Transient expression of thyroid hormone nuclear receptor TRbeta2 sets S opsin patterning during cone photoreceptor genesis.

Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR beta 2, the nuclear thyroid hormone receptor beta isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR beta 2 acts, we compared the spatiotemporal expression of TR beta 2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR beta 2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR beta 2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR beta 2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR beta 2. The mechanism by which TR beta 2 functions was probed in transgenic animals with TR beta 2 ablated, TR beta 2 that is DNA binding defective, and TR beta 2 that is ligand binding defective. These studies show that TR beta 2 is necessary for dorsal repression, but not ventral activation of S opsin. TR beta 2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR beta 2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors.

Competitive antagonism of recombinant P2X(2/3) receptors by 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP).

TNP-ATP has become widely recognized as a potent and selective P2X receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have investigated the ability of TNP-ATP to inhibit alpha,beta-methylene ATP (alpha,beta-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X(2/3) receptors. Pharmacological responses were measured using electrophysiological and calcium imaging techniques. TNP-ATP was a potent inhibitor of P2X(2/3) receptors, blocking both rat and human receptors with IC(50) values of 3 to 6 nM. In competition studies, 10 to 1000 microM alpha,beta-meATP was able to overcome TNP-ATP inhibition. Schild analysis revealed that TNP-ATP was a competitive antagonist with pA(2) values of -8.7 and -8.2. Inhibition of P2X(2/3) receptors by TNP-ATP was rapid in onset, reversible, and did not display use dependence. Although the onset kinetics of inhibition were concentration-dependent, the TNP-ATP off-kinetics were concentration-independent and relatively slow. Full recovery from TNP-ATP inhibition did not occur until >/=5 s after removal of the antagonist. Because of the slow off-kinetics of TNP-ATP, full competition with alpha,beta-meATP for receptor occupancy could be seen only after both ligands had reached a steady-state condition. It is proposed that the slowly desensitizing P2X(2/3) receptor allowed this competitive interaction to be observed over time, whereas the rapid desensitization of other P2X receptors (P2X(3)) may mask the detection of competitive inhibition by TNP-ATP.

The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.

Mice express S and M opsins that form visual pigments for the detection of light and visual signaling in cones. Here, we show that S opsin transcription is higher than that of M opsin, which supports ultraviolet (UV) sensitivity greater than midwavelength sensitivity. Surprisingly, most cones coexpress both S and M opsins in a common cone cell type throughout the retina. All cones express M opsin, but the levels are graded from dorsal to ventral. The levels of S opsin are relatively constant. However, in the far dorsal retina, S opsin is repressed stochastically, such that some cones express M opsin only. These observations indicate that two different mechanisms control M and S opsin expression. We suggest that a common cone type is patterned across the retinal surface to produce phenotypic cone subtypes.

Molecular and functional characterization of human P2X(2) receptors.

P2X receptors are a family of ATP-gated ion channels. Four cDNAs with a high degree of homology to the rat P2X(2) receptor were isolated from human pituitary and pancreas RNA. Genomic sequence indicated that these cDNAs represent alternatively spliced messages. Northern analysis revealed high levels of human P2X(2) (hP2X(2)) message in the pancreas, and splice variants could be detected in a variety of tissues. Two cDNAs encoded functional ion channels when expressed in Xenopus oocytes, a receptor structurally homologous to the prototype rat P2X(2) receptor (called hP2X(2a)) and a variant containing a deletion within its cytoplasmic C terminus (called hP2X(2b)). Pharmacologically, these functional human P2X(2) receptors were virtually indistinguishable, with the P2X receptor agonists ATP, 2-methylthio-ATP, 2' and 3'-O-(4-benzoylbenzoyl)-ATP, and ATP5'-O-(3-thiotriphosphate) being approximately equipotent (EC(50) = 1 microM) in eliciting extracellular Ca(2+) influx. The P2 receptor agonists alpha,beta-methylene ATP, adenosine, adenosine 5'-O-(2-thiodiphosphate), and UTP were inactive at concentrations up to 100 microM. Both hP2X(2a) and hP2X(2b) receptors were sensitive to the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (IC(50) = 3 microM). In contrast to the analogous rat P2X(2) and P2X(2b) receptors, the desensitization rates of the hP2X(2a) and hP2X(2b) receptors were equivalent. Both functional forms of the human P2X(2) receptors formed heteromeric channels with the human P2X(3) receptor. These data demonstrate that the gene structure and mRNA heterogeneity of the P2X(2) receptor subtype are evolutionarily conserved between rat and human, but also suggest that alternative splicing serves a function other than regulating the desensitization rate of the human receptor.

