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PubMed2000

Expression of the organizer specific homeobox gene goosecoid (gsc) in porcine embryos.

The homeobox gene goosecoid is one of the first genes expressed in the organizer region of vertebrates and specifies future dorsal regions along the anterior/posterior axis of the embryo. Goosecoid (gsc) expression marks the posterior end of the anterior/posterior axis and might be a good marker to visualise early events in embryonic axis formation and differentiation processes in the epiblast at the onset of gastrulation. The aim of the present study was to evaluate gsc expression in porcine embryos. For this the homeobox containing region of the porcine gsc was isolated using RT-PCR. The sequence of the PCR product appeared to be highly homologous to the sequence in the mouse, human, and chicken. We concluded that the isolated region represents part of the porcine gsc messenger. Relative levels of gsc expression were estimated in porcine embryos from day 9 to day 12 of pregnancy. Gsc was expressed in embryos of all ages and localisation on one side of the embryoblast was demonstrated with in situ hybridisation on whole- mount embryos at day 10 of pregnancy. In embryos collected at day 13 of pregnancy gsc expression was localised anterior to the primitive streak. The correlation between embryo size and level of gsc expression was low. Levels and pattern of expression varied within and between litters collected at similar days of pregnancy. It is concluded that gsc expression can be used as an early marker of differentiation and to describe embryo diversity in the pig.

Amino Acid Sequence↗
PubMed2002

[Clinical examination of menisci in the era of arthroscopy].

PURPOSE OF THE STUDY: A profound clinical history and examination in case of a soft-tissue injury of the knee joint has a principal significance for the diagnosis and the following treatment. Our aim was to compare the development of surgery of the soft-tissue injury of the knee in our region within a 25-year interval. The most frequently affected structures in the knee joint are the menisci. We have focussed on the evaluation of the extent to which selected clinical history and symptoms are actually associated with the affection of menisci. MATERIAL: First we compared groups of patients operated on for the soft-tissue injury of the knee joint within a 25-year interval. The scope of the care in the out-patient department was in this period almost the same. Then we examined a group of patients with a suspect tear of menisci and focussed on the clinical history and findings which are most often related to the injuries of menisci. METHODS: In a retrospective study aimed at the acquisition of sufficient statistical data and concentrated on the development of the surgery of the soft-tissue knee we compared a group of 91 patients operated on by arthrotomy in the period of 1973-1975 with a group of 780 patients operated on in the years 1996-98 by arthroscopy. In the subsequent prospective study we examined 104 patients sent to arthroscopic examination for a suspect tear of the medial or lateral meniscus. Based on the performed arthroscopy we divided the patients into three group--with the injured medial meniscus, with the injured lateral meniscus and without affection of meniscus. We investigated the diagnostic value of clinical history and clinical symptoms. RESULTS: In the retrospective study comparing the groups of patients in the 25-year interval the number of knee joints operated on increased 9times, the average age decreased by 8 years and the duration of hospitalisation was reduced by 9 days, the period of after-treatment in the out-patient department was reduced by 59 days. In the prospective study focussed on the lesion of meniscus the success rate of clinical history and examinations was 62% in lesions of the medial meniscus, 47% in lesions of the lateral meniscus. Significant in the clinical history of both menisci was a joint effusion and click phenomenon in the knee joint. Of clinical symptoms relating to lesion of the medial meniscus there often (above 60%) occurred pain of the medial joint line, painful bend and positive Steinmann II test, in case of the lateral meniscus painful bend and inability of full bend (nebo flexion--co má autor na mysli termínem "dotazení?) and walking in bend. Their disadvantage is a low specificity. On the contrary lower sensitivity but higher specificity is recorded by the clinical history of the locked knee and the presence of click phenomena during clinical examination. Simultaneous affection of the cartilage of the medial condyle of the femur in the lesion of the medial meniscus was in 64% of cases and of the lateral condyle of the femur in the lesion of the lateral meniscus in 47% of cases. DISCUSSION: Opinion on the clinical history and examination of the injured knee joint has not changed substantially during 25 years as shown by the comparison of the literature, but arthroscopy thanks to its visualisation of the joint cavity and a lower invasiveness contributed to the improvement of the diagnostics of the lesions of the knee joint, a less invasive surgery resulting in a reduced period of treatment. As compared to other authors we achieved in the evaluation of clinical history and examination practically the same success rate of diagnosis in the lesion of the medial meniscus but a lower success rate in the lesion of the lateral meniscus. As concerns clinical history we did not find any significant differences in comparison with other authors, in case of clinical examination we did not achieve such good results in case of McMurray test and as the most significant manoeuvre we consider the Steinmann II test for the medial meniscus. Frequent simultaneous injury of the cartilage and menisci corresponds to the findings of other authors. CONCLUSION: Indication for surgery for the affection of menisci therefore has to be based on a careful analysis of clinical history and examination of the patient, it is not possible to rely on any of the menisci symptoms and their significance is only supportive. As the most significant symptoms we assess the clinical history of the verified knee swelling, click phenomena and locked knee in the knee joint, in the clinical examination pain and click phenomena of the joint lines.

