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PubMed2001

Genasense (Genta Inc).

Genasense (formerly known as G-3139), an antisense oligonucleotide specific for Bcl-2, is under development by Genta as an iv drip infusion for the potential treatment of various cancers including melanoma, prostate, breast and colon cancer [3083751. It is in phase III trials for malignant melanoma, for which it has been awarded Fast Track status 1359044]. Genasense received Orphan Drug status in August 2000 [3782331. In September 2000, the company announced that pivotal phase III trials in multiple melanoma, chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML) would be underway by 2001 [382783]. By January 2001, trials in AML and CLL had been initiated 1396512]. As of February 2001, Genta was planning the initiation of two additional, registration quality trials. Pending positive results from these trials, launch of Genasense is anticipated in 2002 13984111. A phase III trial in patients with advanced multiple myeloma at 65 centers in the US, Canada and Great Britain began in February 2001. The trial will examine whether the addition of Genasense can improve response rates, response duration and quality of life compared with dexamethasone therapy alone 13989081. Genta Inc has been issued a patent (US-05831066) for Genasense 1283005]. The patent provides protection to Genta for the composition of Genasense and its analogs. Furthermore, Genta Inc has also been issued two new patents that cover a series of compounds containing new backbone constructions that enhance the antisense affinity of the drug to the target pre-RNA, while the other patent covers the methods for preparation of antisense oligonucleotides containing the new backbone structures 12896851. Genta has already licensed the rights for the use of Bd-2 as a target for antisense- and gene therapy-based treatments from The University of Pennsylvania. The licensing agreements with Chugai Pharmaceutical Co for worldwide marketing and profit sharing places Genta in a favorable position. In January 2001, Needham & Co expected Genasense to have a potential market of 47,700 malignant melanoma patients in the US. The analysts also expected potential patient market sizes of 50,000 (CLL), 21,000 (AML), 136,000 (non-small cell lung cancer; NSLCC) and 180,000 (prostate cancer) in the US. In addition, the analysts predicted that Genasense would be approved for melanoma in the second quarter of 2002, with approvals to follow for CLL (third quarter of 2002), AML (third quarter of 2002) and myeloma (fourth quarter of 2002) 1399251].

Antineoplastic Agents↗
PubMed1984

Isolation, characterisation and crystallization of deoxyribonuclease I from bovine and rat parotid gland and its interaction with rabbit skeletal muscle actin.

A purification procedure is described yielding DNase I from bovine and rat parotid glands of high homogeneity. The apparent molecular masses of the DNases I isolated have been found by sodium dodecyl sulfate/polyacrylamide gel electrophoresis to be 34 and 32 kDa for bovine and rat parotid DNase I, respectively, and thus differ from the enzyme isolated from bovine pancreas (31 kDa). By a number of different criteria concerning their enzymic behaviour, the isolated enzymes could be clearly classified as DNases I, i.e. endonucleolytic activity preferentially on native double-stranded DNA yielding 5'-oligonucleotides, a pH optimum at about 8.0, the dependence of their enzymic activity on divalent metal ions, their inhibition by 2-nitro-5-thiocyanobenzoic acid and by skeletal muscle actin. Comparison of their primary structure by analysis of their amino acid composition and also two-dimensional fingerprints and isoelectric focusing indicate gross similarities between the enzymes isolated from bovine pancreas and parotid, but distinct species differences, i.e. between the enzymes isolated from bovine and rat parotid. All the DNases I are glycoproteins. From bovine parotid DNase I crystals suitable for X-ray structure analysis could be obtained. The DNases I from both parotid sources specifically interact with monomeric actin forming 1:1 stoichiometric complexes. Their binding constants to monomeric actin differ, being 2 X 10(8) M-1 and 5.5 X 10(6) M-1 for bovine and rat parotid DNase I, respectively. Only the enzyme isolated from bovine sources is able to depolymerize filamentous actin.

Actins↗
PubMed1988

Cloning of authentic human epidermal growth factor as a bacterial secretory protein and its initial structure-function analysis by site-directed mutagenesis.

