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At least 1,189 records · Page 66Linked to original sources
PubMed1998

Amino acid sequence of glutathione S-transferase rGSTM5* from rat testis.

Glutathione S-transferase rGSTM5* was isolated from rat testis with a combination of glutathione affinity column and reverse-phase column chromatography. The protein was digested with Achromobacter protease I or endoproteinase Arg-C. The peptide fragments were isolated for electrospray MS and N-terminal peptide sequencing analyses. The primary amino acid sequence of rGSTM5* comprises 217 residues and has a calculated average molecular mass of 25495.3 Da. The result is identical to that obtained for rGSTM5* with liquid chromatography-MS from a mixture of rat testicular GSTs. Therefore, rGSTM5* has not been post-translationally modified.

Amino Acid Sequence↗
PubMed1987

Fast mapping in normal and language-impaired children.

In this study, the fast mapping skills of a group of 11 normal children (ages 4:0-5:6) were compared to those of a group of 11 language-impaired children (ages 4:1-5:4) exhibiting expressive syntactic deficits. Fast mapping is a hypothesized process enabling children to create lexical representations for new words after as little as a single exposure. Subjects encountered a nonsense word and its novel object referent. Subsequent tasks probed the amount and kinds of information about the new word that the subjects had entered into memory. Normal and language-impaired subjects did not differ in their ability to infer a connection between the novel word and referent, to comprehend the novel word after a single exposure, and to recall some nonlinguistic information associated with the referent. However, the language-impaired subjects were less successful than the normal subjects in producing the new word, recalling significantly fewer of its three phonemes.

Child, Preschool↗
PubMed2001

Person-centred care for people with dementia: a quality audit approach.

This paper addresses the concept of person-centred care for people with dementia by consideration of an audit process using dementia care mapping as the audit tool. It is argued that this tool is best for identifying the lived experiences of the people in receipt of care. As a result it is able to identify the overall culture of care and its level of 'person-centred' approach. The audit was conducted on 12 units, half of which were day units and the others catering for inpatients. Five patients were mapped on each day for a 4-day period. The results give some idea of the quality of care and identify where improvement is necessary. Scores such as well-being values and the Dementia Care Index give clear signposts to the level of person-centred care and highlight where staff development is necessary. Recommendations are given to aid on-going planning.

Dementia↗
PubMed2001

Identification of antigenic domains on the human sodium-iodide symporter which are recognized by autoantibodies from patients with autoimmune thyroid disease.

The sodium-iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease. In the present study we have characterized the antigenic domains on the human symporter which are recognized by autoantibodies from patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH). Deletion derivatives of complementary DNA (cDNA) encoding the Na(+)/I(-) symporter were constructed using polymerase chain reaction (PCR) amplification. These deletion constructs were translated in vitro with the concomitant incorporation of [(35)S]methionine into the protein products. The reactivity of seven GD and six AH sera, which were known to contain symporter-binding antibodies, to each of the radiolabelled modified symporters was then determined in immunoprecipitation experiments. Analyses of the results obtained in the radiobinding assays suggest the existence of multiple antibody binding sites on human NIS (hNIS), including regions between amino acids (aa) 1--134, 191--286, 290--411, 411--520 and 520--588. Computer prediction of the potential B cell epitopes on the symporter revealed that, apart from aa 134--191, all the epitope domains identified overlapped, at least in part, with areas predicted to be highly antigenic. Interestingly, the antigenic domains represented by aa 191--286, 290--411 and 411--520 include regions of the polypeptide which form putative extracellular domains in the secondary structure model of the rat symporter. No correlation between the recognition of specific epitopes on the human symporter and the type of autoimmune thyroid disease was demonstrated.

Adult↗
PubMed2003

Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification.

The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin-related system (URM1-UBA4 ) and KTI11 enhances post-translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co-fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator-nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS- and karyopherin-dependent nuclear import.

Active Transport, Cell Nucleus↗
PubMed1997

Muscle patterning, differentiation and vascularisation in the chick wing bud.

