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M F Walter

Publications and source records attributed to M F Walter.

At least 37 records · Page 2Linked to original sources
PubMed1998

Influence of pretreatment and experimental conditions on electrophoretic mobility and hydrophobicity of Cryptosporidium parvum oocysts.

Surface properties of Cryptosporidium parvum oocysts were investigated by using electrophoretic mobility and hydrophobicity measurements. Oocysts purified from calf feces by several sucrose flotation steps and deionized water (DI) washes (DIS method) had an electrophoretic mobility (neutral surface charge) near 0.0 m2 V-1 s-1 over a pH range of 2 to 10. The mean electrophoretic mobility of oocysts stored in DI containing a mixture of antibiotics had a lower standard deviation (sigma = 0.36) than that of oocysts stored in DI without antibiotics (sigma = 0.53); their electrophoretic mobility remained unchanged up to 121 days after collection. The electrophoretic mobility of oocysts purified on a cold Percoll-sucrose gradient after the feces was defatted with ethyl acetate (EAPS method) varied linearly with pH from 0.0 m2 V-1 s-1 at pH 2.4 to -3.2 x 10(-8) m2 V-1 s-1 at pH 10 (sigma = 0.52), thus displaying the negative surface charge at neutral pH observed by other researchers. The hydrophobicity of oocysts and two types of polystyrene beads was measured as a function of ionic strength by adhesion to polystyrene. Oocysts were purified by the DIS method. The ionic strength of the suspending solution was varied from 0 to 95 mmol liter-1. Two-week-old oocysts exhibited strong adhesion ( approximately 85%) at ionic strengths of 0 to 10 mmol liter-1 and moderate adhesion ( approximately 20%) at ionic strengths of 20 to 95 mmol liter-1. Two-month-old oocysts exhibited high adhesion ( approximately 60 to 80%) at all ionic strengths. These results show that adhesion properties governed by the electrophoretic mobility of purified C. parvum oocysts can be altered by the method of purification and that hydrophobicity can change as oocysts age.

Animals↗
PubMed1997

Cholesterol, calcium and atherosclerosis: is there a role for calcium channel blockers in atheroprotection?

It is well known that the atherogenic dyslipidemias of either elevated serum LDL or reduced HDL levels correlate with the degree and severity of atherosclerosis. However, how this leads to atherogenesis is poorly understood. A role for cellular oxidative stress mediated by oxidized LDL has gained widespread acceptance, but this pathway is unlikely to be the sole atherogenic signal. Recent evidence obtained from arterial smooth muscle cells (SMC) and endothelial cells (EC) is consistent with another pathway that may explain, in part, the early alterations contributing to the initiation of cellular atherogenic modifications. This pathway involves enrichment of the cell plasma membrane with cholesterol. In SMC, in vitro (cell culture) and in vivo (cholesterol feeding) experiments demonstrate that cholesterol enrichment of the SMC membrane occurs rapidly and is associated with an increase in membrane bilayer width, calcium permeability, and cell proliferation. Removal of excess membrane cholesterol with human HDL restores these alterations, suggesting that this membrane structural 'defect' mediates these changes in cell function. In vitro, the increased calcium permeability is inhibitable by calcium channel blockers (CCBs), but in vivo, a calcium 'leak' pathway develops that is virtually uninhibitable. It is not surprising that the literature on the application of CCBs for atheroprotection is not wholly convincing. However, with the advent of the new third generation of CCBs, new hope arises. One of the first CCBs of this generation is amlodipine (Norvasc), a charged dihydropyridine that has a remarkable pharmacologic profile. First, it is markedly lipophilic allowing it to partition readily into cell membranes. Second, in the membrane it has the ability to re-order, or restore, the 'swollen' membrane bilayer back to normal in atherosclerotic SMC. Third, it has potent antioxidant properties. Fourth, it appears to inhibit the expression of a variety of genes implicated in atherogenesis. Fifth, it is a CCB. Amlodipine has demonstrated atheroprotection in both rabbit and subhuman primate models of this disease. We propose that cellular alterations induced by enrichment of the cell membrane with cholesterol, which appears to modulate SMC to the atherosclerotic phenotype, are inhibitable by amlodipine through a combination of its varied pharmacologic properties. The potential for atheroprotection with amlodipine is currently being investigated in a human trial (PREVENT trial) and the results of this trial will determine the relevance of the preclinical findings to humans.

