Identification of New Genes Involved in the Regulation of Yeast Alcohol Dehydrogenase II

Recessive mutations in two negative control elements, CRE1 and CRE2, have been obtained that allow the glucose-repressible alcohol dehydrogenase (ADHII) of yeast to escape repression by glucose. Both the cre1 and cre2 alleles affected ADHII synthesis irrespective of the allele of the positive effector, ADR1. However, for complete derepression of ADHII synthesis, a wild-type ADR1 gene was required. Neither the cre1 nor cre2 alleles affected the expression of several other glucose-repressible enzymes. A third locus, CCR4, was identified by recessive mutations that suppressed the cre1 and cre2 phenotypes. The ccr4 allele blocked the derepression of ADHII and several other glucose-repressible enzymes, indicating that the CCR4 gene is a positive control element. The ccr4 allele had no effect on the repression of ADHII when it was combined with the ADR1-5c allele, whereas the phenotypically similar ccr1 allele, which partially suppresses ADR1-5c, did not suppress the cre1 or cre2 phenotype. Complementation studies also indicated that ccr1 and snf1 are allelic. A model of ADHII regulation is proposed in which both ADR1 and CCR4 are required for ADHII expression. CRE1 and CRE2 negatively control CCR4, whereas CCR1 is required for ADR1 function.     

Identification and Characterization of RBEL1 Subfamily of GTPases in the Ras Superfamily Involved in Cell Growth Regulation

Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236–302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.The Ras superfamily is known to comprise five structurally distinct subfamilies of small GTPases, including Ras, Rho, Rab, Sar1/Arf, and Ran, and each subfamily of these GTPases possess distinct functions in the regulation of a variety of cellular processes such as cell proliferation, cell differentiation, cytoskeletal organization, protein transport, and trafficking (1–4). The Ras subfamily of GTPases (N-, H-, and K-Ras) function predominantly in relaying signals from receptors at the plasma membrane and modulating cell signaling pathways that regulate cell proliferation, differentiation, and survival (5). Ran GTPase, on other hand, is a key regulator of nucleocytoplasmic transport that regulates protein transport across the nuclear pore complex (6, 7). The Rab subfamily is the largest subfamily among the Ras superfamily and contains more than 60 members. The key functions of the Rab GTPases are to regulate protein exocytic and endocytic pathways and modulate intracellular protein transport/trafficking (8–13).In general, the Ras superfamily GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. There are five N-terminal motifs involved in the binding and hydrolysis of GTP that are highly conserved among all GTPases: G1 (GXXXXGK(S/T)), G2 (T), G3 (DXXG), G4 ((N/T)(K/Q)XD), and G5 (EXSAX). Each sequence has particular functions involved in binding nucleotides (GTP or GDP) and facilitating hydrolysis (4, 14, 15). In general, the intrinsic GTPase activity (converting GTP to GDP) and exchange of GDP for GTP are slow processes for these GTPases and thus require regulatory proteins such as GTPase-activating proteins and GDP/GTP exchange factors to facilitate these processes (16–18).For the last two decades, the Ras superfamily has been a major focus in the cancer field as many of the members are either mutated or dysregulated in cancer. The founding members of the Ras superfamily, H-Ras and K-Ras, were first identified as viral oncogenes (1, 4). Later studies demonstrated that mutations of the Ras proteins (H-, N-, and K-Ras) occur frequently in human cancers, and the mutations identified are mostly clustered within the GTP-binding domains of the proteins thus locking Ras proteins in a GTP-bound configuration. GTP-bound Ras is constitutively active; it constantly activates its effector proteins to transduce cell proliferative signals (1, 4). Unlike Ras subfamily genes, mutations occurring in Rab and Rab-like genes are less common, yet alterations in gene expression of a number of Rab genes have been reported in multiple human malignancies. For example, Rab25 overexpression has been linked to prostate cancer progression (19). Rab2 overexpression has been found in lung adenomas and adenocarcinomas (20). In addition, alterations in Rab gene expression have also been linked to cancer drug resistance. For instance, resistance to the anticancer drug doxorubicin in MCF-7 cells has been linked with reduced expression of Rab6C, and introduction of exogenous Rab6C restores drug sensitivity (21).We have recently reported the identification two novel Ras superfamily GTPases, RBEL1A and RBEL1B (22). RBEL1A and RBEL1B are two splice variants of the RBEL1 gene and are highly homologous to the Rab and Ran GTPases within their N-terminal GTP-binding domains (22). Our studies show that both RBEL1A and -B predominantly bind to GTP. A single point mutation (T57N) in the GTP-binding domain of RBEL1A and -B abolishes their ability to bind to both GTP and GDP. Both RBEL1A and RBEL1B localize in the nucleus as well as in the cytosol. Whereas RBEL1A is predominantly cytosolic, RBEL1B is primarily nuclear. Interestingly, our studies also suggested that nucleotide (GTP or GDP)-binding could be important for the nuclear distribution of RBEL1B, because the nucleotide binding-deficient mutant form (T57N) of RBEL1B did not reside in the nucleus but rather became largely cytosolic (22).In our continuous efforts to fully elucidate the function of RBEL1, we have identified two additional splice variants that we have named RBEL1C and RBEL1D. Here we report further characterization of all four RBEL1 splice variants in terms of their GTPase activities, subcellular localizations, regulations, and potential functions. Our results indicate that RBEL1 GTPases, although sharing some common features with other Ras superfamily members, also harbor unique characteristics that are significantly different from other Ras superfamily GTPases. Based on our findings, we suggest that RBEL1 proteins appear to form a novel subfamily of GTPases within the Ras superfamily.     

