Draft genome sequence of the purple photosynthetic bacterium Phaeospirillum molischianum DSM120, a particularly versatile bacterium.

Here we present the draft genome sequence of the versatile and adaptable purple photosynthetic bacterium Phaeospirillum molischianum DSM120. This study advances the understanding of the adaptability of this bacterium, as well as the differences between the Phaeospirillum and Rhodospirillum genera.

E2A-ZNF384 and NOL1-E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia.

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)-PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3' portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5' regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT-PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

Cooperative transformation by MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins generated by the variant t(1;19) in acute lymphoblastic leukemia.

A variant t(1;19)(q23;p13.3) translocation creates reciprocal DAZAP1/MEF2D and MEF2D/DAZAP1 fusion genes that are expressed in acute lymphoblastic leukemia. We used retroviral gene transfer to ectopically express wild-type and chimeric DAZAP1 and MEF2D fusion proteins in NIH 3T3 cells. In soft agar assays, each of the fusion proteins transformed 3T3 cells with a 20-fold increase in colony formation as compared to empty vector or native MEF2D or DAZAP1 proteins. Co-expression of both DAZAP1/MEF2D and MEF2D/DAZAP1 led to a threefold increase in colony formation as compared to either fusion protein alone. Expression of wild-type DAZAP1, MEF2D or DAZAP1/MEF2D allowed 3T3 cells to proliferate under low serum (0.5%) conditions and suppressed apoptosis. In contrast, MEF2D/DAZAP1 expression did not facilitate proliferation in low serum and led to a modest increase in apoptosis. Both MEF2D/DAZAP1 and DAZAP1/MEF2D have oncogenic properties, and co-expression of both fusion proteins is synergistic.

Cloning and functional characterization of MEF2D/DAZAP1 and DAZAP1/MEF2D fusion proteins created by a variant t(1;19)(q23;p13.3) in acute lymphoblastic leukemia.

We analyzed the TS-2 acute lymphoblastic leukemia (ALL) cell line that contains a t(1;19)(q23;p13.3) but lacks E2A-PBX1 fusion typically present in leukemias with this translocation. We found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 (Deleted in Azoospermia-Associated Protein 1) to the 1q23 gene MEF2D (Myocyte Enhancer Factor 2D), leading to expression of reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts. MEF2D is a member of the MEF2 family of DNA binding proteins that activate transcription of genes involved in control of muscle cell differentiation, and signaling pathways that mediate response to mitogenic signals and survival of neurons and T-lymphocytes. DAZAP1 is a novel RNA binding protein expressed most abundantly in the testis. We demonstrate that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable from that of native MEF2D and is a substantially more potent transcriptional activator than MEF2D. We also show that DAZAP1/MEF2D is a sequence-specific RNA-binding protein. MEF2D has been identified as a candidate oncogene in murine retroviral insertional mutagenesis studies. Our data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contribute to leukemogenesis by altering signaling pathways normally regulated by wild-type MEF2D and DAZAP1.

Differential modulation of energy balance by leptin, ciliary neurotrophic factor, and leukemia inhibitory factor gene delivery: microarray deoxyribonucleic acid-chip analysis of gene expression.

Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.

Hypothalamic delivery of doxycycline-inducible leptin gene allows for reversible transgene expression and physiological responses.

Our purpose was to incorporate regulation into the recombinant adeno-associated virus encoding leptin by introducing a tet-inducible promotor. This system, TET-Ob, allows for control of leptin gene expression via doxycycline in drinking water. F344XBN rats (aged 4 months) were given a hypothalamic injection of TET-Ob or control virus. During 34 days of doxycycline (doxy) administration to all rats (STAGE 1), TET-Ob rats gained 50.7% less mass, ate 10.4% less food, and had a 77.5% reduction in serum leptin as compared with controls. Doxy was then withdrawn from half of the TET-Ob rats for 32 days (TET-Ob-OFF), while half continued to receive doxy (TET-Ob-ON) (stage 2). During stage 2, TET-Ob-ON rats gained 44.8% less mass than TET-Ob-OFF and ate significantly less food than both TET-Ob-OFF and controls. Serum leptin increased to 83.4% of control values in TET-Ob-OFF, but remained very low in the in TET-Ob-ON. At death, visceral adiposity was 14.5% of controls in TET-Ob-ON animals, but had risen to 76.9% of controls in TET-Ob-OFF. A reversible increase in both leptin signal transduction in the hypothalamus and uncoupling protein expression in brown adipose was recorded. This system allows for more precise regulation of gene therapy-mediated fat loss.

Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats.

The purpose of this study was to determine if long-term leptin treatment desensitizes leptin signal transduction and the subsequent downstream anorexic and thermogenic responses in normal and leptin-resistant age-related obese rats. To this end, we administered, i.c.v., recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control virus into young and aged-obese rats and after 9 or 46 days, examined food intake, oxygen consumption, body weight, serum leptin, STAT3 phosphorylation, hypothalamic NPY and POMC mRNAs, and UCP1 expression and protein level in brown adipose tissue (BAT). In young rats, rAAV-leptin depleted body fat and both anorexic and thermogenic mechanisms contributed to this effect. Moreover, leptin signal transduction was not desensitized, and there were persistent physiological responses. Similarly, in the aged-obese rats, there was unabated leptin signal transduction, however, both the anorexic and thermogenic responses completely attenuated sometime after day 9. This attenuation, downstream of the leptin receptor, may be contributing to the leptin-resistance and age-related weight gain in these aged-obese rats. Finally, in young rats, although the initial responses to rAAV-leptin were dominated by anorexic responses, by 46 days, the predominant response was thermogenic rather than anorexic, suggesting that energy expenditure may be an important component of long-term weight maintenance.

Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study.

The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.

Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings.

In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.

Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice. Relationship with body weight change.

We investigated the long-term effects of physiological levels of leptin produced by gene therapy on body weight (BW) and expression of genes that encode orexigenic and anorexigenic peptides in the hypothalamus. Recombinant adeno-associated viral vector (rAAV), a non-pathogenic and non-immunogenic vector, encoding leptin (betaOb) was generated and administered iv to ob/ob mice lacking endogenous leptin. Whereas the lowest dose of rAAV-betaOb (6x10(9) particles) was ineffective, the middle dose (6x10(10) particles) curbed BW gain without affecting food consumption for 75 days of observation. A ten-fold higher dose (6x10(11) particles) resulted in increased blood leptin levels and suppressed both BW gain and food consumption throughout the duration of the experiment. rAAV-betaOb doses that either curbed BW without affecting food consumption or evoked BW loss and reduced food intake, decreased the expression of genes encoding the orexigenic peptides, neuropeptide Y and agouti-related peptide in the ARC, and the two doses were equally effective. Concomitantly, the expression of genes encoding the anorexigenic peptide, alpha-melanocyte stimulating hormone and cocaine-and-amphetamine regulatory transcript, was augmented with the latter gene displaying a dose-dependant response. These results document the efficacy of delivering biologically active leptin for extended periods by an iv injection of rAAV-betaOb and show that physiological leptin concentrations simultaneously exert a tonic inhibitory effect on orexigenic and a stimulatory effect on anorexigenic signaling in the hypothalamus. This intricate dynamic interplay induced by leptin regulates BW with or without an effect on food intake in leptin-deficient ob/ob mice. Further, these results suggest that gene therapy is an effective mode of delivery to the hypothalamus of those therapeutic proteins that cross the blood-brain barrier to ameliorate neuroendocrine disorders.

Alteration of the glucocorticoid receptor subcellular localization by non steroidal compounds.

