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Biomedical subjects

A W Linnane

Publications and source records attributed to A W Linnane.

At least 37 records · Page 2Linked to original sources
PubMed1996

The specificity of mitochondrial complex I for ubiquinones.

We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.

Animals↗
PubMed1995

The universality of bioenergetic disease and amelioration with redox therapy.

Overt mitochondrial diseases associated with mitochondrial DNA mutations are characterized by a decline in mitochondrial respiratory function. Similarly, a progressive decline in mitochondrial respiratory function associated with mitochondrial DNA mutations is clearly evidenced in aged human subjects. This communication is concerned with the development of a rat model for the study of bioenergy decline associated with the ageing process and overt mitochondrial diseases. The model involves the treatment of young rats with AZT to induce skeletal and cardiac myopathies. It has shown that there is a decline in soleus muscle function in vivo and that this decline is mirrored in the capacity of heart sub-mitochondrial particles to maintain bioenergy function. Coenzyme Q10 and several analogs were administered with AZT as potential therapeutics for the re-energization of affected tissues. Coenzyme Q10 and especially decyl Q were found to be therapeutically beneficial by both in vivo improvement in soleus muscle function and in vitro cardiac mitochondrial membrane potential capacity. Sub-mitochondrial particles were also prepared from heart mitochondria of young and aged rats. The particles prepared from the aged rats were found to have a decreased ability to maintain membrane potential as compared to those derived from the young rats.

Animals↗
PubMed1995

Resistance of melanoma cell lines to interferons correlates with reduction of IFN-induced tyrosine phosphorylation. Induction of the anti-viral state by IFN is prevented by tyrosine kinase inhibitors.

Clinical and experimental studies examining the action of IFNs on human malignant melanomas and melanoma cell lines have shown that this cancer cell type is frequently IFN resistant. In the present study, the IFN responsiveness of five melanoma cell lines, SK-MEL-28, SK-MEL-3, MM96, HT-144, and Hs 294T, as determined by the levels of IFN-induced expression of the antiviral proteins, 100 kDa 2',5'-oligoadenylate synthetase (OAS) and Mx Ag, was shown to correlate with the IFN responsiveness of the five lines measured in antiproliferative and antiviral assays. Three of the lines, SK-MEL-28 (IFN sensitive), SK-MEL-3 (moderately IFN sensitive), and MM96 (IFN insensitive) were analyzed further to ascertain their relative levels of IFN-activated signal transduction. Pretreatment of the three melanoma cell lines with the tyrosine kinase inhibitors, Herbimycin A or Genistein, produced a dose-dependent inhibition of the antiviral action of IFN-alpha, -beta, and -gamma and the induction of OAS by IFN-beta. Thus, induction of the antiviral state in melanoma cells by IFN requires activation of tyrosine kinase-dependent signaling pathways. Furthermore, the IFN responsiveness of three melanoma cell lines could be correlated with the ability to detect by immunoblotting of SDS-PAGE displays of cell lysates, IFN-induced tyrosine phosphorylated cellular proteins in the range m.w. 80 to 130 kDa. This induction was also sensitive to the tyrosine kinase inhibitors Herbimycin A and Genistein. Based on these results, we propose that the IFN-resistant melanoma cell lines examined contain a deficiency early in the IFN signal transduction pathway resulting in a reduced potential for IFN-induced tyrosine phosphorylation and a lack of responsiveness to IFN.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Constitutive and virus-induced interferon production by peripheral blood leukocytes.

