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Query: UNIPROT:P06889 (Mol)
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Human diploid fibroblasts, strain MRC-5, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) in the S phase of the cell cycle. The frequency of TGR HPRT- cells was increased up to 20-fold in comparison to control untreated cultures. Representative TGR clones were unable to grow in HAT, and these were treated with 5-azacytidine (5-aza-CR). In many cases subsequent growth in HAT medium was observed, but in others it is likely that the cells had run out of growth potential. The results provide the first evidence of the silencing and reactivation of a gene in normal diploid mammalian cells.
Somat Cell Mol Genet 1995 May
PMID:Evidence for gene silencing by DNA methylation in normal human diploid fibroblasts. 748 35

To study the relationships between mutagenesis and carcinogenesis, we compared the mutations and their frequency induced by ultraviolet irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in nucleotide excision repair. Whereas XP-D patients develop early skin tumors, TTD patients do not exhibit abnormal levels of cancers. After verification of UV hypersensitivity and DNA repair defect of the immortalized cell lines XP-D and TTD compared with a wild-type cell line, UV-induced mutagenesis was studied with a new shuttle vector pR2, carrying the target lacZ' gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in chiefly on the cytosine at 5'-TC-3' sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5'-TCPur-3' sites, while mutations in normal and TTD cells were mostly at 5'-TCC-3' sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines. Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.
J Mol Biol 1995 Oct 06
PMID:Characteristics of UV-induced mutation spectra in human XP-D/ERCC2 gene-mutated xeroderma pigmentosum and trichothiodystrophy cells. 756 73

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex, and this expression was clearly increased by a single intraperitoneal administration of calcium chloride solution (5-15 mg Ca/100 g body weight) in rats; this increase was remarkable at 60-120 min after the administration. Thyroparathyroidectomy (TPTX) caused a slight decrease of regucalcin mRNA levels in the kidney cortex. However, the administration of calcium (10 mg/100 g) in TPTX rats produced a clear increase of regucalcin mRNA levels in the kidney cortex. The subcutaneous administration of calcitonin (10-100 MRC mU/100 g) or parathyroid hormone [1-34] (1-10 U/100 g) in TPTX rats which received calcium (10 mg/100 g) administration did not cause an appreciable alteration of regucalcin mRNA levels in the kidney cortex, suggesting that the mRNA expression is not stimulated by calcium-regulating hormones. The administration of trifluoperazine (TFP; 5 mg/100 g), an inhibitor of Ca2+/calmodulin action, completely blocked the expression of regucalcin mRNA stimulated by calcium administration. Now, calcium content in the kidney cortex was significantly elevated by a single intraperitneal administration of calcium (10 mg/100 g) in rats. The present study clearly demonstrates that the expression of regucalcin mRNA in the kidney cortex is stimulated by calcium administration in rats. This expression may be mediated through Ca2+/calmodulin action in the kidney cortex.
Mol Cell Biochem 1995 May 10
PMID:Expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats: the stimulation by calcium administration. 765 81

The involvement of a hypocalcemic hormone calcitonin (CT) in the expression of hepatic Ca(2+)-binding protein regucalcin mRNA was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). A single oral administration of calcium chloride (100 mg Ca/100 g body weight) to rats induced a remarkable increase in the serum calcium concentration and a corresponding elevation of the liver calcium content during 120 min after the administration. Thyroparathyroidectomy (TPTX) did not cause a significant increase in the liver calcium content after calcium administration. Hepatic regucalcin mRNA level was markedly elevated by calcium administration; the level was about 180% of controls at 60 min after the administration. This increase was completely abolished by TPTX. A single subcutaneous administration of CT (synthetic eel CT; 25-100 MRC mU/100 g) to TPTX rats received oral administration of calcium (100 mg/100 g) produced a remarkable increase in hepatic regucalcin mRNA levels; the level was about 280% of controls with the dose of 25 MRC mU CT/100 g. The present finding suggests that the expression of hepatic mRNA is stimulated by CT, and that the hormonal effect is mediated through Ca2+ in rat liver.
Mol Cell Biochem 1994 Jul 13
PMID:Expression of calcium-binding protein regucalcin mRNA in rat liver is stimulated by calcitonin: the hormonal effect is mediated through calcium. 785 30

