A complementary DNA (cDNA) for human cyclophilin C (Cyp-C) was isolated from a human kidney cDNA library. Northern blot experiments with several human tissues and cell lines revealed that Cyp-C is less abundant than Cyp-A. The amount of Cyp-C mRNA was 10-fold lower than that of Cyp-A in kidney. Expression of human Cyp-C in the kidney is not significantly elevated compared to pancreas, skeletal muscle, heart, lung, and liver. This argues against a previously postulated specific role for Cyp-C in the nephrotoxic effects of CsA in humans, based on the studies of its relative abundance in murine kidney. It is present in extremely low concentrations in brain and in the Jurkat T cell line. The binding of recombinant human Cyp-A, -B, and -C to cyclosporin A (CsA) was studied by immunochemical methods. The relative affinity of Cyp-C for CsA is lower by a factor of 2 than that of Cyp-A, which itself is 10-fold lower than that of Cyp-B. Cross-reactivity studies with a series of Cs derivatives showed that Cyp-C binds CsA with a fine specificity similar to that of Cyp-A and Cyp-B. Cs amino acid residues 1, 2, 10, and 11 seemed essential for the interaction with all three Cyp subtypes. However, Cyp-C tolerates a greater variety of structures on Cs at position 2 than Cyp-A does, suggesting that this residue of CsA might not be in tight contact with Cyp-C. This was confirmed by modeling of human Cyp-C on the structure of the complex formed by Cyp-A and CsA. The knowledge of the fine specificity of human Cyps for CsA and of their expression levels may provide better insights into how CsA acts on its different target proteins in vivo.
We have investigated the role of the C-terminal cytoplasmic domain of the human PTH receptor in effector coupling. Following transient expression in COS-1 cells, coupling to both AC and PI-PLC was observed with the full-length receptor. Progressive C-terminal truncations did not dissociate activation of the two signalling systems. In stably transfected 293 cells, however, the full-length receptor as well as the majority of truncated constructs stimulated AC exclusively but failed to activate PI-PLC. Activation of both signalling systems was again observed following stable expression of a severely truncated receptor (R483) in 293 cells. In this case, pertussis toxin was also found to potentiate the cAMP response to hPTH-(1-38) significantly, indicating functional coupling of R483 to Gi proteins. Our results suggest that a core region of the human PTH receptor (first, second, third intracellular loop) can interact promiscuously with different G proteins and that the C-terminus of the full-length receptor directs the receptor towards an interaction with G~.Key words." Parathyroid hormone; Receptor; G protein; Adenylyl cyclase; Phosphoinositide 1, IntroductionPTH is a major regulator of bone and mineral metabolism. In its target cells, which are primarily osteoblasts and kidney proximal tubule cells, the hormone elicits a multitude of responses which are not readily explained solely on the basis of its capacity to stimulate AC (see [1,2] for review). Receptors for PTH have been cloned from opossum, rat, and human cDNA libraries [3][4][5][6]. These receptors bind PTH, PTH-related peptide, and N-terminal fragments of these hormones with nanomolar affinities. Only one PTH receptor gene exists in the mouse, rat, and human genome [7] and so far, there is no evidence lbr receptor subtypes mediating different physiological effects [5].Importantly, the rat and human receptor activate both cAMP and IP production following transient expression in COS cells [4,6]. Stimulation of both signal transduction systems has also been observed with the structurally related calcitonin receptor [8]. Studies presented in abstract form [9] concerning the full-length and a truncated opossum PTH receptor (OK-H, see Fig. 1) had suggested that regions within the C-terminus of the PTH receptor play an important role in the activation of PI turnover, whereas they are dispensable for the coupling to AC.Given these results, we set out to study the coupling specificity of the full-length human PTH receptor, which we had recently cloned from a kidney cDNA library [6], as well as a number of receptor constructs truncated in the C-terminal intracellular tail region. Coupling to AC and PI-PLC was inves-*Corresponding author. Fax: (41) (61) 324 4774.Abbreviations: AC, adenylyl cyclase; Gs and G~, stimulatory and inhibitory G protein of adenytyl cyclase, respectively; Gq, stimulatory G protein of PI-PLC; IP, inositol phosphate; PI, phosphoinositide; PI-PLC, phosphoinositide-specific phospholipase C; PTH, parathyroid hormone; hPTH, human parathyroi...
Background-Published data are contradictory about the importance of K-ras mutations in advanced tumours and are not available for early cancers. Aims-To establish whether specific K-ras mutations are prognostic markers in early stage colorectal adenocarcinoma. Methods-The presence of K-ras exon 1 mutations were correlated with tumour recurrence in two groups of patients: group 1 was a consecutive series of patients with resected colorectal adenocarcinoma at low risk of recurrence; group 2 were patients referred for chemotherapy after relapse of previously resected early stage tumours. K-ras mutations were detected by direct sequencing of whole tissue samples in all patients and in some, the leading edge and centre of the tumour were also microdissected out individually and sequenced. Results-Mutations were present in 26 (26.5%) of 98 patients in group 1; 14 patients developed a recurrence, four (28.5%) of whom had a K-ras mutation. Seventy nine patients have not developed tumour recurrence, 22 (28%) of whom had a mutation (p=0.84). K-ras mutations were present in five of 14 patients in group 2. Microdissection did not increase the number of mutations detected. Conclusions-Individual K-ras genotypes are distributed homogeneously throughout early stage colorectal adenocarcinomas, but detection of a mutation has no apparent prognostic value.
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