The production of interferon-alpha (IFN-alpha) by normal human peripheral blood mononuclear cells (PBMNC) was studied using polyclonal antipeptide antibodies designed to react either with all IFN-alpha subtypes or with individual subtypes IFN-alpha 2 or IFN-alpha 4. In this study, we demonstrate the detection of intracellular IFN-alpha in PBMNC using immunofluorescence staining and flow-cytometric analysis. Virtually all cells of the PBMNC population were shown to produce IFN-alpha reactive with all three antisera after stimulation with Sendai virus. The immunofluorescence studies also demonstrated that IFN-alpha is produced by PBMNC in the absence of known viral stimulation but is not secreted in detectable levels. Double-labeling with specific monoclonal antibodies to T and B lymphocytes confirmed that the entire populations of these two cell types produce IFN-alpha, both constitutively and after virus induction. Polymorphonuclear cells (PMNC) isolated from Ficoll-Paque pellets were also shown to contain intracellular IFN-alpha, both before and after virus induction. The finding that all PBMNC produce IFN-alpha constitutively suggests that IFN-alpha may have important regulatory functions in situations other than during overt viral infections.

B-Lymphocytes↗
PubMed1994

Up-regulation of the plasma membrane oxidoreductase as a prerequisite for the viability of human Namalwa rho 0 cells.

We have studied aspects of the regulatory interrelationship between the plasma membrane oxidoreductase (PMOR) system and the mitochondrial respiratory capacity of human Namalwa cells. Although the role of mitochondria in the maintenance of cellular redox and energetic states is well established, the PMOR system in comparison is a poorly characterized enzyme system whose functions, particularly in relation to cellular metabolism, have not been clearly elucidated. Therefore we compared the PMOR and mitochondrial respiratory activities of human Namalwa cells during the induction by ethidium bromide treatment of rho 0 cells, which lack a functional mitochondrial respiratory system. The plasma membrane NADH-ferricyanide reductase activity of the PMOR system was found to increase in a stepwise manner concomitant with a decline in cellular mitochondrial respiratory activity. Addition of p-chloromercuriphenylsulfonic acid to the culture medium, at a concentration totally inhibiting the plasma membrane NADH-ferricyanide reductase in vitro, leads to cell death of rho 0 but not of rho + cells. Thus, the up-regulation of a functional PMOR system is a necessary phenomenon in maintaining the viability of mammalian rho 0 cells.

4-Chloromercuribenzenesulfonate↗
PubMed1994

Mitochondrial respiratory chain inhibitors induce apoptosis.

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

Animals↗
PubMed1994

Structure-function analysis of human IFN-alpha. Mapping of a conformational epitope by homologue scanning.

The purpose of this study was to map a conformational epitope of a mAb that binds the IFN subtype alpha 4a. Binding of this mAb, designated I-4-A, to IFN-alpha 4a does not block receptor binding, but does neutralize biologic activity by inhibition of signal transduction. A novel strategy was developed, termed homologue scanning, which uses template-coupled polymerase chain reaction to generate hybrid molecules consisting of part (N-terminus) of the reactive IFN-alpha 4a subtype to locate the epitope, and the remainder of the nonreactive IFN-alpha 14 subtype to provide the overall conformation of an IFN-alpha molecule. Hybrid molecules were expressed as 35S-methionine-labeled proteins and tested for immunoreactivity by Western blotting and antiviral activity by cytopathic effect reduction bioassays. Unless an entire IFN-alpha (hybrid) molecule was formed, immunoreactivity and biologic activity were lost, indicating the importance of the C-terminus for correct folding of IFN-alpha molecules. The epitope for I-4-A was localized to the N-terminal 23 residues of IFN-alpha 4a. Furthermore, the immunoreactivity of IFN-alpha 4a analogues, with alterations in the putative receptor-binding region of IFN-alpha 4a residues 30 to 40 was unaffected, in contrast to the biologic activity that was reduced by several orders of magnitude. Thus, the N-terminal 23 residues of IFN-alpha 4a, which probably are not involved in receptor binding, may be important for other interactions of the receptor-bound ligand. In general terms, the novel approach of homologue scanning, using template-coupled PCR to facilitate the generation of hybrid proteins, will have broad application in the mapping of conformational epitopes of proteins that are members of a homologous family. The ability to identify conformational epitopes will increase our understanding important interactions of proteins with antibodies, receptors, and other macromolecules.

Amino Acid Sequence↗
PubMed1994

Immunolocalisation of interferon-alpha in hepatitis C patients and its correlation with response to interferon-alpha therapy.