A synthetic chimeric gene, coding for the human epidermal growth factor fused to the signal peptide of Escherichia coli alkaline phosphatase, was cloned into E. coli under the transcriptional control of the trp-lac (tac) promoter. Following induction with isopropylthiogalactoside, the secretion of the correctly processed protein product into the bacterial periplasm was detected and quantitated by its specific binding to the epidermal growth factor receptor. The purified protein was identical to authentic human epidermal growth factor in size, amino acid composition, primary sequence, receptor binding, and stimulation of receptor protein-tyrosine kinase activity. Based on interspecies homologies, structural considerations, and reported studies with peptide fragments, structure-function analysis was initiated with alterations of targeted amino acid residues by oligonucleotide-directed mutagenesis. The receptor binding affinity of each mutant, relative to the wild type, was measured by both radioreceptor competition and receptor tyrosine kinase stimulation assays. In general, the values obtained by the two methods were in agreement for each species of epidermal growth factor and followed the order: wild type greater than Glu24----Gly greater than Asp27----Gly much greater than Pro7----Thr greater than Tyr29----Gly greater than Leu47----His. The relatively low values obtained with the last two mutants suggest that Tyr29 and Leu47 may be important for the biological activity of human epidermal growth factor.

Alkaline Phosphatase↗
PubMed2004

A genomic predictor of oral squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: The objective was to identify a genomic profile that predicts the likelihood of oral squamous cell carcinoma compared with normal oral mucosa in unknown tissue samples. STUDY DESIGN: Using a training set of tissue samples that were histologically classified as oral squamous cell carcinoma or normal mucosa, the authors used principal component analysis to develop a genomic predictor for oral squamous cell carcinoma. On a separate test set of unclassified samples, the authors used the predictor to classify the samples, then evaluated the performance of the predictor using histological diagnosis. METHODS: The authors used a data set consisting of messenger RNA extracted from 29 oral squamous cell carcinoma and 19 normal oral mucosa tissue samples and hybridized to Affymetrix oligonucleotide microarrays containing probe sets for 7070 genes and expressed sequence tags. The samples were divided into a training set of 15 oral squamous cell carcinoma and 10 normal samples and a test set consisting of the remaining samples. Using principal component analysis on the training set, the authors found a composite gene expression vector (principal component vector), which they used to compute likelihood ratios for oral squamous cell carcinoma on the test set. By calculating the contribution of each gene to the principal component vector, the authors identified genes with the greatest predictive value. RESULTS: Using the likelihood ratio, the authors correctly classified all 23 samples in the test set as either oral squamous cell carcinoma or normal. The authors found that many of the most predictive genes are known to be markers of squamous cell carcinoma or normal mucosa. CONCLUSION: Principal component analysis can be used with genomic microarray data to correctly predict the presence of oral squamous cell carcinoma in unknown tissue samples.

Biomarkers, Tumor↗
PubMed1993

Collaborative study of the molecular epidemiology of Tay-Sachs disease in Europe.

Tay-Sachs disease is a lipidosis due to the deficiency of the lysosomal hexosaminidase A. In order to understand the molecular mechanisms of this enzyme deficiency we studied 42 patients of different ethnic origins diagnosed in Europe. The strategy used consists in HEXA cDNA amplification followed by allele-specific oligonucleotide analysis for the frequent mutations, and by chemical cleavage mismatch and denaturing gradient gel electrophoresis for the detection of new mutations. 90% of alleles were clarified in this way, showing a high heterogeneity of HEXA lesions in Tay-Sachs disease. 28 different mutations were found, 20 being identified for the first time in this group of patients.

Adult↗
PubMed1997

Antisense oligonucleotides induce functional deletion of ligand gated ion channels in cultured neurons and brain explants.

