Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT sub(4) receptor sequence, we have cloned the complete coding region for a human 5-HT sub(4) receptor. ...The sequence shows extensive similarity to the published porcine 5-HT sub(4A) and rat 5-HT sub(4L) receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5-HT sub(4) sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT sub(4) receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT sub(4) receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT sub(4) receptors in guinea pig striatal tissue. super(3)HGR 113808 revealed K sub(D) values of 0.15 plus or minus 0.01 nM for the human receptor and 0.3 plus or minus 0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5-HT sub(4) antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT sub(4)-specific agonists caused an increase in cyclic AMP levels.
In the fission yeast Schizosaccharomyces pombe the rad1 super(+) gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a ...human homologue of the S. pombe rad1 super(+) gene, designated Hrad1, as well as a mouse homologue: Mrad1. Two Hrad1 alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B. Hrad1A has 60% identity to the S. pombe rad1 super(+) sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad1 is located on chromosome 5p13.2-13.3. This region is subject to loss of heterozygosity in several human cancers. Hrad1 also shares homology with the Saccharomyces cerevisiae RAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3' arrow right 5' exonuclease with a C-terminal domain essential for cell cycle checkpoint function. We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3' arrow right 5' exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.
Heterologous expression of cloned receptor subtypes for screening programs has become a real necessity for a modern pharmaceutical company. As the expression levels obtained so far are often low or ...unstable, we addressed this problem by using an inducible promoter system, i.e. the interferon-inducible mouse Mxl promoter. Using the gene coding for chloramphenicol acetyltransferase (CAT) as a reporter gene, we tested the inducibility of this promoter in the murine cell line L929. We found that background expression was low and that a distinct interferon-induced expression could be obtained. CAT expression reached its maximum at approximately 15 ng CAT/mg protein after induction for 24 hr with 1000 U/ml murine interferon-beta; the induction ratio was 150-fold. Next, L929 cells were transfected with four different human serotonin (5HT) receptor cDNAs (5HT1A, 5HT2A, 5HT1D beta and 5HT1E) under the control of the same Mxl promoter fragment. Also in this case well-regulated serotonin receptor-expressing clones were isolated. Bmax values varied from 3100 fmol/mg protein for the 5HT2A receptor, 3300 fmol/mg protein for the 5HT1D beta receptor, 9800 fmol/mg protein for the 5HT1E receptor, and even up to 10,400 fmol/mg protein for the 5HT1A receptor. Furthermore, the expression levels were shown to remain stable during serial propagation for at least one year, demonstrating the usefulness of this expression system. In fact, the 5HT1D beta receptor-expressing cells were used in the characterization of a new antimigraine agent, viz. alniditan.
Objective
The ANP32A gene encodes a tumor suppressor molecule that plays a regulatory role in apoptosis and interferes with canonical Wnt signaling in vitro. We undertook this study to test whether ...genetic variation at ANP32A was associated with osteoarthritis (OA) in women.
Methods
Single‐nucleotide polymorphisms (SNPs) in the ANP32A gene were genotyped in 438 control women, 425 women with total knee replacements (TKRs), and 537 women with total hip replacements (THRs) from the Nottingham case–control study as well as in 820 women from the population‐based Chingford Study cohort for whom hip and knee radiographs were available. The most highly associated SNP was further tested in women from the Rotterdam Study (131 with THRs, 633 with knee OA, and 1,567 controls) and the TwinsUK Study cohort (67 with THRs, 43 with TKRs, and 358 controls), for a total of 2,170 patients with OA and 2,849 controls.
Results
The ANP32A transcript was abundantly expressed in normal and OA articular cartilage. Three SNPs in the ANP32A gene were significantly associated in Nottingham patients with hip OA, but not knee OA. One of these (rs7164503) was associated with hip and knee OA in the Chingford Study cohort and with THR in the TwinsUK Study cohort, but the association was not statistically significant in the Rotterdam Study. When we combined hip data from all 4 cohorts, we found that the minor allele of rs7164503 was associated with a significantly lower risk of hip OA (Mantel‐Haenszel odds ratio 0.67 95% confidence interval 0.53–0.84, P < 3.8 × 10−4) and that a similar trend was observed for knee OA (Mantel‐Haenszel odds ratio 0.87 95% confidence interval 0.73–1.01, P < 0.055).
Conclusion
Our results provide evidence suggesting that ANP32A is involved in the pathogenesis of OA of the hip.