Localised interferon-alpha production was investigated in hepatitis C patients entered into a trial of interferon-alpha-2a therapy. Antibodies capable of reacting specifically with interferon-alpha-2, interferon-alpha-4 or with all interferon-alpha subtypes were used as immunohistochemical and immunofluorescence probes to study interferon-alpha production in liver biopsy tissue, and peripheral blood mononuclear cells prior to and after stimulation with Sendai virus. Measurement of cytoplasmic interferon-alpha, specifically interferon-alpha-2 and interferon-alpha-4, in peripheral blood mononuclear cells isolated from the hepatitis C patients and of total interferon-alpha secreted into culture supernatants by these cells showed interferon-alpha production similar to that of peripheral blood mononuclear cells isolated from normal individuals. Interferon-alpha-positive cells were observed in the infiltrating mononuclear cells of the liver biopsy tissue obtained from 8 of the 14 patients. Lymphocytes, fibroblasts, Kupffer cells, polymorphonuclear cells and monocytes stained positive for interferon-alpha, and specifically interferon-alpha-4, in all of the eight patients. The cytoplasm of hepatocytes also stained weakly positive in three of these patients. Interferon-alpha positive cells showed a good correlation with the degree of histological damage observed in the liver biopsies but not with presence of antibodies towards hepatitis C virus or levels of serum alanine aminotransferase measured prior to interferon-alpha-2a therapy. Interestingly, response to therapy seemed linked to local interferon-alpha production status. Those patients who responded best to therapy displayed no or only low levels of interferon-alpha positive cells in liver biopsy tissue. Thus patients with a lower activation of their endogenous interferon-alpha system may benefit from administration of exogenous interferon-alpha.

Adolescent↗
PubMed1994

Expression of IFN A genes in subpopulations of peripheral blood cells.

The polymerase chain reaction (PCR) was used to detect the expression of IFN A genes in general (with 'universal' primers) and specifically the expression of mRNA transcripts encoding the subtypes IFN-alpha-1, -alpha-2, -alpha-4, -alpha-5 and -alpha-14 (with gene specific primers) in normal human peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. Our examination revealed that all transcripts tested for could be detected not only following induction with inducers such as Sendai virus, Semliki Forest virus and poly(I):poly(C), but also in the absence of induction. IFN A1, IFN A2 and IFN A4 mRNAs were found to constitute the major transcripts of Sendai virus and poly(I):poly(C) induced PBMC. Fractionation of PBMC into T cells, B cells, adherent cells, mononuclear (MN) cells and polymorphonuclear (PMN) cells revealed that these cell populations all contain specific IFN A mRNA transcripts both in the absence of an inducer and following induction with Sendai virus. The proportion of IFN A transcripts detected was dependent on the cell type investigated. IFN A1, IFN A2 and IFN A4 transcripts constituted the major RNA species present in PBMC and PMN cells. In MN cells IFN A5 transcripts were also present as a major IFN RNA species. Expression of the IFN A transcripts tested for in T cells, B cells and adherent cells did not vary significantly. These results emphasize the importance of identifying IFN A subtype expression in order to further our understanding of the biological significance of differential regulation and expression of particular IFN-alpha subtypes.

Antigens, Viral↗
PubMed1994

cDNA sequence identity for the type I interferon receptor subunit from cell lines of widely differing responsiveness to interferon.

Melanoma cell lines exhibit strikingly different sensitivity to the antiproliferative effects of interferon. cDNAs encoding the Type I interferon receptor subunit were amplified by polymerase chain reaction, using as template RNA isolated from three melanoma cell lines displaying greater than 100 fold range in their sensitivity to the antiproliferative effects of IFN-beta. Comparison of the cDNA sequences obtained with the published cDNA sequence from the highly interferon-sensitive lymphoid cell line Daudi revealed only one base change that leads to a conservative amino acid substitution. It is concluded that the cellular differences in responsiveness to interferon, of the melanoma cell lines tested, do not arise from the expression of variants of the cloned Type I interferon receptor subunit.