Amlodipine↗
PubMed1997

Alzheimer's disease amyloid beta peptide 25-35 inhibits lipid peroxidation as a result of its membrane interactions.

The biological activity of the Alzheimer's disease amyloid beta protein may be related to modulation of membrane lipid peroxidation. The effect of amyloid beta protein fragment 25-35 [A beta(25-35)] on lipid peroxidation was examined in liposomes enriched with polyunsaturated fatty acids. The activity of A beta(25-35) was compared to that of A beta(25-35) with either a scrambled sequence [A beta(25-35)scram] or a peptide sequence in which methionine was replaced with leucine [A beta(25-35) met]. A beta(25-35) inhibited lipid peroxidation in a dose- and time-dependent manner. The antioxidant activity of A beta(25-35) was observed at concentrations as low as 10 nM. The relative antioxidant activities of the amyloid beta protein fragments were as follows: A beta(25-35) > A beta(25-35) met > A beta(25-35)scram. The two more potent peptides intercalated into the membrane hydrocarbon core, as determined by small-angle x-ray diffraction approaches. These findings indicate that the amphiphilic A beta(25-35) peptide inhibits lipid peroxidation at low concentrations as a result of physicochemical interactions with the membrane lipid bilayer.

Alzheimer Disease↗
PubMed1997

Drosophila telomere elongation.

Drosophila melanogaster has an unusual telomere elongation mechanism. Instead of short repeats that are synthesized by telomerase, long retrotransposons, HeT-A and TART, transpose to the ends of chromosomes. This mechanism generates tandem arrays of these elements at the chromosome ends, in which all elements are oriented with their oligo(A) tails towards the centromere. Structural features of HeT-A and TART elements may provide clues as to their transposition mechanism. Drosophila telomere length polymorphism is mainly due to terminal retrotransposon arrays that differ between chromosome tips and that change with time. In addition, stable terminal chromosome deletions can be generated that do not contain terminal HeT-A and TART arrays, suggesting that, unlike the equivalent terminal repeats in yeast and humans, the presence and length of terminal arrays in Drosophila may not be critical for cell cycle progression.

Animals↗
PubMed1997

Effect of oxidative stress on membrane structure: small-angle X-ray diffraction analysis.

Free radical damage to cellular membranes appears to underlie alterations in function in aging and various pathological processes, including cardiovascular disease. The objective of this study was to directly characterize changes in the molecular structure of membrane lipid bilayers resulting from oxidative stress. Membrane samples reconstituted from either synthetic or cardiac phospholipids enriched with polyunsaturated fatty acids were examined at high resolution using small-angle x-ray diffraction methods. In this study, Fe2+/ascorbate-induced lipid peroxidation produced significant and dose-dependent alterations in the basic physical structure of the phospholipid bilayer. Electron density profiles (A vs. electrons/A3) calculated from the x-ray diffraction data showed a marked reduction in the hydrocarbon core width of dilinoleoyl phosphatidylcholine (DLPC) bilayers from 36 A to 32 A, and a decrease in overall membrane width, including surface hydration, from 48.7 A to 44.6 A. In addition, a broad decrease in molecular volume was observed +/-3-10 A from the center of the membrane bilayer, along with interdigitation of the terminal methyl segments. Pronounced changes in the lipid bilayer structure following oxidative stress were also observed in membranes reconstituted from cardiac lipids, including a 4 A reduction in hydrocarbon core width from 40 A to 36 A and interdigitation of the terminal methyl segments. These data provide direct evidence for changes in membrane hydrocarbon core width and molecular volume resulting from phospholipid peroxidation, which may contribute to perturbations in membrane structure/function relationships associated with aging and cardiovascular disease.

Aging↗
PubMed1997

Chromosome end elongation by recombination in the mosquito Anopheles gambiae.

