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Biomedical subjects

N Groome

Publications and source records attributed to N Groome.

At least 19 recordsLinked to original sources
PubMed1995

Protein expression of the alpha, gamma, delta and epsilon subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum.

Total protein kinase C (PKC) activity and protein expression of the alpha and delta subspecies of PKC increases markedly as C6 glioma cells grow from low cell density to the contact-inhibited quiescent state (also known as G(o)) in the presence of serum. At the same time protein expression of PKC subspecies gamma and epsilon decreases while the beta I, beta II, iota and zeta subspecies did not change. Serum deprivation of growing C6 glioma cells does not induce the same changes in PKC subspecies protein expression. The findings support the growing view that there are significant differences between the G(o) states brought about by contact inhibition or serum deprivation.

Blood↗
PubMed1995

A novel protein, amyloid precursor-like protein 2, is present in human brain, cerebrospinal fluid and conditioned media.

A monoclonal antibody, 3B11, was raised to a novel protein, amyloid precursor-like protein 2, which did not recognize amyloid precursor protein. Multiple bands were detected in human brain fractions and cell lysate by Western blotting, indicating the presence of isoforms, 3B11 immunoreactivity was also detected in cerebrospinal fluid and conditioned medium, indicating that the protein is secreted. Immunocytochemistry revealed 3B11 immunoreactivity in sections of human brain.

Amino Acid Sequence↗
PubMed1995

Identification of biologically active inhibin in the peritoneal fluid of women.

PURPOSE: Immunoreactive inhibin (i-inhibin) has been reported to be present in the peritoneal fluid of women. The radioimmunoassay employed measures free, biologically inactive alpha-subunits(s) equally as well as dimeric, biologically active inhibin. The present study was designed to determine if biologically active, dimeric inhibin is present in the peritoneal fluid of women. METHODS: Peritoneal fluid of four women was assayed by radioimmunoassay, a sheep pituitary bioassay, and two ELISA procedures which utilized specific monoclonal antibodies for the "capture" of the alpha-subunit (ELISA-A) or the beta-subunit (ELISA-B) of inhibin and subsequent quantification of dimeric inhibin-A. RESULTS: There was a good correlation between the values obtained by radioimmunoassay, bioassay, and both ELISAs; two samples (from the late follicular phase) with relatively high i-inhibin concentrations were positive in all four assays, whereas two samples (from the early follicular phase) with very low i-inhibin concentrations were negative in the bioassay and ELISAs. CONCLUSION: A significant portion of the immunoreactive inhibin in the peritoneal fluid obtained during the late follicular phase of women is dimeric, biologically active inhibin. We speculate that this may have potential implications for oocyte maturation and early embryogenesis within the oviduct.

Animals↗
PubMed1995

Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr98.

Phosphorylation is one of a number of post-translational modifications resulting in charge microheterogeneity of myelin basic protein (MBP). This phosphorylation is claimed to destabilise the compact myelin sheath by decreasing the interaction of membrane bilayers, thereby creating or maintaining pockets of cytoplasm. To further investigate and localise MBP phosphorylation to discrete regions of the myelin sheath we raised a monoclonal antibody with specificity for a known phosphorylation site in MBP. A synthetic peptide was made by Fmoc peptide chemistry and phosphorylation of Thr98 was achieved on the resin by the global phosphorylation methodology, utilising dibenzyl-N,N-diethylphosphoramidite phosphitylation and t-butylhydroperoxide oxidation. The peptide coupled to tuberculin was used to immunise mice for monoclonal antibody production. The selected hybridoma (Clone P12) secreted an IgG2a antibody which reacted strongly with the phosphorylated immunogen and with phosphorylated fractions of bovine MBP obtained by ion exchange chromatography. The antibody had minimal reactivity with the unphosphorylated peptide; the same peptide phosphorylated at another site Ser102; a preparation of unphosphorylated MBP obtained by ion exchange chromatography; and with an irrelevant phosphorylated protein (histone). Similar phosphorylation state-specific monoclonal antibodies could be made to recognise other specific phosphorylation sites in MBP or other proteins. It is planned to use these antibodies to quantify and locate the extent of MBP phosphorylation in normal and multiple sclerosis myelin.