Base Sequence↗
PubMed1994

Age-related human mtDNA deletions: a heterogeneous set of deletions arising at a single pair of directly repeated sequences.

Deletions in mtDNA accumulate during the human aging process, arising from either intramolecular illegitimate recombination or strand slippage during replication, which results in subgenomic mtDNA molecules. We identify here two classes of mtDNA deletions--class A deletions, which are homogeneous at the breakpoints, with all subgenomic molecules therefore being identical in size, and class B deletions, which arise from a less stringent process that gives rise to heterogeneity at the breakpoints, with the subgenomic molecules being of slightly different sizes. A novel approach is described that offers a global overview of the populations of different deletions in individual tissues. It is based on PCR cycle-sequencing reactions that are carried out directly on mtDNA segments, amplified by PCR from total cellular DNA. The results show a clear size homogeneity of the subgenomic mtDNA molecules representative of class A, which carry a commonly detected 4,977-bp deletion occurring at a pair of 13-bp directly repeated sequences. In this case, precisely one copy of the repeat is retained in the subgenomic molecules. We then describe a class B situation comprising a family of at least nine closely related 8.04-kb deletions involving the same pair of 5-bp direct repeats. In this situation, the breakpoints differ at the base-pair level (8,037-8,048-bp deletions); the subgenomic molecules retain > 1 copy, 1 copy, or < 1 copy of the 5-bp repeat. In different tissues from either the same individual or among different individuals, there is a widely variable occurrence of particular deletions in the subgenomic mtDNA population within this class B set. Class B deletions offer a new approach for studying the accumulation of mtDNA deletions, thereby providing insight into the independent somatic origin of mutated mtDNA molecules, both in aging and in mitochondrial diseases. We also report a convenient method for ascertaining whether a given PCR product results from the amplification of a subgenomic mtDNA template, on the basis of the selective degradation of full-length mtDNA molecules prior to PCR.

Adult↗
PubMed1993

Occurrence of a particular base substitution (3243 A to G) in mitochondrial DNA of tissues of ageing humans.

Allele-specific polymerase chain reaction has been used to demonstrate the occurrence, during the human ageing process, of the mutation A to G at nucleotide position 3243 on mitochondrial DNA. An oligonucleotide with a 3'-end specific to this mutation was designed to selectively amplify DNA bearing this mutation under annealing temperature conditions that efficiently discriminate between normal and mutant mitochondrial DNA templates. Seven out of 38 adult tissues tested were found to contain this mutation, whereas the mutation could not be detected in any of the 16 infant tissues analyzed. The present demonstration of an age-related point mutation adds to the growing list of mutations in human mitochondrial DNA, till now almost exclusively large deletions, that accumulate in human tissues as a function of age. The method described here has general application in the detection of other age-related point mutations in human mitochondrial DNA.

Adenine↗
PubMed1993

Detection of human interferon-alpha-encoding gene expression.

We have devised a sensitive method based on the polymerase chain reaction (PCR) to detect expression of human interferon-alpha-encoding genes (IFN-A) in general, and specifically, expression of the IFN-A2 or IFN-A4 genes. The utility of the PCR approach was assessed by analysis of cloned IFN-A genes, as well as genomic DNA and mRNA isolated from peripheral blood mononuclear cells. We demonstrate the specific amplification of sequences encoding IFN subtypes IFN-alpha-2 and IFN-alpha-4 from as little as 0.1 pg of IFN-A mRNA. The PCR technique has potential clinical application for the detection of IFN-A expression and, thus, identification of the IFN-alpha subtypes produced, particularly in small biopsy samples or otherwise, where only low numbers of cells are available.

Base Sequence↗
PubMed1993

Monoclonal antibody 4D3 detects small intestinal mucin antigen (SIMA)--glycoprotein in the serum of patients with colorectal cancer.