Adult muscle is highly vascularised, with blood vessels being essential for adequate oxygenation of the tissue and for supporting increased metabolic demands. Whether this is the case during muscle development has not been examined. Resin histology was used to map the muscle splitting process and conventional transmission electron microscopy to examine early muscle differentiation at the midlimb level or later at the mid radius/ulna level in the chick wing bud from stages 24 (4.5 d) to 36 (10 d) (Hamburger & Hamilton, 1951). Microinjection of India Ink into the extra-embryonic vasculature was used to visualise the patent muscle microcirculation. The results showed that the premuscle masses are present at stage 24 and initial splitting of the muscle masses commences at stage 28. The final muscle pattern is not established until stage 36. At stage 26 the cells within the premuscle masses exhibited a mesenchymal morphology, but at stage 28 overt muscle differentiation was evident with myofibrils present within myoblasts. Undifferentiated mononucleated cells were interspersed with the differentiating myoblasts. The ratio of mononucleated cells:myoblasts decreased and the myoblasts became plumper and increasingly packed with myofibrils with age. There was no evidence of secondary myotube formation at any of the stages examined. Vascular invasion of the limb occurred at stage 35 just prior to the establishment of the final muscle pattern. This was surprising as it was assumed that myogenic differentiation would be both oxygen and nutrient dependent. The results of this study provide descriptions of the splitting of the premuscle masses through to the establishment of the final muscle pattern at the midlimb or mid radius/ulna level of the chick wing bud together with the differentiation of the myogenic cells within the developing muscles. However, the relationship between muscle patterning at the tissue level and muscle differentiation at the cellular level with vascularisation remains unclear. It is hoped that the results of the study may provide the basis for future investigations into mechanisms involved in muscle patterning and the signalling mechanisms for vascular invasion.

Animals↗
PubMed2005

UV photodissociation of the van der Waals dimer (CH3I)2 revisited: pathways giving rise to ionic features.

The CH(3)I A-state-assisted photofragmentation of the (CH(3)I)(2) van der Waals dimer at 248 nm and nearby wavelengths has been revisited experimentally using the time-of-flight mass spectrometry with supersonic and effusive molecular beams and the "velocity map imaging" technique. The processes underlying the appearance of two main (CH(3)I)(2) cluster-specific features in the mass spectra, namely, I(2)(+) and translationally "hot" I(+) ions, have been studied. Translationally hot I(+) ions with an average kinetic energy of 0.94+/-0.02 eV appear in the one-quantum photodissociation of vibrationally excited I(2)(+)((2)Pi(32,g)) ions (E(vib)=0.45+/-0.11 eV) via a "parallel" photodissociation process with an anisotropy parameter beta=1.55+/-0.03. Comparison of the images of I(+) arising from the photoexcitation of CH(3)I clusters versus those from neutral I(2) shows that "concerted" photodissociation of the ionized (CH(3)I)(2)(+) dimer appears to be the most likely mechanism for the formation of molecular iodine ion I(2)(+), instead of photoionization of neutral molecular iodine.

Journal Article↗
PubMed2003

Quantitative modeling of sensitivity in bacterial chemotaxis: the role of coupling among different chemoreceptor species.

We propose a general theoretical framework for modeling receptor sensitivity in bacterial chemotaxis, taking into account receptor interactions, including those among different receptor species. We show that our model can quantitatively explain the recent in vivo measurements of receptor sensitivity at different ligand concentrations for both mutant and wild-type strains. For mutant strains, our model can fit the experimental data exactly. For the wild-type cell, our model is capable of achieving high gain while having modest values of Hill coefficient for the response curves. Furthermore, the high sensitivity of the wild-type cell in our model is maintained for a wide range of ambient ligand concentrations, facilitated by near-perfect adaptation and dependence of ligand binding on receptor activity. Our study reveals the importance of coupling among different chemoreceptor species, in particular strong interactions between the aspartate (Tar) and serine (Tsr) receptors, which is crucial in explaining both the mutant and wild-type data. Predictions for the sensitivity of other mutant strains and possible improvements of our model for the wild-type cell are also discussed.

Bacterial Proteins↗
PubMed2003

Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor.