Epileptic temporal and parietal cortices, removed from 6 patients with therapy-resistant (intractable) partial epilepsy (TRPE) during neurosurgery, were studied. Neurons (40-50 in each slice) in laminae I-VI and white matter were injected with Lucifer Yellow (LY). Samples were examined in a confocal laser scanning microscope (BioRad [Richmond, CA] MRC 600), and individual cells were scanned at 0.1-2 microns incremental levels. 2D maximal linear projection was used for overview. Frames (50-60) of scanned neurons were transformed into 3D volumes, using VoxelView software on a Silicone Graphics workstation, and rotated. All samples contained pyramidal neurons with duplicated apical dendrites, additional basal dendrites, or were misplaced in a horizontal position in the white matter. Rarely were such cells observed in normal cases. The relation between the observations and the disease is discussed. The attempt to simultaneously apply immunofluorescence was successful concerning synaptic vesicle antigens. This approach will be used for a detailed study of the synaptology of this disease.
Mol Neurobiol
PMID:Morphological aberrations in therapy-resistant partial epilepsy (TRPE). Confocal laser scanning and 3D reconstructions of Lucifer Yellow injected atypical pyramidal neurons in epileptic human cortex. 788 1

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.
Mol Cell Biol 1993 Feb
PMID:Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70. 842 8

I describe the current version of the sequence analysis package developed at the MRC Laboratory of Molecular Biology, which has come to be known as the "Staden Package." The package covers most of the standard sequence analysis tasks such as restriction site searching, translation, pattern searching, comparison, gene finding, and secondary structure prediction, and provides powerful tools for DNA sequence determination. Currently the programs are only available for computers running the UNIX operating system. Detailed information about the package is available from our WWW site: http:@www.mrc-lmb.cam.ac.uk/pubseq/.
Mol Biotechnol 1996 Jun
PMID:The Staden sequence analysis package. 883 29

Our laboratory has previously characterized a keratan sulfate proteoglycan, named claustrin, and shown by molecular cloning that claustrin and the mouse MAP1B protein share high homology, with claustrin representing a 5'-truncated fragment of MAP1B. In the present study, we examine further the relationship between claustrin and MAP1B, and also describe the isolation of a cDNA encoding the 3'-region of MAP1B, which shares 3'-untranslated sequence, but not coding sequence, with claustrin. We call this partial cDNA 3'-MAP1B-related clone (3'-MRC), since it is homologous to the 3'-region of the mouse MAP1B sequence. We show by Northern analysis that distinct mRNAs are recognized by the claustrin and 3'-MRC cDNAs, and by RT-PCR that mRNAs encoding these distinct MAP1B-related molecules are present in embryonic chick brain and cardiac and smooth muscle. Our data also suggest a higher level of expression of claustrin mRNA in astrocyte cultures, when compared to 3'-MRC. Our data therefore provide new evidence that alternatively spliced variants of MAP1B are expressed in brain, and that at least one of these variants encodes the claustrin proteoglycan.
J Mol Neurosci 1997 Dec
PMID:An alternatively spliced, 5'-truncated MAP1B isoform is expressed in the developing chick nervous system. 948 19

Dialysis of rhodopsin isolated from bovine rod outer segments resulted in the formation of a new two-dimensional crystal form suitable for electron crystallography. The crystals obtained were tubular or single layers and showed p22121 symmetry (a=60.6(+/-0.8) A, b=86.3(+/-1.6) A). For the first time the size and order of the crystals allowed us to take electron diffraction patterns showing spots to a resolution of about 3.5 A. Images were recorded at liquid nitrogen temperature using a high voltage field emission electron microscope. Out of a large number of images 20 crystalline areas were selected and processed with the MRC image processing software. A projection structure of bovine rhodopsin to 5 A resolution was calculated using amplitudes and phases extracted from these images. The achieved resolution exceeds the resolution of all previously obtained structures of frog, bovine and squid rhodopsin crystals. In this map small differences are observed compared to the previous maps. Helix 5 seems to be even more highly tilted and between the arc-shaped feature and helix 5 a peak is present suggesting that helix 3 is prolonging this feature towards helix 5. These observations are in agreement with the latest model for the three-dimensional arrangement of rhodopsin. The resolution achieved as well as the availability of electron diffraction data suggest that there is a good possibility to collect data from tilted crystals and calculate an improved three-dimensional structure of rhodopsin.
J Mol Biol 1998 Oct 09
PMID:Characterisation of an improved two-dimensional p22121 crystal from bovine rhodopsin. 975 49

Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.
Mol Immunol 1998 Jun
PMID:Human complement factor I: its expression by insect cells and its biochemical and structural characterisation. 980 78


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