Amino Acid Sequence↗
PubMed1995

Inhibin as a marker for ovarian cancer.

Inhibin is a polypeptide hormone produced by the granulosa cells of the ovary, and is present in body fluids as dimers of various sizes each comprising an alpha- and beta-subunit. Free forms of the alpha-subunit also circulate, and the presently available radioimmunoassay (Monash assay) cannot distinguish these from biologically active dimeric inhibin. Recently we described a new two-site enzyme immunoassay able for the first time to measure the levels of dimeric inhibin throughout the human menstrual cycle. The sensitivity limit of this assay is 2 pg ml-1 in human serum with cross-reactivity against activin of 0.05%. The normal range of inhibin in post-menopausal women is < 5 pg ml-1, in pre-menopausal women 2-80 pg ml-1 (2-10 pg ml-1 in the follicular phase, 40-80 pg ml-1 in the luteal phase). This assay was used to determine inhibin levels in sera from 15 (five pre-menopausal and ten post-menopausal) patients with granulosa cell tumours of the ovary. It was raised in a pre-menopausal patient preoperatively (261 pg ml-1), in six post-menopausal patients (32, 43, 54, 66, 24 and 58 pg ml-1) and one pre-menopausal patient with recurrent tumour, (237 pg ml-1), all confirmed clinically. Inhibin was normal in six patients in remission. Oestradiol levels were normal in all patients. Serial levels of inhibin predicted recurrence before overt clinical relapse in two patients. In 29 patients with malignant epithelial ovarian tumours inhibin levels were modestly elevated in nine and normal in the rest. Three patients with endometrioid histology, two with undifferentiated tumours, three with mucinous adenocarcinoma and one with clear cell carcinoma had elevated inhibin levels. Functional inhibin is secreted by all granulosa cell tumours of the ovary studied and can be used as a tumour marker to determine response to therapy and predict recurrence and is superior to oestradiol. A more detailed analysis of the levels of inhibin, and its subunits in epithelial ovarian cancer is needed to identify the molecular forms of the immunoreactive material before optimised assays can be applied to this more common tumour.

Adult↗
PubMed1994

Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice.

A recombinant protein corresponding to the Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) and synthetic 15-mer peptides of the whole MOG molecule with eight amino acid overlaps were screened for their ability to induce experimental allergic encephalomyelitis (EAE) in Biozzi AB/H (H-2dq1) and SJL (H-2S) mice. Clinical and histologic evidence of EAE developed after sensitization with the recombinant MOG protein in both AB/H and SJL mice. In AB/H mice at least three MOG epitopes within residues 1-22, 43-57, and 134-148 induced clinical and histologic EAE, whereas only the sequence 92-106 was encephalitogenic in SJL mice. Histologically, the inflammatory response in the central nervous system consisted of perivascular accumulations of CD5+ T cells and F4/80+ macrophage/microglia cells equally distributed in the brain and spinal cord. The subpial/meningeal infiltration, characteristic of mouse EAE induced with spinal cord homogenate, was only observed in cases of severe clinical disease in SJL mice in which the cellular infiltrates predominated in the spinal cord. In spite of the presence of histologic lesions in AB/H mice immunized with MOG, clinical disease either rapidly resolved or was clinically silent. In contrast to immunization of SJL mice with recombinant MOG, sensitization to MOG 92-106 induced severe clinical paralysis. After recovery these animals relapsed and exhibited demyelinated lesions. This study is the first to describe encephalitogenic epitopes of MOG that induce both clinical and histologic signs of EAE in mice. These and previous findings implicating MOG as a target Ag for Ab-mediated attack in EAE suggest that such autoreactivity to MOG may be significant in the development of human demyelinating diseases such as multiple sclerosis.