Recently, p53 was demonstrated to affect the expression of the insulin-like growth factor 1 receptor (IGF-1R), a receptor tyrosine kinase that plays a crucial role in growth and survival of cancer cells. However, the underlying mechanisms for interaction between p53 and IGF-1R are still not fully understood. One of the challenging questions remaining to be answered is why the wild-type p53, which per se represses the transcription of the IGF-1R gene, in overexpressed form is necessary for a high IGF-1R expression. In this study, we show that inhibition of p53 causes ubiquitination and down-regulation, through increased degradation, of the IGF-1R in human malignant melanoma cells. This effect, which was independent of the p53 status (i.e., wild type or mutated), was prevented if Mdm2 was coinhibited. Similar results were obtained in UV-irradiated human melanocytes (harboring wild-type p53), in which level of the IGF-1R increased after up-regulation of p53. Interestingly, the basal ubiquitination of the IGF-1R in untreated cells also depended on Mdm2. We could prove that Mdm2 physically associates with IGF-1R and that Mdm2 causes IGF-1R ubiquitination in an in vitro assay. Taken together our data provide evidence that Mdm2 serves as a ligase in ubiquitination of the IGF-1R and thereby causes its degradation by the proteasome system. Consequently, by sequestering Mdm2 in the cell nuclei, the level of p53 may indirectly influence the expression of IGF-1R. This role of Mdm2 and p53 represents an unexpected mechanism for the regulation of IGF-1R and cell growth.

3T3 Cells↗
PubMed2003

Promotion of G alpha i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP.

We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucine-rich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G protein-coupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an approximately 94-kDa membrane form. GIPN shows Zn2+- and E1/E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous G alpha i3 in HEK293 cells and reduces the half-life of overexpressed G alpha i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G alpha i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G alpha subunits through its RGS domain and to GIPN through its cysteine string motif.

Amino Acid Motifs↗
PubMed2002

Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway.

Precise regulation of the evolutionarily conserved hypoxia-inducible transcription factor (HIF) ensures proper adaptation to variations in oxygen availability throughout development and into adulthood. Oxygen-dependent regulation of HIF stability and activity are mediated by hydroxylation of conserved proline and asparagine residues, respectively. Because the relevant prolyl and asparginyl hydroxylases use O(2) to effect these posttranslational modifications, these enzymes are implicated as direct oxygen sensors in the mammalian hypoxic response pathway. Here we present the structure of factor-inhibiting HIF-1 (FIH-1), the pertinent asparaginyl hydroxylase involved in hypoxic signaling. Hydroxylation of the C-terminal transactivation domain (CTAD) of HIF by FIH-1 prevents CTAD association with transcriptional coactivators under normoxic conditions. Consistent with other structurally known hydroxylases, FIH-1 is comprised of a beta-strand jellyroll core with both Fe(II) and the cosubstrate 2-oxoglutarate bound in the active site. Details of the molecular contacts at the active site of FIH-1 have been elucidated and provide a platform for future drug design. Furthermore, the structure reveals the presence of a FIH-1 homodimer that forms in solution and is essential for FIH activity.

Amino Acid Sequence↗
PubMed2002

Studies on the function of oligosaccharyl transferase subunits: a glycosylatable photoprobe binds to the luminal domain of Ost1p.

Oligosaccharyl transferase (OT) is a complex multisubunit enzyme that, in the case of Saccharomyces cerevisiae, contains nine different transmembrane proteins. One of our goals is to identify the OT subunit(s) responsible for recognizing the consensus sequence, -Asn-X-ThrSer-, and catalyzing the oligosaccharide transfer reaction. By using a substrate-based photoprobe, earlier we found that Ost1p was specifically linked to the radiolabeled photoprobe. We have now examined Ost1p in more detail. Deletion of the cytoplasmic tail of Ost1p caused no defects in growth and glycosylation. In addition, replacement of the transmembrane domain with other hydrophobic amino acids did not impair growth. In contrast, a construct containing only the luminal domain of Ost1p did not support cell growth. Given these observations, we concentrated on studying the luminal domain of Ost1p and localized the photoprobe attachment region within a sequence of nine amino acid residues. Because mutations in the photoprobe attachment region did not cause any severe growth or glycosylation defects, we conclude that this region is not involved in the recognition of the N-glycosylation site. By further mutagenesis of the conserved residues of Ost1p we conclude that the luminal domain mediates interactions with other subunits of OT and becomes labeled because of its proximity to the recognition andor catalytic subunit in the OT complex, Stt3p.