The in situ application of the antisense technique for the study of ligand gated channels is discussed here. Using antisense oligodeoxynucleotides to downregulate a gene of interest means being confronted with a number of choices that will determine the success. These include choosing a target sequence, considering chemical modifications of the oligo as well as its length and estimation of the turnover of the target protein in order to set up the treatment schedule. In this paper a short overview of technical aspects of the antisense approach on primary cultured neurons and brain slice cultures is presented. In addition, the effects of antisense oligos on the expression of neuronal nicotinic acetylcholine receptors and GABA(A) receptors are discussed: Patch-clamp recordings of neurons treated with specific antisense oligos targeted at individual subunits showed a clear downregulation of the expression of native ligand gated channels. Moreover, in a number of experiments novel channel types with altered properties were observed following antisense treatment. Thus, non-targeted channel subunits that remain expressed after antisense deletion, may aggregate to form novel channel types that are normally not present. Alternatively, the translational arrest of a protein may be accompanied by compensatory changes in the synthesis and/or targeting of other channel subunits to the cell surface. The antisense technique enables identification of the functional contribution of individual channel subunits to endogenous channel activity in the central nervous system. As such it paves the way to the elucidation of in vivo channel-subunit composition and channel functions, of post- as well as pre-synaptic ligand gated channel receptors.

Brain↗
PubMed2006

Global analysis of gene expression in Xenopus hindlimbs during stage-dependent complete and incomplete regeneration.

Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large-scale screen of gene expression in the regeneration-complete, stage 53 (st53), and regeneration-incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration-complete and -incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration-competent and deficient tissues.

Animals↗
PubMed1991

Translational control of transposition activity of the bacterial insertion sequence IS1.

The experiments reported here provide strong evidence indicating that the transposition frequency of the bacterial insertion sequence IS1 is determined principally by two IS1-specified proteins. The first, InsA, was previously shown to bind to the ends of the element and to act as a repressor. We present both physical and genetic evidence which reveals that the second, the InsAB' transposase, is a fusion of InsA with the product of a downstream reading frame, InsB'. Synthesis of this protein occurs by a -1 frameshift between the insA and insB' frames. It requires the presence of an intact retroviral-like frameshift signal composed of an A6C motif and a downstream region able to form several alternative secondary structures. In vivo studies show that IS1 transposition activity depends on the relative rather than on the absolute levels of InsA and InsAB'. The ratio is determined primarily at the translational level by frameshifting and appears to be relatively insensitive to large variations in levels of transcription. This novel homeostatic control could therefore protect IS1 from activation as a consequence of insertion into active transcription units.

Base Composition↗
PubMed1990

Functional expression of the mutants of the chloroplast tRNA(Lys) gene from the liverwort, Marchantia polymorpha, in Escherichia coli.

The anticodon of the tRNA(Lys) gene (trnK) in the liverwort, Marchantia polymorpha, was artificially converted to an amber anticodon. This mutant tRNA(Lys) (CTA) gene carrying either the intron of the C27-C43 mismatch at the anticodon-stem is not functional in Escherichia coli, but without both of them, it does work as a tRNA(Lys) amber suppressor.

Base Composition↗
PubMed2005

Characterization of the DNA binding features of Saccharomyces castellii Cdc13p.

The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand. Moreover, the minimal binding site for scasCdc13p is the octamer 5'-GTGTCTGG-3' of the S.castellii telomeric sequence. The scasCdc13p displayed a high affinity binding, where four individual nucleotide residues were found to be of most importance for the sequence specificity. Nonetheless, scasCdc13p binds the telomeric repeats from various other species, including the human. In spite of considerable divergence in telomere repeat length and sequence between these species, a conserved Cdc13p binding motif was detected. Among the budding yeasts this conserved Cdc13p binding site overlaps the Rap1p binding site. Together, these data implicate scasCdc13p as a telomere end-binding protein with a potential role in the regulation of telomere maintenance in vivo. Moreover, the results suggest that Rap1p and Cdc13p act together to preserve the conserved core present within the otherwise highly divergent btelomeric sequences among a wide variety of yeasts.

Amino Acid Sequence↗
PubMed1996

A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro.

A minor class of introns with noncanonical splice (AT-AC) and branch site sequences exists in metazoan protein coding genes. We have established a HeLa cell in vitro system that accurately splices a pre-mRNA substrate containing such an intron from the human P120 gene. Splicing occurs via a lariat intermediate whose branch site A residue is predicted to bulge from a duplex formed with the low abundance U12 small nuclear ribonucleoprotein (snRNP), which we confirm by psoralen cross-linking. Native gel electrophoresis reveals that U11, U12, and U5 snRNPs assemble onto the P120 pre-mRNA to form splicing complexes. Inhibition of P120 splicing by 2'-O-methyl oligonucleotides complementary to U12 or U5 demonstrates that U12 and U5 snRNPs perform essential roles in the AT-AC spliceosome.