Adolescent↗
PubMed2001

A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands.

The majority of biologically active proteins are glycosylated, therefore any approach to proteomics which fails to address the analysis of oligosaccharides is necessarily incomplete. To appreciate the structure of a glycoprotein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are available at the low microgram level or less has involved increasing the sensitivity of the technology for glycan analysis. Since one protein may have many different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at one N-linked glycosylation site. Applications of recently developed technology suggest that it is now becoming realistic to extend the proteomics analysis of glycoproteins to include details of glycosylation. This is achieved by releasing the N-glycans from the protein in a gel by optimised peptide-N-glycosidase F digestion. The released glycans are then tagged with the fluorophore, 2-amino benzamide. The labelled glycan pools (containing 50-100 femtomoles of glycans) are resolved by predictive normal phase high performance liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary matrix-assisted laser desorption/ionization-mass spectrometry, which requires 10-20 times as much sugar for a single run, can be used where there is sufficient material. This provides a composition analysis but not linkage information.

Amidohydrolases↗
PubMed2000

Affinity profiles for human somatostatin receptor subtypes SST1-SST5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use.

In vivo somatostatin receptor scintigraphy using Octreoscan is a valuable method for the visualisation of human endocrine tumours and their metastases. Recently, several new, alternative somatostatin radioligands have been synthesised for diagnostic and radiotherapeutic use in vivo. Since human tumours are known to express various somatostatin receptor subtypes, it is mandatory to assess the receptor subtype affinity profile of such somatostatin radiotracers. Using cell lines transfected with somatostatin receptor subtypes sst1, sst2, sst3, sst4 and sst5, we have evaluated the in vitro binding characteristics of labelled (indium, yttrium, gallium) and unlabelled DOTA-[Tyr3]-octreotide, DOTA-octreotide, DOTA-lanreotide, DOTA-vapreotide, DTPA-[Tyr3]-octreotate and DOTA-[Tyr3]-octreotate. Small structural modifications, chelator substitution or metal replacement were shown to considerably affect the binding affinity. A marked improvement of sst2 affinity was found for Ga-DOTA-[Tyr3]-octreotide (IC50 2.5 nM) compared with the Y-labelled compound and Octreoscan. An excellent binding affinity for sst2 in the same range was also found for In-DTPA-[Tyr3]-octreotate (IC50 1.3 nM) and for Y-DOTA-[Tyr3]-octreotate (IC50 1.6 nM). Remarkably, Ga-DOTA-[Tyr3]-octreotate bound at sst2 with a considerably higher affinity (IC50 0.2 nM). An up to 30-fold improvement in sst3 affinity was observed for unlabelled or Y-labelled DOTA-octreotide compared with their Tyr3-containing analogue, suggesting that replacement of Tyr3 by Phe is crucial for high sst3 affinity. Substitution in the octreotide molecule of the DTPA by DOTA improved the sst3 binding affinity 14-fold. Whereas Y-DOTA-lanreotide had only low affinity for sst3 and sst4, it had the highest affinity for sst5 among the tested compounds (IC50 16 nM). Increased binding affinity for sst3 and sst5 was observed for DOTA-[Tyr3]-octreotide, DOTA-lanreotide and DOTA-vapreotide when they were labelled with yttrium. These marked changes in subtype affinity profiles are due not only to the different chemical structures but also to the different charges and hydrophilicity of these compounds. Interestingly, even the coordination geometry of the radiometal complex remote from the pharmacophoric amino acids has a significant influence on affinity profiles as shown with Y-DOTA versus Ga-DOTA in either [Tyr3]-octreotide or [Tyr3]-octreotate. Such changes in sst affinity profiles must be identified in newly designed radiotracers used for somatostatin receptor scintigraphy in order to correctly interpret in vivo scintigraphic data. These observations may represent basic principles relevant to the development of other peptide radioligands.