Binding Sites↗
PubMed1987

Identification of an acetylation site of Chlamydomonas alpha-tubulin.

An acetylation site of Chlamydomonas axonemal alpha-tubulins was identified near, or within, the binding site of 6-11B-1, a monoclonal antibody specific for posttranslationally acetylated alpha-tubulins. In a first approach, axonemal proteins were hydrolyzed by formic acid, cyanogen bromide, or chymotrypsin and analyzed with immunoblots. The smallest alpha-tubulin peptide retained on nitrocellulose and containing antibody-binding site(s) was found to span amino acids 37-138 (alpha 37-138). A smaller antibody-binding peptide, identified as alpha 25-50, was obtained by complete digestion of alpha-tubulin with chymotrypsin. This fragment was purified by reversed-phase HPLC and assayed by its ability to bind 6-11B-1 in solution. Determination of the amino acid sequences of alpha 37-138 and alpha 25-50 showed that residue 40 in axonemal alpha-tubulin is epsilon N-acetyllysine. A sequence very similar to Chlamydomonas alpha 25-50 is found in the majority of alpha-tubulins analyzed so far. However, the corresponding region is markedly divergent in some alpha-tubulin isoforms from chicken, Drosophila, and yeast.

Acetylation↗
PubMed1990

Posttranslational modification of class III beta-tubulin.

The charge heterogeneity of class III beta-tubulin (beta III) during neural development was analyzed by high-resolution isoelectric focusing/two-dimensional polyacrylamide gel electrophoresis in combination with site-specific proteolytic digestion and immunological detection. The number of beta III isoforms (charge variants) gradually increases from one in embryonic brain to seven in adult brain. All of the charge heterogeneity is due to posttranslationally modified sites located within the extreme C-terminal region of the beta III polypeptide. One beta III isoform is present in testis, the only other tissue in which this isotype is expressed. The testis beta III isoform cofocuses with the earliest-appearing embryonic brain beta III charge variant. Our results indicate that the posttranslational modifications of beta III are developmentally regulated, occur at more than one site, and are neuron-specific. The location of these modifications within the extreme C-terminal domain suggests that their function is to modulate the interaction of tubulin with microtubule-associated proteins.

Aging↗
PubMed1991

Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.

Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major products, named gp35 and gp70, were shown to be transported into microsomes and heavily glycosylated, suggesting that the processing events are partly mediated by the signal peptidase of the endoplasmic reticulum. The other two products, p19 and p21, were probably associated with the outer surface of the microsomal membrane. Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino-terminal amino acid sequences of gp35 and gp70 indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH2-p22-gp35-gp70-p19. Both gp35 and gp70 could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.

Acetylglucosaminidase↗
PubMed1991

Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.

Animals↗
PubMed1991

Characterization of a posttranslational fucosylation in the growth factor domain of urinary plasminogen activator.

A posttranslational modification site in natural and recombinant urinary-type plasminogen activators (urokinases; EC 3.4.21.31) has been localized to Thr-18, in the growth factor domain of the molecule. This is the region of urinary plasminogen activator responsible for its specific receptor binding. An unusual carbohydrate-protein linkage, a single monosaccharide, fucose, covalently attached directly to threonine in the peptide, is described here. The glycan moiety and the site of modification have been identified with mass spectrometry and confirmed by carbohydrate composition analysis, Edman degradation, and one- and two-dimensional NMR studies. This type of modification is normally not detected without mass spectrometry because the fucose-threonine bond is hydrolyzed under standard acidic conditions of the amino acid analysis and Edman sequencing. This modification may be widely found in other proteins.

Amino Acid Sequence↗