Base Composition↗
PubMed1991

Sequence specificity in triple-helix formation: experimental and theoretical studies of the effect of mismatches on triplex stability.

The specificity of a homopyrimidine oligonucleotide binding to a homopurine-homopyrimidine sequence on double-stranded DNA was investigated by both molecular modeling and thermal dissociation experiments. The presence of a single mismatched triplet at the center of the triplex was shown to destabilize the triple helix, leading to a lower melting temperature and a less favorable energy of interaction. A terminal mismatch was less destabilizing than a central mismatch. The extent of destabilization was shown to be dependent on the nature of the mismatch. Both single base-pair substitution and deletion in the duplex DNA target were investigated. When a homopurine stretch was interrupted by one thymine, guanine was the least destabilizing base on the third strand. However, G in the third strand did not discriminate between a C.G and an A.T base pair. If the stretch of purines was interrupted by a cytosine, the presence of pyrimidines (C or T) in the third strand yielded a less destabilizing effect than purines. This study shows that oligonucleotides forming triple helices can discriminate between duplex DNA sequences that differ by one base pair. It provides a basis for the choice of antigene oligonucleotide sequences targeted to selected sequences on duplex DNA.

Base Composition↗
PubMed1989

Effects of functional group changes in the EcoRV recognition site on the cleavage reaction catalyzed by the endonuclease.

Oligodeoxynucleotides have been prepared which contain changes in the functional group pattern present in the EcoRV recognition site d(GATATC). These modifications involve the deletion of specific functional groups or the reversal of the relative positions of functional groups within the canonical six base pair recognition site. The duplex stability of these modified oligodeoxynucleotides has been assessed by determining the thermodynamic parameters characterizing helix formation. Steady-state kinetic parameters have been used to characterize the interaction of the modified oligodeoxynucleotides with the EcoRV endonuclease. The enzyme is very sensitive to the deletion of either of the adenine amino or thymine methyl groups, or the reversal of the relative positions of the adenine amino group and thymine carboxy group which form an interstrand hydrogen bond in the major groove of the B-DNA helix. Conversely, deletion of the guanine amino group had only minimal effects upon the measured kinetic parameters. Deletion of the exocyclic amino group from the "inner" dA-dT base pair resulted in the fragment which interacted with the enzyme on the basis of observed inhibition experiments but was not cleaved. The results suggest that the endonuclease interacts with its recognition sequence via contacts in the major groove of the B-DNA helix and that both hydrogen bonding to the adenine amino groups and also hydrophobic interactions with the thymine methyl groups are involved.

Adenine↗
PubMed1998

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA).

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.

Antibodies↗
PubMed1992

Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3], plays an important role in the regulation of mineral ion homeostasis. As well as being the major steroid hormone that regulates calcium metabolism, 1,25(OH)2D3 suppresses transcription of the gene encoding parathyroid hormone, a peptide that plays a dominant role in regulating extracellular calcium levels. To identify DNA sequences that may mediate this transcriptional repression, nuclear extracts containing the 1,25(OH)2D3 receptor were examined for binding to sequences in the 5'-flanking region of the human parathyroid hormone gene. A 25-base-pair (bp) oligonucleotide containing the sequences from -125 to -101 from the start of exon I binds nuclear proteins recognized by monoclonal antibodies against the 1,25(OH)2D3 receptor. The sequences in this region contain a single copy of a motif (AGGTTCA) homologous to the motifs repeated in the up-regulatory 1,25(OH)2D3-response elements. When placed upstream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediate transcriptional repression in response to 1,25(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. This down-regulatory element, therefore, differs from the up-regulatory 1,25(OH)2D3-response elements both in sequence composition and in the requirement for particular cellular factors other than the 1,25(OH)2D3 receptor for repressing transcription.

Animals↗
PubMed2001

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Antigens, Bacterial↗
PubMed2005

Complete genome sequence and lytic phase transcription profile of a Coccolithovirus.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Apoptosis↗