Amino Acid Sequence↗
PubMed1994

Rat brain glial cells in primary culture and subculture contain the delta, epsilon and zeta subspecies of protein kinase C as well as the conventional subspecies.

We raised polyclonal antibodies against the C-terminal peptides of protein kinase C (PkC) subspecies alpha, beta 1, beta 2, gamma, delta, epsilon, and zeta and checked their specificity against brain extracts using Western immunoblot analysis. With equal amounts of protein applied to gels PkC subspecies beta 1, delta, epsilon and zeta were detected in primary cultures of mixed glial cells: bands for the alpha and beta 2 subspecies were less prominent. PkC gamma was not detected in primary glial cultures. The epsilon and zeta subspecies of PkC were detected in subcultures of type 1 astrocytes with weaker bands for the alpha, beta 1 and beta 2 subspecies. Blots of O-2A-lineage glia contained PkCs delta and zeta as prominent bands: the alpha, beta 1 and epsilon subspecies were also present. All PkC subspecies including PkC gamma were detected in C6 glioma cells.

Amino Acid Sequence↗
PubMed1994

The occurrence of three calcium-independent protein kinase C subspecies (delta, epsilon and zeta) in retina of different species.

The localisation and immunochemical identification of three different forms of calcium-independent protein kinase C (PKC-epsilon, PKC-delta and PKC-zeta) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE/Western blotting, respectively. More than one component of different molecular weights reacted with the polyclonal antibodies in all retinal samples though in all instances a component of molecular weight corresponding to the individual PKCs was recognised and could be eliminated or reduced by preincubating the primary antibodies with the peptides used to generate the antibodies. PKC-zeta immunoreactivity was exclusively associated with the inner segments of the photoreceptors in both mammalian (guinea-pig, rabbit, rat) and non-mammalian (goldfish, chick) retinas. PKC-epsilon immunoreactivity is present in bipolar cells, particularly in their terminals of mammalian and goldfish retinas. In the chick retina immunoreactivity for this enzyme and for PKC-delta was with the inner segments of the photoreceptors. The Müller cells in mammalian retinas and a sub-population of ganglion cells in the goldfish retina exhibited positive immunoreactivity for PKC-delta. The immunoreactivities for all the PKC isoenzymes were eliminated or drastically reduced when the primary antibodies were first preincubated with the peptides used to generate the antibodies.

Amino Acid Sequence↗
PubMed1994

Exploring myelin basic protein for HLA class I-binding sequences.

In view of the increasing evidence of the involvement of CD8+ T cells in the pathogenesis of multiple sclerosis (MS), we have scanned the sequence of the myelin basic protein (MBP), using 162 overlapping nonapeptides, for HLA-class I binding sites. Peptide binding was measured using the recently reported HLA class I alpha-chain-refolding assay, and the following HLA allelic products were analyzed: HLA-A2 (*0201, *0204), B27 (*2705), B35, B51 and B62. A considerable number of binding peptides were distinguished for each of the allelic products tested. In addition, three interesting points emerged. The first was the identification of several binding peptides which did not contain the known anchor motifs. The second was the evidence that several peptides showed a promiscuous binding profile, being able to bind to different HLA class I molecules that were either allelic or non allelic. The third was that in several cases two consecutive peptides could bind to the same HLA molecule.

Amino Acid Sequence↗
PubMed1994

Changes in protein kinase C subspecies protein expression and activity in a series of multidrug-resistant human KB carcinoma cell lines.