Direct amplification of Escherichia coli O157 vero toxin genes from human faeces by the polymerase chain reaction.

Direct amplification of DNA from clinical specimens, such as blood and faeces, by polymerase chain reaction (PCR) is most often hindered by endogenous inhibitory substances, including haemoglobin and bile acids. We tested whether Ampdirect A (Shimadzu), a novel reagent cocktail that has been shown to suppress the inhibitors in blood, is also useful for faecal samples, and found that the vero toxin genes (VT1 and VT2) of Escherichia coli O157 could be efficiently amplified from the supernatant of boiled faeces by PCR in the presence of this cocktail without prior extraction of DNA. We compared the efficiency of amplification with and without the cocktail, using the supernatant of boiled normal faeces supplemented with E. coli O157. PCR without the cocktail failed to amplify the vero toxin genes from the supernatant diluted < 6400-fold or containing > 0.02% (final concentration) of boiled faeces. By contrast, PCR with Ampdirect A amplified the toxin genes in the mixture containing as much boiled faeces as 0.5% and as few E. coli as 4 to 8 colony-forming units (CFU). The minimum limit for E. coli O157 detection by this method was estimated to be about 10(4) CFU/g faeces. The results obtained by this direct method agreed well with those obtained by the indirect method using DNA pre-extracted from patients' faeces (the detection limit being 10(3) CFU/g faeces).

Basic fibroblast growth factor induces cyclooxygenase-2 expression in endothelial cells derived from bone.

Although histological studies have suggested that endothelial cells in bone (BDECs) are associated with some osteolytic bone diseases, it is still unclear how BDECs contribute to bone remodeling. Here we examined the response of BDECs to basic fibroblast growth factor (bFGF, FGF-2) using primary and cloned murine BDECs isolated from the femurs of BALB/c mice. Treatment of primary and cloned BDECs with bFGF induced cyclooxygenase-2 (COX-2) mRNA and protein expression. Furthermore, bFGF promotes the production of prostaglandin E2 (PGE2), which is known to be a potent stimulator of bone resorption and to induce osteoclast formation. Because the secretion of PGE2 was suppressed by COX-2 specific inhibitor NS-398 and by COX-2 antisense oligodeoxynucleotides, bFGF promotes the synthesis of PGE2 in a COX-2-dependent manner. Therefore, endothelial cells in bone are associated with bone remodeling by controlling COX-2 expression and consequently PGE2 production.

A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript.

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes.

Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein-Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

A remarkable improvement of clinical manifestations in a breast cancer patient with widespread bone metastases after administration of pamidronate.

A breast cancer patient with bone metastases showed a marked response to treatment with a bisphosphonate, an inhibitor of osteoclastic bone resorption. The patient was admitted to our hospital with hypercalcemia, widespread bone metastases and severe disseminated intravascular coagulation (DIC). We treated her conservatively with pamidronate and gabexate mesilate, because the patient had refused any anti-cancer chemotherapy. She showed marked improvement in performance status, hypercalcemia, DIC and tumor markers, whereas splenomegaly due to metastasis progressed. These results suggest that pamidronate has the potential to suppress metastatic tumor growth selectively in bone.

Adenosine as an endogenous mediator of hypoxia for induction of vascular endothelial growth factor mRNA in U-937 cells.

Adenosine induced by hypoxia exerts various effects via different types of receptors. Recently, hypoxia was shown to be a strong inducer of vascular endothelial growth factor, a secreted endothelial cell specific mitogen. In this report, we studied on effects of adenosine on inducibility of VEGF and possible mediation of hypoxia for its induction in U-937 cells. Hypoxia induced expression of VEGF mRNA with an early peak at 1 hour. 5'-N-ethylcarboxamidoadenosine, an adenosine analog, strongly induced VEGF mRNA, which was inhibited by 3,7-dimethyl-1-propargylxanthine (DMPX), an A2-antagonist. The hypoxic induction was inhibited by adenosine deaminase, 7-(beta-hydroxyethyl)theophylline, a non-selective adenosine receptor antagonist and DMPX. These results suggest that the hypoxic induction of VEGF mRNA is mediated by adenosine via A2-receptor in U-937 cells.

High incidence of mitral and tricuspid regurgitation in patients with Graves' disease detected by two-dimensional color Doppler echocardiography.