Stromal Fibroblasts in Cancer: A Novel Tumor-Promoting Cell Type

Tumors are highly complex tissues composed of neoplastic cells and, in the case of carcinomas, stromal cell compartments containing a variety of mesenchymal cells, notably fibroblasts, myofibroblasts, endothelial cells, pericytes, and a variety of inflammatory cells associated with the immune system. Fibroblasts and myofibroblasts often represent the majority of the stromal cells within various types of human carcinomas, yet the specific contributions of these cells to tumor growth are poorly understood. Recent work has demonstrated that stromal fibroblast fractions, named carcinoma-associated fibroblasts (CAFs), that have been extracted from a number of invasive human breast carcinomas are more competent to promote the growth of mammary carcinoma cells and to enhance tumor angiogenesis than are comparable cells derived from outside of these tumor masses. CAFs include large populations of myofibroblasts that secrete elevated levels of stromal cell-derived factor 1 (SDF-1), also called CXCL12, which plays a central role in the promotion of tumor growth and angiogenesis; CAF-derived SDF-1 not only stimulated carcinoma cell growth directly through the CXCR4 receptor displayed on tumor cells but also served to recruit endothelial progenitor cells (EPCs) into tumors, thereby furthering neoangiogenesis. In this review, we highlight the importance of this SDF-1-CXCR4 signaling pathway in the tumor microenvironment and discuss the mechanisms by which stromal fibroblasts within mammary carcinomas enhance tumor growth.     

B细胞稳态的信号调节

在人类,65%的骨髓产生的B细胞是自身反应性的,它们大部分在骨髓中被克隆删除了。但有些B细胞通过免疫无力的方式逃脱了这种克隆删除到达外周,产生抗自身的抗体。研究表明,在鼠和人类中,B细胞存活时间过长是引发自身性免疫病的原因之一。B细胞的过度活化将导致自身反应性B细胞的产生和破坏自身免疫耐受,引起自身免疫性疾病或肿瘤;但B细胞的活化不足将使B细胞数量大大减少,抗原应答能力降低,从而使适应性免疫应答失衡。细胞因子和其他信号分子对B细胞稳态的调节是十分严密的,它们或调节B细胞的发育、成熟和分化,或调节B细胞向外周的迁移,或通过调节B细胞周期而使B细胞停留在特定时期,从而使B细胞避免凋亡,或通过调节抗凋亡蛋白或凋亡蛋白而决定B细胞的生存或死亡。本文就细胞因子、转录因子、蛋白激酶等信号分子对B细胞稳态的调节做一综述。     

Spinal and Peripheral Mechanisms Involved in the Enhancement of Morphine Analgesia in Acutely Inflamed Mice

The analgesic effect induced by opiates is often potentiated during experimental inflammatory processes. We describe here that lower doses of systemic morphine are necessary to increase thermal withdrawal latencies measured in both hind paws of mice acutely inflamed with carrageenan than in healthy ones. This bilateral potentiation seems mediated through spinal opioid receptors since it is inhibited by the intrathecal (i.t.), but not intraplantar (i.pl.) administration of the opioid receptor antagonist naloxone-methiodide, and also appears when morphine is i.t. administered. Furthermore, the i.pl. administration of the nitric oxide (NO) synthase inhibitor, l-NMMA, or the K_ATP⁺-channel blocker, glibenclamide, to carrageenan-inflamed mice inhibits the enhanced effect of systemic morphine in the paw that receives the injection of the drug, without affecting the potentiation observed in the contralateral one. The i.pl. administration of l-NMMA also partially antagonised the analgesic effect induced by i.t. morphine in inflamed mice. Finally, the increased analgesic effect evoked by the i.pl. administration of the NO donor SIN-1 either in the inflamed or in the contralateral paw of carrageenan-inflamed mice suggests that enhanced responsiveness to the peripheral analgesic effect of NO may be also underlying the bilateral potentiation of morphine-induced analgesia in acutely inflamed mice.     

