, and mitogen-activated protein (MAP) kinase pathways were inhibited using pharmacological approaches. RESULTS AND CONCLUSIONS: Rac1 and Cdc42 activities are required for maximal activity of the collagen X promoter, a hypertrophic marker, in primary chondrocytes, suggesting essential roles of these GTPases in chondrocyte hypertrophy. Overexpression of Rac1 or Cdc42 in chondrogenic ATDC5 cells results in reductions in cell numbers and marked acceleration of hypertrophic differentiation, thus opposing the effects of the related GTPase RhoA. Rac1 and Cdc42 also induce accelerated chondrocyte apoptosis, as shown by TUNEL and caspase activity assays and changes in cell morphology and actin organization. Rac1 and Cdc42 overexpression results in activation of the p38 MAP kinase pathway in ATDC5 cells, and pharmacological inhibition of p38 signaling blocks the effects of Rac1 and Cdc42 overexpression on hypertrophy and apoptosis. Our results therefore suggest that Rac1 and Cdc42 signaling accelerates progression through the chondrocyte life cycle in a p38-dependent fashion and antagonizes RhoA signaling pathways in chondrocyte proliferation, hypertrophy, and apoptosis.
Actins↗
Role of cascades in converting oscillatory signals into stationary step-like responses.
In biological signal transduction pathways intermediates are often oscillatory and need to be converted into smooth output signals at the end. We show by mathematical modelling that protein kinase cascades enable converting oscillatory signals into sharp stationary step-like outputs. The importance of this result is demonstrated for the switch-like protein activation by calcium oscillations, which is of biological importance for regulating different cellular processes. In addition, we found that protein kinase cascades cause memory effects in the protein activation, which might be of a physiological advantage since a smaller amount of calcium transported in the cell is required for an effective activation of cellular processes.
Calcium Signaling↗
Biological response to phorbol ester determined by alternative G1 pathways.
A plethora of extracellular signals is known to induce a common set of immediate early genes. The immediate early response, therefore, must not be sufficient to determine the biological outcome. An example of this is found with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). A potent activator of protein kinase C, TPA can either stimulate or inhibit cell proliferation, depending on the cell type. This cell context-dependent response to TPA is observed with two subclones of NIH 3T3 cells, the P- and the N-3T3 clones. TPA is a mitogen for the P-3T3 but an antimitogen for the N-3T3 cells. The immediate early pathway is activated by TPA in both cell types, indicating that this pathway alone does not activate DNA synthesis. The delayed induction of cyclin D1 expression by TPA is observed only in the P-3T3 cells, correlating with mitogenesis. N-Acetylcysteine does not affect the immediate early pathway but can inhibit the TPA-mediated induction of cyclin D1 and DNA synthesis. In the N-3T3 cells, TPA causes an inhibition of the cyclin E-associated kinase at the G1/S transition, correlating with growth inhibition. The growth-inhibitory activity of TPA is not affected by N-acetylcysteine. Thus, the two TPA-regulated G1 pathways can be distinguished by their sensitivity to N-acetylcysteine. These results demonstrate that TPA can activate alternative G1 pathways. Moreover, the selection of the alternative G1 pathways is determined by the cell context, which, in turn, dictates the biological response to TPA.
3T3 Cells↗
Glial cell line-derived neurotrophic factor promotes survival and induces differentiation through the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathway respectively in PC12 cells.
PC12-GFRalpha1 cells, a clonal cell line engineered to express glial cell line-derived neurotrophic factor receptor alpha1 were constructed. Given glial cell line-derived neurotrophic factor could induce the differentiation and promote the survival of PC12-GFRalpha1 cells at low concentrations, the cells provide an unlimited source of monoclonal cells for studies on the signal transduction pathway of glial cell line-derived neurotrophic factor. To characterize the involvement of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in the biological effect of glial cell line-derived neurotrophic factor, we used the mitogen-activated protein kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor LY294002. PD98059 blocked glial cell line-derived neurotrophic factor-induced PC12-GFRalpha1 cells neurite formation in a dose-dependent manner, without significantly altering cell viability. LY294002 reversed the survival-promoting effect of glial cell line-derived neurotrophic factor on the PC12-GFRalpha1 cells in serum-deprived medium. The present study demonstrates that phosphatidylinositol 3-kinase pathway seems to mediate the survival-promoting effect of glial cell line-derived neurotrophic factor on PC12-GFRalpha1 cells, while the activation of mitogen-activated protein kinase pathway could be an important step in mediating PC12-GFRalpha1 cells differentiation induced by glial cell line-derived neurotrophic factor. Therefore, it is inferred that similar intracellular signaling components are used by distinct growth factors toward a common biological effect.
Animals↗
An integrative genomic approach to uncover molecular mechanisms of prokaryotic traits.