We have investigated the relationship between protein kinase C (PKC), levels of resistance and drug used for selection in a series of human KB carcinoma cell lines by comparing protein kinase C activity and PKC alpha, beta I, beta II, gamma, delta, epsilon, and zeta subspecies protein expression. PKC alpha protein expression was increased by 600% and 375% in KB-A1 and KB-C1 lines respectively over the parent KB-3-1 line; only KB-A1 cells showed increased PKC delta expression. Expression of other PKC subspecies was equal to that of KB-3-1 cells. There was considerable variation between the different KB cell lines in total cytosolic PKC activity, the KB-A1 and KB-C1 lines showing 400% and 350% increases respectively, KB-V1 and KB-8-5-11 about 180%, and KB-8-5 no increase relative to the parent KB-3-1 line. For calcium-independent PKC activity, the KB-C1 and KB-A1 lines only were increased over the KB-3-1 line. Immunoprecipitation with antisera to PKC subspecies confirmed that the increase in KB-A1 cytosolic total PKC activity was due largely to PKC alpha and partially to PKC delta. Membrane-associated PKC activity was increased by 500% and 350% in KB-A1 and KB-C1 lines respectively, by 250% and 270% in KB-V1 and KB-8-5-11, and not increased in KB-8-5 cells relative to the KB-3-1 cells. For KB-C1, KB-8-5-11, and KB-8-5 lines, which show decreasing resistance to colchicine, our results suggest a correlation between PKC and multidrug resistance in cells selected for resistance to this drug. There is no correlation between PKC and multidrug resistance for cells selected in different drugs. Our study therefore suggests that specific PKC subspecies are associated with the MDR phenotype of some KB cell lines, but that the extent of PKC involvement depends on the type of drug used for selection and its concentration.

Amino Acid Sequence↗
PubMed1993

Immunoassays for inhibin and its subunits. Further applications of the synthetic peptide approach.

We describe the preparation of a new rat monoclonal antibody (CRC1) to the N-terminal sequence of the 43 kDa subunit of human ovarian inhibin, and its use together with other anti-peptide monoclonal antibodies, in two-site immunoassays for the detection of inhibin-related material in biological fluids. The Fab fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the 20 kDa alpha subunit, coupled to alkaline phosphatase, was used for detection, and either CRC1 or a monoclonal antibody (E4) to the beta-A subunit were used as capture antibodies. The E4/R1 combination, expected to measure dimeric bioactive inhibin, could detect less than 2 pg/ml of recombinant inhibin in diluent, gave good recovery of activity spiked into human blood, and could measure significant levels of immunoreactivity in sera from women undergoing ovulation induction, and in some normal women. Sera from post-menopausal women contained undetectable levels. Apparent inhibin levels in human follicular fluid were increased six-fold by pretreatment with 8 M urea, suggesting masking of epitopes in this fluid. Activin cross-reactivity in the assay was 0.05%. The R1/CRC1 assay, expected to measure only large molecular weight forms of inhibin or its alpha subunit, could detect immunoreactivity in human FF diluted 50,000-fold, and in all sera tested, although the levels in the hyperovulated women were higher. By contrast to the E4/R1 assay much of the immunoreactivity was labile during the clotting process, or subsequent assay, and reliable measurements on blood with this assay will require special sample collection procedures. These results demonstrate the value of anti-peptide monoclonal antibodies in the study of inhibin, and the results obtained with CRC1 show that antibodies useful for immunoassays can sometimes be obtained without the purified target molecule being available for immunization or screening.

Amino Acid Sequence↗
PubMed1993

Identification of a major encephalitogenic epitope of proteolipid protein (residues 56-70) for the induction of experimental allergic encephalomyelitis in Biozzi AB/H and nonobese diabetic mice.