Animals↗
PubMed1998

Visualisation of non-glycosylated somatostatin receptor two (ngsst2) immunoreactivity in the rat central nervous system.

The biological actions of the neuropeptides somatostatin-14 and -28 are receptor-mediated. To date, five G protein-coupled receptors sst1 to sst5 have been characterised pharmacologically and their genes have been cloned. In this study, we used an affinity-purified polyclonal antibody (AS-68) raised against a specific N-terminal peptide sequence of sst2 to localise N-terminal sst2-immunoreactive regions in the rat brain and the cervical spinal cord. The specificity of the antiserum was demonstrated by Western and slot blotting experiments using a N-terminal sst2 fusion protein. Further blotting experiments with a sst2(A)-transfected cell line and rat CNS membrane proteins showed that the antibody detected the non-glycosylated and/or non-sialated receptor. A strong signal using an sst2(A)-transfected CHO-K1 cell line was obtained only if the cells had been treated with N-Glycosidase F prior to the immunochemical detection. Two variants of sst2 (sst2(A) and sst2(B)) have been identified by cloning procedures and gene expression studies in the rodents. They differ in their carboxy-termini: AS-68 would, however, be able to recognise the non-glycosylated form of both these variants. We present here the central nervous system distribution of non-glycosylated sst2-immunoreactivity in the rat using this N-terminal antibody. The sst2 non-glycosylated N-terminal like immunoreactivity was distributed throughout the brain with cells and processes labelled in the cerebral cortex and the basal ganglia (neostriatum, substantia nigra), in the limbic system (hippocampal formation, amygdala), in the diencephalon (epithalamus, thalamus, hypothalamus), the superior colliculus, the periaqueductal grey matter and some of the reticular formation nuclei. The distribution of the non-glycosylated sst2-like immunoreactivity detected here was consistent with that predicted from the localisation of sst2 mRNA and SRIF-ligand binding studies.

Amino Acid Sequence↗
PubMed2001

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody.

Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 x 10(-2) s-1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (gamma 1) hinge region and CH3 domain. This "minibody" was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting.

Amino Acid Sequence↗
PubMed1995

Interaction of human adrenomedullin 13-52 with calcitonin gene-related peptide receptors in the microvasculature of the rat and hamster.