With mounting availability of genomic and phenotypic databases, data integration and mining become increasingly challenging. While efforts have been put forward to analyze prokaryotic phenotypes, current computational technologies either lack high throughput capacity for genomic scale analysis, or are limited in their capability to integrate and mine data across different scales of biology. Consequently, simultaneous analysis of associations among genomes, phenotypes, and gene functions is prohibited. Here, we developed a high throughput computational approach, and demonstrated for the first time the feasibility of integrating large quantities of prokaryotic phenotypes along with genomic datasets for mining across multiple scales of biology (protein domains, pathways, molecular functions, and cellular processes). Applying this method over 59 fully sequenced prokaryotic species, we identified genetic basis and molecular mechanisms underlying the phenotypes in bacteria. We identified 3,711 significant correlations between 1,499 distinct Pfam and 63 phenotypes, with 2,650 correlations and 1,061 anti-correlations. Manual evaluation of a random sample of these significant correlations showed a minimal precision of 30% (95% confidence interval: 20%-42%; n = 50). We stratified the most significant 478 predictions and subjected 100 to manual evaluation, of which 60 were corroborated in the literature. We furthermore unveiled 10 significant correlations between phenotypes and KEGG pathways, eight of which were corroborated in the evaluation, and 309 significant correlations between phenotypes and 166 GO concepts evaluated using a random sample (minimal precision = 72%; 95% confidence interval: 60%-80%; n = 50). Additionally, we conducted a novel large-scale phenomic visualization analysis to provide insight into the modular nature of common molecular mechanisms spanning multiple biological scales and reused by related phenotypes (metaphenotypes). We propose that this method elucidates which classes of molecular mechanisms are associated with phenotypes or metaphenotypes and holds promise in facilitating a computable systems biology approach to genomic and biomedical research.
Algorithms↗
Central projections of the sciatic, saphenous, median, and ulnar nerves of the rat demonstrated by transganglionic transport of choleragenoid-HRP (B-HRP) and wheat germ agglutinin-HRP (WGA-HRP).
The central projections of the rat sciatic, saphenous, median, and ulnar nerves were labeled by injecting each nerve with 0.05 mg B-HRP, or 0.5 mg WGA-HRP, or a mixture of both. The B-HRP labeled large dorsal root ganglion cells (30-50 microns) and, correspondingly, 98% of axons labeled in a rootlet were meyelinated; although all sizes of myelinated axons were labeled, a greater proportion fell in the large ranges (2-6.5 microns axon diameter) than in the small ranges (0.5-2 microns). Primary afferents labeled with B-HRP were distributed in laminae I, III, IV, and V of the dorsal horn and extended into the intermediate grey and the ventral horn; Clarke's column and the respective dorsal column nuclei were also densely labeled. Motoneurons of the nerve were densely labeled by B-HRP, including extensive regions of their dendritic trees. In contrast, WGA-HRP labeled small dorsal root ganglion cells (15-25 microns) and in the dorsal rootlets, 84% of the labeled axons were nonmyelinated; the small population of labeled myelinated afferents mainly fell within the smaller ranges (0.5-2.0 microns). Terminal fields of WGA-HRP labeled afferents were restricted to the superficial dorsal horn (laminae I-III), and to limited regions in the dorsal column nuclei. Sciatic nerve projections traced by labeling with B-HRP alone or in combination with WGA-HRP were more extensive than previously described when using either native HRP or WGA-HRP. Afferents to the dorsal horn extended from L1-S1, to Clarke's nucleus from T8-L1, to the ventral horn from L2-L5, and extended throughout the medial and dorsal region of the gracilie nucleus. Motoneurons were found from L4-L6. Using the same tracers, saphenous projections extended in the superficial dorsal horn from caudal L1 to rostral L4, in the deep dorsal horn to mid L4 and along the length of the central part of the gracilie nucleus. The median nerve projected to the internal basilar nucleus from C1-C6, the dorsal horn from C3-T2, Clarke's nucleus from T1-T6, the external cuneate nucleus, and a large central area throughout the length of the cuneate nucleus. Motoneurons were located in dorsolateral and ventrolateral nuclear groups from C4 through C8. The ulnar nerve projections were less extensive but also included the internal basilar nucleus from C1-C6, the medial region of the dorsal horn from C4-T1, Clarke's nucleus from T1-T6, the external cuneate nucleus, and the medial part of the cuneate nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)
Afferent Pathways↗
Rostrocaudal somatotopy in the neural connections between the lateral hypothalamus and the dorsal periaqueductal gray of the rat brain.
1. The lateral hypothalamus (LH) and the dorsal periaqueductal gray area (dPAG) are two important brain structures involved in central cardiovascular control. 2. In the present study, we searched for possible rostrocaudal somatotopy in the neural connections from the three subdivisions of the LH (anterior-LHa; tuberal-LHt and posterior-LHp) to the different rostrocaudal portions of the dPAG. 3. The bidirectional neuronal tracer biotinylated-dextran-amine (BDA) was microinjected into different rostrocaudal coordinates of the dPAG (AP 3.4-2.7 mm) of male Wistar rats. One week later, animals were sacrificed and brain slices were processed and analyzed to detect neuronal efferent projections from the LH to the dPAG. 4. Neuronal cell body staining was observed along all the rostrocaudal axis of the LH when BDA was microinjected in more rostral dPAG coordinates. When the BDA was microinjected into more caudal dPAG regions, labeled neurons were observed only in the caudal portion of the LH. 5. Efferent projections from the LHa were directed only to the rostral portion of the dPAG. Projections from the rostral and medial portions of the LHt were also directed to the rostral dPAG, whereas both rostral and caudal dPAG received projections from the caudal portion of the LHt. Efferent projections from the anterior portion of the LHp were directed to both rostral and caudal dPAG, whereas projections from the caudal LHp were only directed to the rostral portion of the dPAG.6. The results suggest a somatotopic correlation in LH projections to the dPAG with main connections to the rostral dPAG, which are efferent from the three divisions of the LH. More caudal regions of the dPAG received afferents only from posterior sites in the LH. 7. Moreover, the results point out to extensive and complex neural somatotopic projections from all LH subdivisions to different rostrocaudal portions of the dPAG, reinforcing the idea of significant functional interactions between the brain structures.
Afferent Pathways↗