Native proteolipid (PLP) and synthetic 15- or 16-mer peptides of the whole PLP molecule, with eight amino acid overlaps, were screened for their ability to induce experimental allergic encephalomyelitis in Biozzi AB/H (H-2dq1) mice. Clinical and histological evidence of experimental allergic encephalomyelitis developed after sensitization with native PLP and with the PLP sequence 56-70 (DYEYLINVIHAFQYV) but not with the other synthetic PLP peptides used. Nonobese diabetic mice that share similar MHC determinants (H-2Anod) with Biozzi AB/H mice, could be induced to develop experimental allergic encephalomyelitis after sensitization with either mouse spinal cord homogenate or the PLP 56-70 peptide. Although the PLP 56-70 overlaps with the encephalitogenic epitope of PLP (residues 43-64) in PL/J (H-2u) mice the Biozzi AB/H mice did not exhibit disease with either PLP 43-64 peptide or the nonapeptide PLP 56-64 that overlaps both the Biozzi AB/H and PL/J encephalitogenic peptides. The identification of a novel major encephalitogenic epitope of PLP for Biozzi AB/H mice, increases the repertoire of encephalitogenic epitopes of PLP and supports a role for PLP as a target Ag in autoimmune demyelinating diseases.

Amino Acid Sequence↗
PubMed1993

Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation.

Members of the activin family have been suggested to act as mesoderm-inducing factors during early amphibian development. Little is known, however, about mesoderm formation in the mammalian embryo, and as one approach to investigating this we have studied activin expression during early mouse development. Activins are homo- or heterodimers of the beta A or beta B subunits of inhibin, itself a heterodimer consisting of one of the beta subunits together with an alpha subunit. Our results indicate that the oocyte contains mRNA encoding all three subunits, and antibody staining demonstrates the presence of both alpha and beta protein chains. From the fertilized egg stage onwards, alpha subunit protein cannot be detected, so the presence of beta subunits reflects the presence of activin rather than inhibin. Maternal levels of activin protein decline during early cleavage stages but increase, presumably due to zygotic transcription (see below), in the compacted morula. By 3.5 days, only the inner cell mass (ICM) cells of the blastocyst express activin, but at 4.5 days the situation is reversed; activin expression is confined to the trophectoderm. Using reverse transcription-PCR, neither beta A nor beta B mRNA was detectable at the two-cell stage but transcripts encoding both subunits were detectable at the morula stage, with beta B mRNA persisting into the blastocyst. We have also analyzed activin and inhibin expression in ES and EC cells. Consistent with the observation that activins are expressed in the ICM of 3.5-day blastocysts, we find high levels of beta A and beta B mRNA in all eight ES cell lines tested. F9 EC cells express only activin beta B, together with low levels of the inhibin alpha chain. When ES and EC cells are induced to differentiate, levels of activin fall dramatically. These results are consistent with a role for activins in mesoderm formation and other steps of early mouse development.

Activins↗
PubMed1992

Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.

Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Ultrasensitive two-site assays for inhibin-A and activin-A using monoclonal antibodies raised to synthetic peptides.

Monoclonal antibodies produced to synthetic peptides of the alpha and beta A subunits of inhibin were used to develop the first highly sensitive two-site assays for recombinant human inhibin-A and activin-A. The assay for inhibin-A could detect as little as 5 pg/ml. Activin-A gave a 5.2% cross-reactivity in the inhibin assay. The assay for activin-A could detect 0.1 ng/ml and inhibin-A showed a 5.3% cross-reaction in the activin assay. Determination of inhibins and activins in biological fluids is complicated by the presence of multiple molecular forms and an excess of free alpha subunits. The present study demonstrates the potential of antibodies raised to synthetic peptides to configure more specific two-site assays for inhibins and activins which could facilitate research on these molecules. The monoclonal antibody used as the labelled antibody in these studies was made by immunizing mice with a peptide corresponding to an internal sequence of the beta A subunit. It appears likely that similar high affinity anti-peptide monoclonals suitable for use in ultrasensitive two-site assays could be made to other molecules if a rigorous screening of the fusion supernatants on the native molecule was carried out. The use of anti-peptide monoclonal antibodies in the development of ultrasensitive immunoassays may be more widely applicable than commonly realised.

Activins↗