Adverse events in patients with blood loss: a pooled analysis of 51 clinical studies from the celecoxib clinical trial database.

BACKGROUND: Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with an increased risk of gastrointestinal (GI) toxicity, including occult blood loss and the development of clinically significant anemia. The aim of this study was to investigate the clinical importance of clinically significant anemia/blood loss. METHODS: Pooled analysis of 51 blinded, controlled clinical studies ≥4 weeks duration from the celecoxib clinical trial database, comparing celecoxib with NSAIDs or placebo. The adverse event (AE) profile in patients with clinically significant anemia/blood loss (defined as decreases in hemoglobin ≥2 g/dL and/or hematocrit by ≥10% from baseline) was compared with the AE profile in patients without blood loss. Events that occurred in <0.5% of patients were excluded from any comparisons. A threefold difference between groups was defined arbitrarily as being markedly higher. RESULTS: Overall 932/51,048 patients experienced clinically significant anemia/blood loss. Baseline demographics were similar in both groups. The incidence of AEs was markedly higher in patients who experienced clinically significant anemia/blood loss than those who did not; the majority of these differences were for GI AEs or their likely sequelae. The incidence of the following non-GI related AEs was also markedly higher in patients with blood loss: coronary artery disease (1.2% vs 0.3%), myocardial infarction (0.6% vs 0.2%), and pneumonia (1.7% vs 0.4%). Withdrawals due to AEs were more common among patients who experienced blood loss (16.7% vs 10.4%). CONCLUSIONS: Clinically significant anemia/blood loss may have clinically important adverse consequences beyond the sequelae previously known to be associated with NSAID-related GI effects.

Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping and tissue expression.

We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.

Identification and characterization of a novel member of the fibroblast growth factor family.

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.

Cloning, characterization, and mapping of human homolog of mouse T-cell death-associated gene.

To establish immunologic autotolerance, self-reactive immature thymocytes are eliminated by negative selection during T-cell development in the thymus. Self-reactive clones undergo apoptosis after stimulation via the T-cell receptor (TCR). The process of cell selection is determined by the dedication of the TCR for tolerogenic antigen/major histocompatibility complex. We have cloned a novel human gene that is highly homologous in the transmembrane and G protein-coupling domains to mouse T-cell death-associated gene 8 (TDAG8). The gene, human TDAG8 (hTDAG8), which belongs to the G protein-couple receptor superfamily, encodes a protein of 337 amino acids. An expressed sequence tag (EST) corresponding to hTDAG8 was identified from a human thyroid cDNA library and subsequently used to isolate a full-length genomic clone. Northern blot analysis revealed that the hTDAG8 gene is expressed predominantly in lymphoid tissues, including peripheral blood leukocytes, spleen, lymph nodes, and thymus. Stably transfected mammalian CHO cells were generated, and heterologous expression of hTDAG8 was confirmed by Northern blot analysis. Fluorescent in situ hybridization (FISH) revealed that hTDAG8 maps to human chromosome 14q31-32.1, a region in which abnormalities associated with human T-cell lymphoma or leukemia are found. Taken together, these data implicate the hTDAG8 gene in T-cell-associated diseases in humans, but its actual physiological and pathological role in the human immune system needs further investigation.

A novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer.

We have isolated a prostate-specific gene (NKX3.1) in humans that is homologous to the Drosophila NK homeobox gene family. Northern blot analyses indicate that this gene is expressed at high levels in adult prostate and at a much lower level in testis, but is expressed little or not at all in several other tissues. In an androgen-dependent prostate carcinoma line, LNCaP, NKX3.1 mRNA is expressed at a basal level that was increased markedly upon androgen stimulation; the NKX3.1 mRNA was undetectable in several other human tumor cell lines including two androgen-independent prostate carcinoma lines. The NKX3.1 gene maps to chromosome band 8p21, a region frequently reported to undergo a loss of heterozygosity associated with tissue dedifferentiation and loss of androgen responsiveness during the progression of prostate cancer. Based on these data we propose that NKX3.1 is a candidate gene for playing a role in the opposing processes of androgen-driven differentiation of prostatic tissue and loss of that differentiation during the progression of prostate cancer.

The human transcriptional adaptor genes TADA2L and GCN5L2 colocalize to chromosome 17q12-q21 and display a similar tissue expression pattern.

The chromosomal locations and the tissue expression patterns of the human transcriptional adaptors TADA2L and GCN5L2 have been determined. Northern blot analysis across a range of human tissues revealed that both the TADA2L and the GCN5L2 mRNAs are expressed to varying degrees in all tissue types. Furthermore, in most tissue types, the genes are expressed at relatively similar levels, suggesting coordinated regulation of TADA2L and GCN5L2 transcription. Chromosomal mapping by fluorescence in situ hybridization indicated that these genes reside near each other on chromosome 17; TADA2L mapped within bands 17q12-q21, while GCN5L2 mapped distally within band 17q21.

Isolation, characterization, and mapping of two human potassium channels.

Two novel human genes encoding putative potassium channels, kH1 and kH2, were identified from a human fetal brain cDNA library. Sequence analysis showed that kH1 and kH2 are homologous to rat IK8 and rat K13, respectively. The kH1 encodes a polypeptide of 495 amino acids, which shares 88% and 95% identity to IK8 at the nucleotide and amino acid level, respectively. The kH2 encodes a polypeptide of 515 amino acids with 86% and 92% identity to K13 at the nucleotide and amino acid level, respectively. Northern blot studies revealed that one mRNA species, approximately 5kb, of the kH1 was expressed abundantly in tissues examined, including the heart, skeletal muscle, and less abundant in the brain, liver, kidney, and pancreas. Interestingly, an alternative spliced form of 2.4 kb mRNA species of the kH1 was also found in the brain. Unlike kH1, 2.4 kb of kH2 was expressed predominantly in the brain, placenta, and the skeletal muscle where it shared a differently spliced form of the kH2 mRNA, approximately 2.0 kb. Fluorescence in situ hybridization localized kH1 to the human chromosome 2p25 and kH2 to the human chromosome 20q13.

Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.

Nucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causative mutations and strongly reduced levels of encoded protein were identified in XP-F patients. The XPF protein was purified from mammalian cells in a tight complex with ERCC1. This complex is a structure-specific endonuclease responsible for the 5' incision during repair. These results demonstrate that the XPF, ERCC4, and ERCC11 genes are equivalent, complete the isolation of the XP genes that form the core nucleotide excision repair system, and solve the catalytic function of the XPF-containing complex.

Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.

The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.

Genomic organization of the human PMS2 gene family.

The hPMS2 gene (HGMW-approved symbol PMS2) encodes a mutL homolog that causes hereditary non-polyposis colon cancer (HNPCC) when inherited in mutant form. We have here characterized the genomic structure of the hPMS2 gene to facilitate its analysis in HNPCC kindreds. The hPMS2 genomic locus was found to encompass 16 kb and consist of 15 exons. During its analysis, we identified a family of hPMS2-related genes located on chromosome 7 at bands 7p12-p13, 7q11, and 7q22. Exons 1 through 5 of these homologs shared a high degree of identity with hPMS2. We present the sequence of seven novel genes that represent the hPMSR (hPMS2-related) gene family. The similarity and number of these genes made specific amplification of hPMS2 problematic, but knowledge of them aided the successful design of oligonucleotides for this purpose.

Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination.

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.

Two subfamilies of murine retrotransposon ETn sequences.

Early transposon (ETn) elements are 5.7-kb retrotransposons found in the murine genome. We have sequenced large portions of two ETn elements that have apparently transposed within the DNA of a murine myeloma cell line, P3.26Bu4. One of the transposed ETn elements has 5' and 3' long terminal repeats (LTRs) that are exact duplicates of each other and has a 6-bp target site duplication. These results suggest that this element, which inserted into an immunoglobulin gamma 1 switch region, moved by a retrotransposition process. Our nucleotide sequences confirm that individual ETn elements are very similar to one another and lack open reading frames. However, the ETn sequences reported here and those previously described differ significantly near their 5' LTRs, including 200 bp of weak similarity and 240 bp of complete disparity. Southern hybridization analysis suggests that both subfamilies of ETn sequences are represented many times in the mouse genome. The possibility that the disparate sequences have a role in transposition by ETn elements is discussed.

Interruption of two immunoglobulin heavy-chain switch regions in murine plasmacytoma P3.26Bu4 by insertion of retroviruslike element ETn.

A number of moderately reiterated murine genetic elements have been shown to have structures like those of retroviral proviruses. These elements are thought to be transposons, although little evidence of their transposability exists. Two members of one of these families of reiterated elements, the ETn family, have inserted into separate immunoglobulin heavy-chain switch regions in the plasmacytoma P3.26Bu4. Switch regions are those DNA segments associated with each immunoglobulin heavy-chain gene in which the somatic recombinations that accompany the heavy-chain switch occur. This role in somatic recombination may be relevant to the ETn insertions into the switch regions in P3.26Bu4 DNA. P3.26Bu4 and a number of other B-lineage cells contain ETn transcripts.

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions.

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.

A prospective study of shoulder disability resulting from radical and modified neck dissections.

A prospective longitudinal study of shoulder function after 103 neck dissections involving either preservation or sacrifice of the spinal accessory nerve is presented. The postoperative evolution and course of trapezius muscle denervation and resultant shoulder dysfunction were objectively determined for both radical and modified nerve sparing neck dissections. All patients were enrolled in a program of physical therapy aimed at maintaining range of motion at the shoulder joint. Shoulder function was examined preoperatively and for 12 months postoperatively with manual muscle strength testing, range of motion measurements, and electrodiagnostic testing. Results indicate that modified nerve sparing dissections are followed on the average by a significant, but temporary and reversible phase of shoulder dysfunction. By comparison, radical neck dissection is followed by profound and permanent trapezius muscle weakness and denervation.

DNA sequences near the site of reciprocal recombination between a c-myc oncogene and an immunoglobulin switch region.

The chromosomal translocations found in many B-cell tumors result in the joining of a c-myc oncogene with an immunoglobulin heavy chain switch region. This finding is striking because the natural function of switch regions is to mediate DNA rearrangements important to the maturation of immune responses. These normal switch rearrangements are probably mediated by specific enzymes. In this paper we report the isolation of the two reciprocal products of a recombination between a c-myc gene on murine chromosome 15 and an immunoglobulin switch region (S mu S gamma 2b) on chromosome 12. We have determined the sequences of these DNA molecules near the recombination sites and show that the recombination is nearly perfectly reciprocal, with a seven-nucleotide deletion. An examination of the sequences reported in this paper, and of sequences published by other authors, shows a correlation between the points of recombination for c-myc-S segment rearrangements and for normal heavy chain switches. We suggest that this correlation implies a role for switch recombination enzymes in creating substrates for the c-myc recombination. The c-myc gene also seems to share some limited homology to sequences thought to be important in heavy chain switching. Finally, we discuss a working model that accounts for some characteristics of c-myc-S segment recombinations. The model also suggests a mechanism for increased transcriptional activity of the rearranged c-myc oncogene in B-cell tumors.