1. Adrenomedullin (ADM), a recently discovered circulating hypotensive peptide, shares limited sequence homology with the sensory nerve-derived vasodilator, calcitonin gene-related peptide (CGRP). This study compared the vasodilator effect of sequence 13-52 of human adrenomedullin (ADM13-52) with that of human alpha CGRP (CGRP), in the microvasculature of the hamster cheek pouch and rat skin in vivo. 2. Single arterioles (20-40 microns diameter) in the hamster cheek pouch were visualised by intravital microscopy and video recording, and measured by image analysis. Both ADM13-52 (1 pmol-0.4 nmol) and CGRP (0.1 pmol-1 nmol) evoked dose-related increases in the diameter of preconstricted arterioles (n = 6). ADM13-52 (ED50 14 pmol) was 20 fold less active than CGRP (ED50 0.71 pmol). The kinetics of onset and decline of vasodilator responses to both peptides were similar, with vasodilator responses to both peptides reaching a maximum at ca. 2 min, and reversing after 10-15 min (n = 5-7). The submaximal increase in blood flow evoked by ADM13-52 was significantly inhibited (P < 0.05; n = 6) by the CGRP1 receptor antagonist, CGRP8-37, at a dose (300 nmol kg-1, i.v.) that we have previously shown to inhibit significantly equivalent vasodilator responses to CGRP in this preparation. 3. In experiments measuring changes in local blood flow in rat skin by a 133xenon clearance technique, intradermal injection of both ADM13-52 (3-300 pmol) and CGRP (0.1-30 pmol) evoked dose-related increases in local blood flow. ADM13-52 (ED50 27 pmol) was 17 fold less potent than CGRP (ED501.6 pmol) (n = 6). The submaximal increase in blood flow evoked by both peptides was significantly inhibited (P<0.02; n = 5) by CGRP837 (100 nmol kg-1, i.v.).4. We conclude that ADM13-52 is a potent vasodilator in the microvasculature of the hamster and rat invivo. It mediates its vasodilator effect by arteriolar dilatation and this effect is due, at least in part, to the stimulation of CGRPI receptors.

Adrenomedullin↗
PubMed2006

Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy.

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T(166), that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.

Amino Acid Sequence↗
PubMed2003

A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription.

In vertebrates free messenger ribonucleoprotein (RNP) particles and polysomes contain an abundant Y-box protein called p50 (YB-1), which regulates translation, presumably by affecting the packaging of the RNA. Here, we have identified a p50-like protein in the dipteran Chironomus tentans and studied its relation with the biogenesis of mRNA in larval salivary glands. The salivary gland cells contain polytene chromosomes with the transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate a transcription product, a large RNP particle, which can be visualised (with the electron microscope) during its assembly on the gene and during its transport to and through the nuclear pores. The p50-like protein studied, designated Ct-p40/50 (or p40/50 for short), was shown to contain a central cold-shock domain, an alanine- and proline-rich N-terminal domain, and a C-terminal domain with alternating acidic and basic regions, an organisation that is characteristic of p50 (YB-1). The p40/50 protein appears in two isoforms, p40 and p50, which contain 264 and 317 amino acids, respectively. The two isoforms share the first 258 amino acids and thus differ in amino-acid sequence only in the region close to the C-terminus. When a polyclonal antibody was raised against p40/50, western blot analysis and immunocytology showed that p40/50 is not only abundant in the cytoplasm but is also present in the nucleus. Immunolabelling of isolated polytene chromosomes showed that p40/50 appears in transcriptionally active regions, including the BRs. Using immunoelectron microscopy we revealed that p40/50 is added along the nascent transcripts and is also present in the released BR RNP particles in the nucleoplasm. Finally, by UV crosslinking in vivo we showed that p40/50 is bound to both nuclear and cytoplasmic poly(A) RNA. We conclude that p40/50 is being added cotranscriptionally along the growing BR pre-mRNA, is released with the processed mRNA into the nucleoplasm and probably remains associated with the mRNA both during nucleocytoplasmic transport and protein synthesis. Given that the p40/p50 protein, presumably with a role in translation, is loaded onto the primary transcript concomitant with transcription, an early programming of the cytoplasmic fate of mRNA is indicated.

Amino Acid Sequence↗
PubMed2005

Isolation of C15: a novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow.