The glucocorticoid receptor (GR) engages transient or stable interactions with chaperones (hsp90, hsp70), co-chaperones (p60/hop, hsp40) and several other polypeptides such as immunophilins (Cyp40, FKBP59) and p23 to achieve a high affinity ligand binding state. This complex dissociates in response to hormonal stimuli and holo-GR translocates into the nucleus, where it regulates the activity of glucocorticoid-sensitive genes. GR activity is controlled through its ligand binding domain by steroids displaying either agonistic or antagonistic activity. An alternative approach to modulate GR activity is to target receptor-associated proteins (RAPs), and several non steroidal compounds binding to RAPs affect GR transcriptional activity. We have studied the effect of such drugs on the intracellular localization of a EGFP-GR fusion protein, which has wild type GR pharmacological properties. Agonist and antagonist binding induced nuclear translocation of GR, whereas rifampicin was found to be inactive in our system. Immunosuppressants FK506 and cyclosporin A were able to induce partial nuclear translocation of GR, suggesting that potentiation of glucocorticoid action by these compounds may also proceed through enhanced GR nuclear transfer. Short treatment of cells with the hsp90 inhibitor geldanamycin (GA) did not prevent nuclear translocation of GR. However, longer treatments, in parrallel to the inhibition of GR transcriptional activity, strongly perturbed GR subcellular localization concomitantly to the disruption of the actin network, and caused GR aggregation and down-regulation. The GA-induced transcriptional shutdown was also observed for other nuclear receptors which do not interact stably with hsp90. Thus RAP-binding compounds may exert their effects at least in part through perturbation of the GR cytosol to nucleus partitioning, and identify these proteins as valuable therapeutic targets to control nuclear receptor activity.

Growth protocols for etiolated soybeans germinated within BRIC-60 canisters under spaceflight conditions.

As part of the GENEX (Gene Expression) spaceflight experiment, protocols were developed to optimize the inflight germination and subsequent growth of 192 soybean (Glycine max cv McCall) seeds during STS-87. We describe a method which provided uniform growth and development of etiolated seedlings while eliminating root and shoot restrictions for short-term (4-7 day) experiments. Final seedling growth morphologies and the gaseous CO2 and ethylene levels present both on the last day in space and at the time of recovery within the spaceflight and ground control BRIC-60 canisters are presented.

[The effect of 2',5'-oligoadenylates on the expression of the cellular proto-oncogene c-myc during the development of Svec's leukemia in rats]. / Vliianie 2',5'-oligoadenilatov na ékspressiiu kletochnogo protoonkogena c-myc pri razvitii leikoza Shvetsa u krys.

The effects of 2',5'-oligoadenilates (2,5A) on the survival of Wistar rats inoculated with Svec leukemia cells and the proliferation of tumoral cells were investigated. An agreement between the expression of c-myc protooncogene and inhibition of leukemic cell proliferation by 2,5A was found.

[Cycle dependent expression of the gene coding for B2mRNA]. / Tsikolzavisimaia ékspressiia gena, kodiruiushchego B2mRNK.

The relative contents of individual tissue specific RNA B2mRNAx was studied in the population of nuclear and poly(A)+ cytoplasmic RNA from the livers of intact, falsely operated and having suffered the partial liver resection rats. Dot-hybridization technique was used to study this transcript containing the transcribed copy of the B2 repetitive genetic element of rats. The expression of the gene coding for B2mRNAx takes place at the lower level in the liver induced to proliferation as compared with the one in the liver of intact animals. It is changed reproducibly in antiphase with the c-fos RNA and with major inclinations at the moments of cellular phases switch.

[Effect of monovalent cations on the ratio of RNA transcripts from DNA with different repetitive sequences in leukemia P-388 cells]. / Vliianie monovalentnykh kationov na sootnoshenie RNK-transkriptov s raznykh po povtorennosti posledovatel'nostei DNK v kletkakh leikemii P-388.