Identification of a Terminal Rhamnopyranosyltransferase (RptA) Involved in Corynebacterium glutamicum Cell Wall Biosynthesis

A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum, with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactan-peptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, rhamnopyranosyltransferase A (rptA). Furthermore, an analysis of base-stable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.A common feature of members of the Corynebacterineae is that they possess an unusual cell wall dominated by a heteropolysaccharide termed an arabinogalactan (AG), which is linked to both mycolic acids and peptidoglycan, forming the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex (5, 10, 12, 15, 24, 25, 34). The formation of the arabinan domain in the mAGP complex, consisting mainly of α1→5, α1→3, and β1→2 glycosyl linkages, results from the subsequent addition of arabinofuranose (Araf) from the lipid-linked sugar donor β-d-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) by a set of unique membrane-bound arabinofuranosyltransferases (5, 7, 12, 18, 34).The deletion of Corynebacterium glutamicum emb (emb_Cg) (4) and a chemical analysis of the cell wall revealed a novel truncated AG structure possessing only terminal Araf residues with a corresponding loss of cell wall-bound mycolic acids (4). The presence of a novel enzyme responsible for “priming” the galactan domain for further elaboration by Emb_Cg proteins led to the identification of AftA, which belongs to the glycosyltransferase C (GT-C) superfamily (5). Recently, additional GT-C enzymes have been identified, termed AftB, which is responsible for the attachment of terminal β(1→2) Araf residues (34), and AftC, which is involved in AG branching (12) before decoration with mycolic acids, both of which are conserved within the Corynebacterineae (12, 34). It is clear that additional glycosyltransferases involved in both AG and lipoarabinomannan biosynthesis still remain to be identified. Indeed, Liu and Mushegian (22) identified 15 members of the GT-C superfamily residing in the Corynebacterineae, representing candidates involved in the biosynthesis of cell wall-related glycans and lipoglycans (22). We have continued our earlier studies (5, 12, 34) to identify genes required for the biosynthesis of the core structural elements of the mAGP complex by studying mutants of C. glutamicum and the orthologous genes and enzymes of Mycobacterium tuberculosis.A particularly interesting feature of C. glutamicum is the presence of terminal rhamnopyranose (t-Rhap) residues attached to the C2 position of α(1→5)-linked Araf residues in the arabinan domain of AG (4). The biological function of these residues remains to be clarified; nevertheless, they are a feature of the corynebacterial cell wall, and the biosynthesis of which needs to be addressed. The current paradigm of AG biosynthesis follows a linear pathway which is built upon a decaprenyl pyrophosphate lipid carrier. The unique disaccharide linker and galactan domain is synthesized by a variety of GT-A and GT-B family glycosyltransferases, all of which utilizing a nucleotide diphosphate-activated sugar substrate for transferase activity. It has been hypothesized by us (3, 5) and others (8) that a major shift in the biosynthetic machinery takes place upon the initiation of arabinan polymerization. AftA, Emb, AftC, and AftB all belong to the GT-C family of glycosyltransferases, all of which utilize DPA as the sole lipid-activated phosphosugar donor for arabinose transfer into the cell wall. Since t-Rhap residues are present in the arabinan component of the cell wall, the enzyme(s) responsible for its addition is likely to belong to the GT-C family of glycosyltransferases and, as determined through deduction, is one which utilizes a lipid-phosphate-derived rhamnose substrate similar to DPA. Herein, we present the putative protein NCgl0543 as a distinct t-Rhap of the GT-C superfamily, which is responsible for the transfer of t-Rhap residues to the arabinan domain to form the branched 2,5-linked Araf motifs of C. glutamicum. In addition, we have identified a novel decaprenyl-monophosphorylrhamnose and discuss its role in substrate presentation for AG biosynthesis in C. glutamicum.     