In order to explore the sensitivity of spatially resolved 1H and 31P NMR spectroscopy on a whole-body NMR instrument, cerebral metabolic changes in human volunteers were measured during ...hyperventilation provocation. During hyperventilation the flow velocity in the middle cerebral artery decreased significantly and the EEG showed a marked increase in slow activity. 1H NMR spectra revealed an increase in cerebral lactate concentration. 31P NMR spectra showed no changes in ATP or PCr peak heights, but a shift toward tissue alkalosis was derived from changes in Pi chemical shift. During subsequent recovery, lactate concentration decreased and a slight intracellular acidosis was detected. In three experiments broadening of the lactate resonance peak resulted in separation into two components at 1.32 and 1.48 ppm, in which the latter signal possibly arose from alanine.
Objective
Male DBA/1 mice are known to spontaneously develop arthritis in the hind legs. The present study was undertaken to investigate the role of endogenous interferon‐γ (IFNγ) in the pathogenesis ...of this ankylosing enthesopathy.
Methods
The role of IFNγ was studied by examining the development of arthritis in IFNγ receptor–knockout (IFNγR‐KO) DBA/1 mice as compared with wild‐type mice, and by treatment of wild‐type mice with monoclonal anti‐IFNγ antibody. IFNγ‐disrupted and wild‐type mice were mixed and housed in the same cage, and clinical symptoms of arthritis were assessed weekly for at least 9 weeks. Histologic examination was performed at the end of the experiment.
Results
In DBA/1 wild‐type mice, 70% of the animals developed clinical symptoms of spontaneous arthritis, such as redness and swelling of the proximal interphalangeal joints, toe stiffness, and ankylosis. As evident from microscopic evaluation, the arthritis was mainly characterized by formation of new cartilage and bone, originating at the entheses and leading to ankylosis. The incidence and severity of arthritis, both clinically and histologically, were significantly reduced in IFNγR‐KO mice. In wild‐type mice, neutralizing anti‐IFNγ antibody inhibited the occurrence of the disease for the duration of treatment.
Conclusion
The results suggest that endogenous IFNγ plays an important role in the initial stages of spontaneous arthritis, and that the inflammatory components in its pathogenesis are more prominent than has been believed. In view of the similarity between this disease and spondylarthropathies in humans, the data suggest that endogenous IFNγ may also play a disease‐promoting role in the human condition and thus may serve as a target for therapy.
The cellular and molecular basis of bone development and its regulation by differentiation and growth factors is an exciting area of current research. This article briefly reviews the historical ...progress in the isolation of osteogenin, a novel bone differentiation factor, and its modulation by well known growth factors. Endochondral bone development is a multistep sequential cascade and the process must be operationally dissected. It has been accomplished with the demineralized bone matrix-induced bone formation model. The reproducible development of cartilage and bone in an extraskeletal site permits the study of the initiation of the first cycle of endochondral bone formation and mineralization. Recent progress in the isolation of osteogenin, a specific bone differentiation factor, by heparin affinity chromatography permits the further investigation of the commitment and clonal expansion of the putative osteoprogenitor stem cells. Once initiated, bone formation is promoted by growth factors such as platelet derived growth factor, fibroblast growth factor, insulin like growth factor, transforming growth factor β and a plethora of non specific cytokines. Finally bone development is further modulated by systemic hormones and nutrition and a host of physical signals including electrical, gravitational and mechanical forces.
Time-domain model function fitting techniques were applied to improve the reconstruction of metabolite maps from the data sets obtained from in vivo 1H spectroscopic imaging (SI) experiments. First, ...residual water-related signals were removed from the SI data sets by using SVD-based linear time-domain fitting based upon the HSVD (State Space) approach. Second, peak integrals of the metabolites of interest were obtained by quantifying the proton spin-echoes of the voxels by means of non-linear time-domain fitting based upon the maximum likelihood principle. Third, in order to save computational time, interpolation of the metabolite images (from size 32 x 32 to 128 x 128) was performed in the image-domain by applying one-dimensional cubic splines. It was found that the residual water signals can be almost completely removed from the SI data sets by applying the linear HSVD fitting method. Furthermore, it was found that voxel dependency of certain NMR parameters (e.g., variations of the spin-echo offset frequencies and/or phase factors) can be accounted for automatically by applying the nonlinear time-domain fitting technique. For that purpose it appeared to be essential to employ prior knowledge of the NMR spectral parameters.
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