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M Lowe

Publications and source records attributed to M Lowe.

At least 19 recordsLinked to original sources
PubMed1997

The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner.

The docking of transport vesicles with their target membrane is thought to be mediated by p115. We show here that GM130, a cis-Golgi matrix protein, interacts specifically with p115 and so could provide a membrane docking site. Deletion analysis showed that the N-terminus binds to p115, whereas the C-terminus binds to Golgi membranes. Mitotic phosphorylation of GM130 or a peptide derived from the N-terminus prevented binding to p115. The peptide also inhibited the NSF- but not the p97-dependent reassembly of Golgi cisternae from mitotic fragments, unless it was mitotically phosphorylated. Together, these data provide a molecular explanation for the COPI-mediated fragmentation of the Golgi apparatus at the onset of mitosis.

Amino Acid Sequence↗
PubMed1997

Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

Animals↗
PubMed1996

In vivo assembly of coatomer, the COP-I coat precursor.

Coatomer, a seven-subunit hetero-oligomeric complex, is the major component of the COP-I membrane coat of transport vesicles of the early secretory pathway. We have followed the assembly of this complex in vivo by pulse-chase experiments and immunoprecipitation of native coatomer subunits and found that it is an ordered process that takes 1-2 h to complete. During assembly, direct interactions between alpha-, beta'- and delta-COP, beta- and delta-COP, and gamma-, zeta-, and delta-COP occur. Coatomer, once it has assembled, is stable with a half-life of approximately 28 h. No significant amounts of partial coatomer complexes have been detected. The only subunit to exist at steady state out of the complex is zeta-COP, which has a similar half-life to coatomer subunits within the complex. Assembly is inhibited by brefeldin A, suggesting that it may be a regulated process. These results describe for the first time in vivo assembly of a coat protein complex involved in membrane traffic and extend our knowledge of how coatomer is structured.

Animals↗
PubMed1996

Biochemical heterogeneity and phosphorylation of coatomer subunits.

The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membrane-associated coats thought to mediate vesicular transport between the endoplasmic reticulum and the Golgi complex, between adjacent Golgi cisternae, and possibly in the endocytic pathway. We investigated whether this heterogeneity in the sites of COPI action might be reflected in biochemical heterogeneity of one or more COPI subunits. A simplified method was devised to purify the cytosolic COPI precursor complex, coatomer, from rat liver cytosol. The individual subunits were analyzed by high resolution two dimensional gel electrophoresis and mass spectroscopic analysis of tryptic peptides. Considerable charge heterogeneity was observed, particularly for the beta-COP and delta-COP subunits. The multiple species detected, however, did not appear to reflect the presence of distinct translation products but rather a significant degree of protein phosphorylation. The observed pI of beta-COP was sensitive to alkaline phosphatase digestion. Moreover, isolation of coatomer from metabolically labeled tissue culture cells demonstrated directly that both beta-COP and delta-COP, but no other coatomer subunits, were serine-phosphorylated. COPI phosphorylation may regulate coatomer assembly, membrane recruitment, or the specificity of coatomer-organelle interaction.

Animals↗
PubMed1995

In vitro assembly and disassembly of coatomer.

Coatomer, a complex of seven proteins, is the major component of the non-clathrin (COP I) membrane coat. We report here the first system to reversibly disassemble and reassemble this complex in vitro. Coatomer disassembles at high salt concentrations and reassembles when returned to a more physiological buffer. Using this system, we show that alpha-, beta'-, and epsilon-COP interact directly and that gamma-COP interacts with zeta-COP. A partial complex comprising alpha-, beta'-, and epsilon-COP, obtained after coatomer disassembly, can bind to membranes in vitro. This binding is, at least in part, mediated by interactions with cytoplasmic KKXX motifs of proteins normally retained in or retrieved to the endoplasmic reticulum. Using coatomer disassembly and epitope-specific antibodies, we also demonstrate that the N- and C-terminal domains of beta-COP are buried within the native coatomer complex. These results provide the first insights into how the coatomer is structured.

Amino Acid Sequence↗
PubMed1995

Competent patients, incompetent decisions.

We describe the epidemiologic aspects of dialysis withdrawal, the role of patient autonomy and of medical beneficence in the decision to discontinue dialysis, and the conditions under which limits may be placed on patient autonomy. A case is presented that provides insight into the issues involved in making such decisions and that should provoke further discussion on the role of "medical guidelines" for dialysis refusal and withdrawal.

Beneficence↗
PubMed1995

The behaviour of the f2-f1 acoustic distortion product: lack of effect of brainstem lesions in anaesthetized guinea pigs.

Two tones of frequency f1 and f2 (the primary tones), when presented simultaneously to the ear, generate acoustic distortion products in the external ear canal. One of these distortion products, of frequency f2-f1, has been shown to undergo a reversible change in amplitude when the primary tones generating distortion are presented continuously to the test (ipsilateral) ear (Brown, 1988; Kirk and Johnstone, 1993). The effect is apparent for low and moderate primary tone intensities and has been postulated to be caused by the action of a neural feedback loop via the superior olivary complex. We have carried out a series of studies of this phenomenon in anaesthetized guinea pigs, making brainstem lesions positioned so as to interrupt the known medical and/or lateral efferent projections to the cochlea from the superior olivary complex. We could not demonstrate any consistent change after lesioning, either in the baseline level of f2-f1 or in the alteration of f2-f1 caused by continuous monaural primary tones. These results are not consistent with the suggestion by others that a neural feedback loop involving either the medial (Brown, 1988) or lateral (Kirk and Johnstone, 1993) olivocochlear efferents may be responsible for the effect. We therefore conclude that either 1) the changes in f2-f1 produced by continuous low-level primary tones reflect the operation of intrinsic hair cell mechanisms and do not involve efferent feedback via brainstem nuclei or 2) a neural feedback loop does play a role, but this loop involves an unknown pathway that was not interrupted by our lesions.