We have developed a sensitive ELISA using MAb 4D3 for the detection of a novel epitope on Small Intestinal Mucin Antigen (SIMA) and report here that SIMA is present in the serum of patients with colorectal cancer. SIMA has been shown to occur in tissue from a high proportion of patients with colorectal cancer. SIMA derived from serum was similar to tissue-derived SIMA: both eluted in the void volume of a Superose 6 column indicating a molecular weight above 5,000 kDa and they exhibited similar buoyant densities on CsCl gradients. The ELISA was most reliable after pre-treatment of serum with 0.4 M perchloric acid to remove interfering substances. The upper limit for SIMA in normal serum was set as the mean plus 2 standard deviations determined from a group of 97 healthy control subjects. In a sample of 113 patients with colorectal cancer, SIMA serum levels were elevated in 15% of patients with Dukes' Stage A, 38% with Stage B, 32% with Stage C and 75% with Stage D colorectal cancer. SIMA serum levels were compared with those of the widely used tumor marker, carcinoembryonic antigen (CEA). The SIMA assay detected a significant number of sera that were not detected by the test for CEA. We propose that SIMA will prove to be a valuable serological tumor marker, in combination with CEA and other tumor markers, for the detection of colorectal cancer.

Adenocarcinoma↗
PubMed1993

Differentiation-associated changes in mucin glycoprotein antigenicity in mucosa adjacent to rare gastrointestinal tract tumours of non-mucosal origin.

We have investigated changes in mucin antigenicity and morphology in the perineoplastic mucosa adjacent to rare, predominantly non-mucosal gastrointestinal (GI) tumours. Twenty-nine tumours of small and large intestine, including primary mesenchymal and ectodermal tumours, were examined immunohistochemically using 11 monoclonal antibodies (MAbs) raised against SIMA and LIMA (small and large intestinal mucin antigens). Non-epithelial GI tumours were essentially non-reactive, while adjacent mucosa showed altered mucin expression and morphology, in particular, features of transitional mucosa (TM). Combinations of different SIMA epitopes were detected adjacent to all colorectal tumours, and, similarly, LIMA epitopes adjacent to small intestinal tumours. Specific patterns adjacent to certain tumours may reflect influences of factors produced by individual tumours on mucin composition. Altered antigenicity and morphology in TM thus appear to be reactive changes in response to a wide range of GI tumours, presumably as a consequence of factors secreted by the tumour and/or a host response to the tumour.

Autoantigens↗
PubMed1993

Expression of large intestinal mucin antigen (LIMA) epitopes in the normal and neoplastic gastrointestinal tract.

This study has identified the expression in normal and neoplastic gastrointestinal (GI) tract of epitopes on the colonic mucin LIMA (large intestinal mucin antigen), which are unique markers of normal colonic differentiation. Six anti-LIMA monoclonal antibodies (MAbs) (22D4, 9B5, 2C3, 23B2, 46A2, and 10B3) were studied immunohistochemically in normal GI tract, colorectal adenomas, and colorectal and gastric cancers. All MAbs showed specificities consistent with distinct epitopes, five of which were neuraminidase-resistant and four periodate-sensitive. Each reacted with mucin in 60-100 per cent normal colons--MAbs 10B3 and 23B2 also with small intestinal mucin--but none with gastric mucin. Five MAbs showed crypt and regional gradients in normal colon, MAbs, 22D4, 9B5, and 2C3 showing a hierarchy of reactivities in the crypt. Individual adenomas showed decreasing goblet cell (GC) LIMA expression with increasing size. However, 30 per cent of familial adenomatous polyposis (FAP) patients had generalized background losses of 9B5 and 2C3 GC reactivity, retaining 22D4, whilst 44 per cent of non-FAP patients lost 22D4 GC reactivity, regaining 9B5 and 2C3--evidence for polymorphism of mucin expression. All colorectal cancers expressed LIMA epitopes (frequently weaker than normal), and three MAbs (22D4, 9B5, and 2C3) showed deeper than normal staining in adjacent crypts. Eighty-five per cent of gastric cancers also expressed LIMA epitopes.

Adenoma↗