One of the functions of telomeres is to counteract the terminal nucleotide loss associated with DNA replication. While the vast majority of eukaryotic organisms maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, other mechanisms such as the transposition of retrotransposons or recombination can also be used in some species. Chromosome end regression and extension were studied in a medically important mosquito, the malaria vector Anopheles gambiae, to determine how this dipteran insect maintains its chromosome ends. The insertion of a transgenic pUChsneo plasmid at the left end of chromosome 2 provided a unique marker for measuring the dynamics of the 2L telomere over a period of about 3 years. The terminal length was relatively uniform in the 1993 population with the chromosomes ending within the white gene sequence of the inserted transgene. Cloned terminal chromosome fragments did not end in short repeat sequences that could have been synthesized by telomerase. By late 1995, the chromosome ends had become heterogeneous: some had further shortened while other chromosomes had been elongated by regenerating part of the integrated pUChsneo plasmid. A model is presented for extension of the 2L chromosome by recombination between homologous 2L chromosome ends by using the partial plasmid duplication generated during its original integration. It is postulated that this mechanism is also important in wild-type telomere elongation.

Animals↗
PubMed1996

Catecholamine metabolism and in vitro induction of premature cuticle melanization in wild type and pigmentation mutants of Drosophila melanogaster.

The major pathway leading to adult cuticle melanization in Drosophila melanogaster has been investigated by a combination of biochemical and genetic approaches. By comparing catecholamine pools in newly emerged flies and in frass (excreta) collected 1 to 4 days after eclosion from wild type with those obtained from several pigmentation mutants, the major flow of catecholamines through the pathway to an unidentified final catabolite was determined. We also demonstrate that incubation with dopamine in vitro induces premature melanization in wild type unpigmented pharate adults several hours before the developmentally programmed onset of melanization, supporting the hypothesis that the availability of catecholamines may be the limiting factor determining the onset of melanization and that the major enzymatic activities that act downstream of dopa decarboxylase in the pathway are deposited into the cuticle before pigmentation begins. In vitro melanization studies with various pigmentation mutants that are associated with critical enzymatic steps in Drosophila catecholamine metabolism are consistent with their proposed function and suggest a central role of N-beta-alanyldopamine in adult cuticle pigmentation.

Alleles↗
PubMed1996

Molecular characterization of the Anopheles gambiae 2L telomeric region via an integrated transgene.

A Drosophila P-element derivative (pUChsneo) integrated into the telomeric region of the left arm of the second chromosome of Anopheles gambiae was used to clone the proximally flanking An. gambiae sequences. Molecular analyses revealed that the pUChsneo construct was partially duplicated and had integrated into a subterminal minisatellite. This satellite has a repeat unit of 820 bp and is located exclusively at the tip of 2L. No sequence similarity to subterminal minisatellites from other dipterans was detected, but some structural features such as tandem subrepeats are shared. The end of the chromosome was mapped with respect to restriction sites in pUChsneo at approximately generation 100 after the integration event. Considering inevitable terminal nucleotide loss due to incomplete DNA replication, we conclude that the chromosome end must have undergone a dramatic elongation process since it was mapped in generation 23.

Animals↗
PubMed1995

DNA organization and polymorphism of a wild-type Drosophila telomere region.

Telomeres at the ends of linear chromosomes of eukaryotes protect the chromosome termini from degradation and fusion. While telomeric replication/elongation mechanisms have been studied extensively, the functions of subterminal sequences are less well understood. In general, subterminal regions can be quite polymorphic, varying in size from organism to organism, and differing among chromosomes within an organism. The subterminal regions of Drosophila melanogaster are not well characterized today, and it is not known which and how many different components they contain. Here we present the molecular characterization of DNA components and their organization in the subterminal region of the left arm of chromosome 2 of the Oregon RC wild-type strain of D. melanogaster, including a minisatellite with a 457bp repeat length. Two distinct polymorphic arrangements at 2L were found and analyzed, supporting the Drosophila telomere elongation model by retrotransposition. The high incidence of terminal chromosome deficiencies occurring in natural Drosophila populations is discussed in view of the telomere structure at 2L.