Adult bone marrow stroma contains a source of mesenchymal stem cells (MSC) that have the capacity to self-renew and differentiate into multiple stromal lineages. These rare cells can be visualised indirectly by the formation of heterogeneous colonies, containing stem cells and their differentiated progeny in long-term culture. If MSC and their associated progenitor and precursor populations are to reach their full therapeutic potential, markers will be required to identify and characterize specific bone marrow stromal subsets. We sought to use phage display to generate antibodies against bone marrow mononuclear cells (BMMNC) enriched for colony forming cells. Initially, we identified our target cell population by comparing the colony forming efficiency (CFE) of CD49a-positive, STRO-1-positive and CD45-negative BMMNC subpopulations with unseparated BMMNC. Selection with anti-CD49a gave the greatest enrichment (19-fold) of colony forming cells and in light of these findings, we generated phage antibodies against CD49a-positive BMMNC by simultaneous positive/negative selection. A dominant clone (C15), generated after 3 rounds of selection, has been isolated and sequenced, then characterized for cell and tissue specificity. Sequence analysis showed that the V(H) and V(L) gene segments of C15 aligned most closely to the VH26/DP-47 and IGLV3S1/DPL16 germline V segments found in the synthetic repertoire. C15 bound to 4% of freshly isolated BMMNC and localized to osteoblastic cells and proximal marrow cells in areas of active bone formation in sections of osteophyte. C15 binding was upregulated in cultured bone marrow stromal cells (BMSC) and was also detected on bone-derived cell lines. This report demonstrates that phage display is a powerful tool for the isolation of antibodies against rare cell populations, and provides a platform for the future application of this technology in the search for antigens on MSC and other rare cell populations.

Adult↗
PubMed1999

Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression.

AIMS: To identify the NOV protein detected by immunofluorescence in the nucleus of human cancer cell lines to establish whether targeting to the nucleus reflects dual paracrine and intracrine biological functions of NOV, as has been reported previously for several signalling peptides and proteins. METHODS: Nuclear and cytoplasmic fractions were prepared from 143 and HeLa cells in which nuclear NOV protein was detected. Western blotting analysis of NOV proteins in both types of fractions was performed using two NOV specific antibodies. Confocal microscopy was used to visualise the nuclear NOV protein in HeLa and 143 cells. A yeast two hybrid screening system was used to isolate cDNAs encoding proteins able to interact with the human NOV protein. RESULTS: A 31/32 kDa doublet of NOV protein was identified in the nuclear fraction of 143 and HeLa cells. Because the antibodies were directed against the C-terminus of NOV, the 31/32 kDa NOV isoform is probably truncated at the N-terminus and might correspond to the secreted 32 kDa NOV isoform detected in cell culture medium. Confocal microscopy indicated that in addition to the cytoplasmic NOV protein already identified, a nuclear NOV protein was present in both the nucleoplasm and nucleoli of Hela and 143 cells. Screening of cDNA libraries prepared from HeLa cells, Epstein-Barr virus transformed lymphocytes, and normal human brain showed that the NOV protein interacts with the rpb7 subunit of RNA polymerase in a yeast two hybrid system. CONCLUSIONS: The NOV protein detected in the nucleus of 143 and HeLa cells is probably an N-terminus truncated isoform of the secreted 48 kDa NOV protein. A growing body of evidence suggests that novH expression is closely associated with differentiation in normal human tissues and that the nov gene encodes a signalling protein that belongs to an emerging family of cell growth regulators. The nuclear localisation of a NOV isoform potentially provides an additional degree of signalling specificity. The interaction of the NOV protein and the rpb7 subunit of RNA polymerase II in the two hybrid system suggests that NOV might be involved in regulating gene expression at the transcriptional level. As has already been suggested for several other nuclearly located cytokines, the NOV protein does not contain a typical nuclear localisation signal. Therefore, it is possible that it combines with either a receptor or a chaperone during its translocation. Disruption of the balance between the secreted and nuclear NOV isoforms might affect the putative autocrine and paracrine functions of NOV and might be of considerable importance in the development of cancers in which the expression of novH has been shown to be impaired.

Base Sequence↗