Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy.

The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumor-induced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-gamma. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad-P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy.

An optical supernova associated with the X-ray flash XRF 060218.

Long-duration gamma-ray bursts (GRBs) are associated with type Ic supernovae that are more luminous than average and that eject material at very high velocities. Less-luminous supernovae were not hitherto known to be associated with GRBs, and therefore GRB-supernovae were thought to be rare events. Whether X-ray flashes--analogues of GRBs, but with lower luminosities and fewer gamma-rays--can also be associated with supernovae, and whether they are intrinsically 'weak' events or typical GRBs viewed off the axis of the burst, is unclear. Here we report the optical discovery and follow-up observations of the type Ic supernova SN 2006aj associated with X-ray flash XRF 060218. Supernova 2006aj is intrinsically less luminous than the GRB-supernovae, but more luminous than many supernovae not accompanied by a GRB. The ejecta velocities derived from our spectra are intermediate between these two groups, which is consistent with the weakness of both the GRB output and the supernova radio flux. Our data, combined with radio and X-ray observations, suggest that XRF 060218 is an intrinsically weak and soft event, rather than a classical GRB observed off-axis. This extends the GRB-supernova connection to X-ray flashes and fainter supernovae, implying a common origin. Events such as XRF 060218 are probably more numerous than GRB-supernovae.

Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.

The cosmic microwave background radiation temperature at a redshift of 2.34.

The existence of the cosmic microwave background radiation is a fundamental prediction of hot Big Bang cosmology, and its temperature should increase with increasing redshift. At the present time (redshift z = 0), the temperature has been determined with high precision to be T(CMBR)(0) = 2.726 +/- 0.010 K. In principle, the background temperature can be determined using measurements of the relative populations of atomic fine-structure levels, which are excited by the background radiation. But all previous measurements have achieved only upper limits, thus still formally permitting the radiation temperature to be constant with increasing redshift. Here we report the detection of absorption lines from the first and second fine-structure levels of neutral carbon atoms in an isolated cloud of gas at z = 2.3371. We also detected absorption due to several rotational transitions of molecular hydrogen, and fine-structure lines of singly ionized carbon. These constraints enable us to determine that the background radiation was indeed warmer in the past: we find that T(CMBR)(z = 2.3371) is between 6.0 and 14 K. This is in accord with the temperature of 9.1 K predicted by hot Big Bang cosmology.

Pregnenolone esterification in Saccharomyces cerevisiae. A potential detoxification mechanism.

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs. The apparent Km for pregnenolone is approximately 0.5 microm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.

11 beta-hydroxylase activity in recombinant yeast mitochondria. In vivo conversion of 11-deoxycortisol to hydrocortisone.

In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.

Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes.

The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5'-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive pUB110 replicon and the resulting plasmid was introduced into B. sphaericus IFO3525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in trans on both bioXWF and bioDAYB 5'-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.

Biotin formation by recombinant strains of Escherichia coli: influence of the host physiology.

Strains of Escherichia coli were transformed with different plasmids bearing the gene clusters bioXWF and bioDAYB isolated from the Gram positive bacterium Bacillus sphaericus. These genes encode for the enzymes involved in the metabolic pathway which synthesizes biotin from the precursor pimelic acid. Transformed E. coli strains were grown in bioreactors to reach a biomass of 18 g l-1 cell dry weight in 1 litre batch culture with substrate feeding and approximately 50 g l-1 in 10 l fed batch culture. Improved yields of total vitamers and biotin formed in these processes were achieved after a comparative analysis of different culture conditions, medium compositions, host strains and expression systems. Production of 27 mg l-1 of biotin and 200 mg l-1 of vitamers was achieved in 1 litre batch culture. Using a 10-1 fed batch process, biotin and vitamer concentrations reached maximum values of 45 mg l-1 and 350 mg l-1, respectively.

Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli.

Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography. The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358). Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358. Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another. The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases. The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease.

Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the metabolism of oltipraz in the mouse, rat and monkey and in man. Distribution of the metabolites in each species.

4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (35 972 R.P., oltipraz) and its metabolites were extracted from human urine and from mouse, rat and monkey urine using Amberlite XAD4 resin. The metabolites were identified by GLC, TLC and HPLC and isolated by preparative TLC or HPLC. The structures of 11 compounds were determined by spectroscopic examination (MS, IR, NMR). Six of the principal metabolites isolated in sufficient quantity from human urine were administered to the mouse, confirming the metabolic pathways of oltipraz.

Familial occurrence of mitral valve prolapse in X-linked muscular dystrophy.

A high incidence of mitral valve prolapse (MVP) has been reported in patients with X-linked Duchenne muscular dystrophy. In our study MVP was present in six of 22 Duchenne dystrophy cases (27%) followed in the Maryland General Hospital Muscular Dystrophy Clinic. In addition, seven carriers of Duchenne and X-linked benign (Becker) dystrophy had evidence of MVP. Autosomal dominant transmission of MVP was present in four families. The unusually high prevalence of MVP in families with X-linked muscular dystrophy may have potential value in the recognition of the carrier trait.