With two-dimensional (2D) color Doppler echocardiography, the cardiac and valvular function of 24 consecutive patients with a history of Graves' disease (17 were hyperthyroid and 7 were euthyroid at the time of the examination) were evaluated. The incidences of mitral regurgitation (MR), tricuspid regurgitation (TR) and MR plus TR were significantly higher in the patients with Graves' disease than in the age-matched control group of patients without this disease. In the patients who had signs of congestive heart failure (CHF) while they were hyperthyroid, a significantly higher incidence of severe TR was observed. This is the first report of a 2D color Doppler echocardiography study on the incidences of TR and/or MR in patients with Graves' disease. Our data indicate that in Graves' disease valvular dysfunction can be caused by systemic disorders and that severe TR is a possible risk factor for CHF.

Evolution of avian metallothionein: DNA sequence analyses of the turkey metallothionein gene and metallothionein cDNAs from pheasant and quail.

The turkey metallothionein gene (tkMT) was isolated from a phage lambda-turkey genomic DNA library by virtue of high identity with chicken MT cDNA. The nucleotide sequences of the proximal 240 bp of the 5'-flanking region, of each of the three exons, and of the intron/exon boundaries were determined. Comparisons of the nucleotide sequences of the tkMT and cMT genes revealed (1) absolute conservation of intronic DNA immediately flanking each respective intron/exon boundary, (2) high conservation (95.6%) of exonic DNA encoding translated regions of the mRNA, and (3) high conservation (95%) of exonic DNA encompassing the putative transcription start point and polyadenylation signals. Sequence comparisons of the tkMT and cMT promoters regions near the TATA box revealed that both promoters contain a highly conserved proximal metal-responsive enhancer (MRE-enhancer) motif. The deduced amino acid sequence (63 amino acids) of tkMT was identical with that of cMT. In order to further explore the degree of conservation of the protein coding regions of avian MT genes, partial MT cDNAs from turkey, quail (qMT), and pheasant (pMT) were amplified using the reverse transcriptase-polymerase chain reaction (RT-PCR) and primers corresponding to the amino- and carboxyl-terminal coding regions of cMT mRNA. RT-PCR reaction products were cloned and the DNA sequences of multiple cDNA clones from each species were determined. The results suggest the existence of a single MT mRNA in zinc-treated liver from turkey and pheasant and the existence of a major and possibly a minor MT mRNA in quail.(ABSTRACT TRUNCATED AT 250 WORDS)

Metal ions induce expression of metallothionein in pancreatic exocrine and endocrine cells.

Northern blot hybridization established that metallothionein (MT) mRNA levels were dramatically elevated in the rat pancreas following injection of Cd or Zn salts. To determine which pancreatic cell types express the MT gene, Northern blot hybridization analysis of RNA from preparations enriched for acini, in situ hybridization, and immunocytochemistry were used. RNA from pancreatic acini of Zn-treated rats contained high levels of MT mRNA. In control rats, in situ hybridization suggested very low levels of MT mRNA in both exocrine and endocrine cells in the pancreas, but these levels were dramatically increased in both these cell populations following metal injection. In contrast, levels of insulin-I mRNA in the endocrine cells were not affected by metal injection. A similar result with MT mRNA was obtained in mouse and chicken pancreas using Northern blot and in situ hybridization. Immunocytochemistry detected MT in the rat acinar cell cytoplasm following metal injection. Although inconsistent with in situ hybridization studies and immunocytochemical analysis of exocrine cells, immunocytochemistry for MT indicated a uniform staining pattern of islet cells that was unaffected by metal treatment. These results establish that metal ion induction of the MT genes in pancreas occurs in both endocrine and exocrine cells, which suggests that this protein has diverse physiologic functions in this organ.

Collagen-induced arthritis in rats. Assessment by serial magnification radiography.

Serial whole body and lateral hindpaw magnification radiographs were obtained on 15 rats with collagen-induced arthritis. The radiographs were evaluated for findings of inflammatory arthritis. Soft tissue swelling and juxta-articular osteopenia usually developed two weeks after immunization. The swelling remained stable for the remaining seven weeks of the study, but the osteopenia apparently improved. Three weeks after immunization, erosions and periostitis began to develop in ten of the rats. These latter radiographic changes rapidly worsened over the next three weeks and then stabilized. The most frequent and severe changes were seen in the intertarsal, tibiotalar, and metatarsophalangeal joints. The toes, forepaws, and knees were infrequently involved. Other peripheral joints and the axial skeleton were spared. Five rats developed soft tissue swelling but had no articular erosions or periostitis. Serial radiography has potential usefulness for monitoring the effects of treatment in this experimental model for inflammatory arthritis.