Melatonin is Involved in Regulation of the Expression of Neural Cell Adhesion Molecules in the Rat Brain

Cell adhesion molecules play a diverse role in neural development, signal transduction, structural linkage to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Neural cell adhesion molecules (NCAM) are members of the immunoglobulin superfamily and are involved in synaptic rearrangements in the mature brain. There are three major NCAM isoforms: NCAM 180, NCAM 140, and NCAM 120. Several studies reported that NCAM play a central role in memory formation. We investigated the effects of melatonin on the expression of NCAM in the hippocampus, cortex, and cerebellum of rats. The levels of NCAM isoforms were determined by Western blotting. After administration of melatonin for 7 days, the expression of NCAM 180 increased both in the hippocampus and in the cortex, as compared with the control. In contrast, in rats exposed to constant illumination for 7 days (a procedure that inhibits endogenous production of melatonin), levels of NCAM 180 dropped in the hippocampus and became undetectable in the cortex and cerebellum. Levels of NCAM 140 in the hippocampus of light-exposed rats also decreased. There was no change in the expression of NCAM 120 in any brain region. This is the first report indicating that melatonin exerts a modulatory effect on the expression of NCAM in brain areas related to realization of cognitive functions. Melatonin may be involved in structural remodeling of synaptic connections during memory and learning processes.     

New Type of Oxygenase Involved in the Metabolism of Propane and Isobutane

Nocardia paraffinicum (Rhodococcus rhodochrous), a hydrocarbon-degrading microorganism, was used in a study of propane and isobutane metabolism. The bacterium was able to utilize propane or isobutane as a sole source of carbon, and oxygen was found to be essential for its metabolism. Gas chromatographic analysis showed that n-propanol was the major compound recovered from the metabolism of propane by resting cells, although trace amounts of isopropanol and acetone were detected. When a mixture of propane and isobutane was used, drastic inhibition (72 to 88%) of hydrocarbon utilization by resting cells occurred. The ratio of hydrocarbon to oxygen consumed was found to be approximately 2:1 during the metabolism of propane or isobutane by resting cells when these substrates were provided individually to the organism. Gas chromatographic-mass spectrometric analysis of products formed from ¹⁸O₂ confirmed that the initial oxidative step in the metabolism of these substrates involved molecular oxygen. The proportion of the alcohol containing ¹⁸O was the same as that of ¹⁸O₂ in the gas mixture. Only a negligible amount of ¹⁸O was detected in the alcohol when H₂¹⁸O was incorporated into the system. The observed 2:1 ratio of hydrocarbon to oxygen consumption suggests that the oxygenase in N. paraffinicum, unlike the conventional mono- or dioxygenases, requires two hydrocarbon-binding sites for each of the oxygen-binding sites and is therefore an intermolecular dioxygenase. The newly described oxygenase, which catalyzes the reaction of two molecules of propane with one molecule of oxygen to yield two molecules of a C₃ alcohol, is proposed as the initial oxidation step of the hydrocarbon substrate.     

Immunofluorescent Identification of Melanocytes in Murine Hair Follicles

Summary Immunocytochemical identification of skin cells are difficult due to numerous endogenous autofluorescent components within the cell and the environment. This is particularly evident in hair follicles. This paper reports on a serendipitous modification to an existing method which results in a drastically reduced background fluorescence. Immediately after antigen retrieval, sections exposed to 0.3% hydrogen peroxide in methanol for 30 min at room temperature exhibited low background fluorescence, increased antigenicity and revealed quantifiable numbers of melanocytes. This method is applicable to both human and mouse melanocytes particularly in the hair follicle.     

Identification, Characterization, and Regulation of a Cluster of Genes Involved in Carbapenem Biosynthesis in Photorhabdus luminescens

The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics. We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P. luminescens. The cpm cluster differs in several crucial aspects from other car operons. The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P. luminescens genome. Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production. The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon. Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase. The importance of this carbapenem production in the ecology of P. luminescens is discussed.     

Factors Involved in the Regulation of a Ligninase Activity in Phanerochaete chrysosporium

The regulation of an H₂O₂-dependent ligninolytic activity was examined in the wood decay fungus Phanerochaete chrysosporium. The ligninase appears in cultures upon limitation for nitrogen or carbohydrate and is suppressed by excess nutrients, by cycloheximide, or by culture agitation. Activity is increased by idiophasic exposure of cultures to 100% O₂. Elevated levels of ligninase and, in some cases, of extracellular H₂O₂ production are detected after brief incubation of cultures with lignins or lignin substructure models, with the secondary metabolite veratryl alcohol, or with other related compounds. It is concluded that lignin degradation (lignin → CO₂) by this organism is regulated in part at the level of the ligninase, which is apparently inducible by its substrates or their degradation products.     

The Cannabinoid Receptor Type 2 as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.     

Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces Cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.