Acoustic Stimulation↗
PubMed1995

'These sorts of people don't do very well': race and allocation of health care resources.

Recent literature has highlighted issues of racial discrimination in medicine. In order to explore the sometimes subtle influence of racial determinants in decisions about resource allocation, we present the case of a 53-year-old Australian Aboriginal woman with end-stage renal failure. The epidemiology of renal failure in the Australian Aboriginal population and amongst other indigenous peoples is discussed. We show that the use of utilitarian outcome criteria for resource allocation may embody subtle racial discrimination where consideration is not given to issues of justice, race, culture and gender. It is only where the processes by which resources are allocated are transparent, clearly defined and based upon consultation with individual patients that issues and justice are likely to be adequately addressed.

Americas↗
PubMed1995

COPs regulating membrane traffic.

Cytosolic coat proteins (COPs) regulate membrane traffic in eukaryotic cells. Three classes of coat protein complexes have so far been identified: clathrin and its adaptor proteins, coatomer (COPI), and COPII. Coatomer (composed of seven different subunits) and ADP-ribosylation factor (ARF), which form the COPI coat, are required for budding of coated vesicles from membranes. COPI has been implicated in several steps of transport from the intermediate compartment to the cis-Golgi network, through cisternae of the Golgi stack, and is essential for retrieval to the endoplasmic reticulum (ER) of membrane proteins containing the carboxy-terminal dilysine ER-retention motif. A family of structurally and functionally related COPs may regulate all membrane traffic steps in eukaryotic cells.

ADP-Ribosylation Factors↗
PubMed1992

Structure of the human pancreatic cholesterol esterase gene.

The gene for human pancreatic cholesterol esterase consists of 11 exons and 10 introns and is 9.2 kb in length. The last and longest exon (841 nucleotides) is unique to the human gene. Functional amino acids are encoded on separate exons. The leader sequence is encoded by a single exon which carries two additional N-terminal amino acids of the mature functional protein. A positive TATA element is identified 43 nucleotides from the start codon. Pulse-field gel electrophoresis and hybridization with various cDNA probes and direct sequence data revealed the existence of a CEase-like gene. Partial sequence analysis of this gene from a human cosmid library and human genomic DNA showed a premature stop signal in exon 10, shortly after the codon for the active-site histidine. Both the functional gene and the CEase-like gene have a polyadenylation signal in the 3'-untranslated region. Thus, the complex gene structure for this intestinally active enzyme may provide in part a potential molecular explanation for the well-known heterogeneity of the intestinal absorption of cholesterol.

Base Sequence↗
PubMed1992

Preliminary report: penile vein occlusion therapy: selection criteria and methods used for the transcatheter treatment of impotence caused by venous-sinusoidal incompetence.

Penile vein occlusion therapy is a fluoroscopic guided technique developed for treating venous-sinusoidal incompetence in patients with erectile dysfunction. We report our experience with 22 patients who have been followed for greater than 1 year. Catheterization of the veins draining the penis was technically possible in 20 of 22 patients (91%). Of these 20 patients 5 (25%) were cured and 9 (45%) had significantly improved erectile function. All 5 patients (100%) with venous-sinusoidal incompetence plus normal cavernous arterial flow, no glans or corpus spongiosal shunts, and bilateral complete coil occlusion of the crural and common crural veins were cured. No deterioration of function was noted at 1 and 2 years. Patient selection and bilateral occlusion of the penile veins are essential to achieve successful penile vein occlusion therapy.

Adult↗
PubMed1991

Assessment of normal and abnormal erectile function: color Doppler flow sonography versus conventional techniques.

The penile arteries were studied with color Doppler flow sonography in 10 subjects with normal and 39 patients with abnormal erectile function. The relationships of systolic and diastolic velocities to spectral waveform changes in the penile arteries in response to tumescence were studied before and after intracorporal injection of vasoactive medications that induce erection. In normal subjects, a characteristic spectral waveform pattern corresponded to increasing intracorporal pressure. Patients with abnormal arterial inflow and/or abnormal venous sinusoidal leakage demonstrated deviation from the patterns noted in normal subjects. Patients with abnormal arterial inflow had lower mean peak systolic velocities than normal subjects. Patients with severe venous sinusoidal incompetence had an arrest of waveform progression with evolution to but not beyond phases 1 or 2 (diastolic flow remained positive). Patients with abnormal arterial inflow and abnormal venous sinusoidal outflow had waveform changes that reflected both processes. Systolic/diastolic velocity and waveform relationships can be used to define the integrity of both the cavernosal artery inflow and venous sinusoidal outflow occlusion mechanisms.

Adult↗
PubMed1990

Molecular cloning and expression of a cDNA encoding the membrane-associated rat intestinal alkaline phosphatase.

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A) + RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1-474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476-491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.

Alkaline Phosphatase↗