Using leukemic P-388 cells, it was demonstrated that alterations of the intracellular Na+/K+ ratio results in qualitative changes of newly synthesized mRNA, which manifests itself as changes in the kinetics of hybridization of heterogeneous nuclear RNA (hnRNA) with DNA. The decrease of the Na+/K+ value from 3.8:1 to 1:1 leads to inhibition of mRNA synthesis in mRNA cells hybridized at 10(3) greater than Dot greater than 10(4), but weakly affects the transcription of mRNA sequences hybridized with DNA within the Dot interval of 10(3.4)-10(3.8). A similar phenomenon is observed during hybridization of hnRNA with homogeneous fractions of unique and averagely repeating sequences of DNA. The hybridization rate constants of hnRNA synthesized by cells at different values of Na+/K+ and the limit values of hybridization (H infinity) were calculated. It was shown that the rate constants for RNA hybridization with DNA decrease by more than two orders of magnitude during the transition of the averagely repeating DNA fraction to the unique one; however, in both cases these constants give equal values for the RNA synthesized by cells at different cationic balance. The H infinity values for the RNA synthesized by cells at higher Na+ ratio appeared to be 1.5-2.0 times as high as compared with those for the RNA in the cells, in which the Na+/K+ ratio was 1:1. Thus, the monovalent cation ratio seems to exert a strong influence on the expression of sequences of different repeatedness in the genome and to play a role in the regulation of proliferative activity of the cell.

[Isolation and study of the physicochemical properties of the DNA of the iridescent virus from the mosquito Aedes cantans]. / Vydelenie i izuchenie fiziko-khimicheskikh svoistv DNK virusa raduzhnosti komara Aedes cantans.

Molecules of DNA of Aedes cantans mosquito iridescent virus were found to be of linear shape, about 150 micron in length. The temperature of melting, sedimentation coefficient, molecular weight, and buoyant density of DNA were determined as well as the content of GC pairs in it.

[Features of nuclear genome transcription at early steps of liver regeneration: expression of DNA fractions with different repeating sequence frequencies]. / Osobennosti transkriptsii iadernogo genoma na rannikh stadiiakh regeneratsii pecheni. Ekspressiia fraktsii DNK s razlichnoi chastotoi povtoreniia posledovatel'nostei.

The mode of hybridization of dRNA fraction from normal and regenerating rat liver with purified fractions of repeating and unique sequences of DNA was studied. In order to repress the reassociation of DNA the nucleic acids were annealed in a solution of 2 x SSC--80% formamide at 48 degrees. The differential hybridization curves suggest that the amount of transcripts with a high DNA sequence frequency increases, while the complexity of transcripts of unique DNA sequences decreases 3 hrs after partial hepatectomy. Simultaneously the amount of RNA transcribed from the unique and moderately repeating DNA fractions remains practically unchanged.

[Reassociation of rat DNA fractions]. / Analiz reassotsiatsii fraktsii DNK krysy.

A method for direct registration of the first derivative of DNA reassociation kinetics in spectrophotometric cuvettes is proposed. Using this method, the rat liver DNA obtained by chromatography on hydroxyapatite was studied. The differential curves of reassociation and changes in hyperchromic spectra revealed the presence in the rat genome of 4 types of repeating sequences differing in their GC-content. A scheme of preparative separation of DNA with respect to the reassociation rate was developed. Three types of sequences repeating 3.10(5), 600 and 1 times in the genomes with the GC-content of 50, 42, and 33%, respectively, were obtained in a homogeneous state. The advantages of the differential method of registration of the kinetic curves for the study of DNA structure and calculation of fractionation schemes are discussed.

Macromolecular structure of nuclear polyhedrosis virus genome.

DNA preparations from nuclear polyhedrosis virus (NPV) of Galleria mellonella L. (GmL) were fractionated in high ionic strength neutral sucrose gradient. This procedure allowed a separation of supercoiled infectious DNA molecules with contour length of 48--52 microns from infectious open ring DNA molecule, and noninfectious linear DNA molecules of the same size. In addition a heterogeneity of supercoiled DNA molecules was detected. Covalently closed DNA molecules did not contain protein or ribonucleotide ligands which could be digested by pronase or pancreatic RNase treatment. It is concluded from data on the infectivity of different molecular forms of DNA and reassociation kinetics studies, that the genome of GmL NPV is a unique ring nucleotide sequence with a molecular weight of about 90--100 X 10(6).