Animals↗
PubMed1991

Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development.

The yellow (y) gene of Drosophila is required for the formation of black melanin and its deposition in the cuticle. We have studied by immunohistochemical methods the temporal and spatial distribution of the protein product of the y gene during embryonic and pupal development and have correlated its expression with events of cuticle synthesis by the epidermal cells and with cuticle sclerotization. Except for expression in early embryos, the y protein is only found in the epidermal cells and may be secreted into the cuticle as it is being deposited. The amount of y protein in various regions of the embryo and pupa correlates directly with the intensity of melanization over any section of the epidermis. Expression of the y gene begins in the epidermal cells at 48 hr after pupariation and is well correlated with the beginning deposition of the adult cuticle. At this stage the adult cuticle is unsclerotized and unpigmented and dopa decarboxylase levels, a key enzyme in catecholamine metabolism which provides the crosslinking agents as well as the precursors for melanin, is low. As a separate event 26 hr after the onset of y gene expression, the first melanin deposition occurs in the head bristles and pigmentation continues in an anterior to posterior progression until eclosion. This melanization wave is correlated with elevated dopa decarboxylase activity. Crosslinking of the adult cuticle also occurs in a similar anterior to posterior progression at about the same time. We have shown by imaginal disc transplantation that timing of cuticle sclerotization depends on the position of the tissue along the anterior-posterior axis and that it is not an inherent feature of the discs themselves. We suggest that actual melanization and sclerotization of the cuticle by crosslinking are initiated at this time in pupal development by the availability of the catecholamine substrates which diffuse into the cuticle. Intensity of melanization and position of melanin pigment is determined by the presence or absence of the y protein in the cuticle, thus converting the y protein prepattern into the melanization pattern.

Animals↗
PubMed1990

Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos.

Heat shock has a dramatic effect on the organization of the cytoplasm, causing the intermediate filament cytoskeleton to aggregate at the nucleus. This has previously been shown in cultured Drosophila and mammalian cells. In this paper we analyze the heat lability of the intermediate filament cytoskeleton in early Drosophila embryos by indirect immunofluorescence. At all stages of embryogenesis tested, the intermediate filament cytoskeleton, which is maternally provided, is severely disturbed by 30 min heat shock at 37 degrees C. After the nuclei have migrated to the subcortical cytoplasm, it collapses around them. Nuclei in all heat-shocked embryos are considerably enlarged and become displaced. Embryos before cellular blastoderm stage, in which heat shock protein synthesis is not inducible, are irreversibly arrested in development by heat shock. Embryos at or after cellular blastoderm, which do synthesize heat shock proteins in response to stress, are also immediately arrested in development but continue development when returned to 25 degrees C. We discuss the possibility that cytoplasmic events such as the intermediate filament cytoskeleton rearrangement may be involved in heat shock-mediated phenocopy induction.

Animals↗
PubMed1987

A non-filamentous configuration of intermediate-sized filament proteins in Drosophila Kc tissue culture cells.

Using monoclonal antibodies against the major intermediate filament (10 nm) cytoskeletal proteins of Drosophila tissue culture cells, we showed by indirect immunofluorescence and immuno-electron microscopy that this cytoskeletal material also occurs in a non-filamentous configuration. Patches of fine granular material are detected in the cytoplasm of Kc cells but are absent in another Drosophila cell line (Schneider, line 2). These patches are surrounded by membranes with bound ribosomes, resembling endoplasmic reticulum, and are found throughout the cytoplasm. We suggest that these aggregates are caused by overproduction of intermediate filament material in the Kc cell line.

Animals↗
PubMed1986

Liposome-mediated delivery of antibody to a Drosophila cell line.

Large, unilamellar vesicles composed of equimolar amounts of acidic phosopholipids and phosphatidylethanolamine were able to deliver fluorescent dye [5(6)-carboxyfluorescein] or a monoclonal antibody directed against intermediate-filament proteins to a Drosophila cell line (Kc cells). Millimolar Ca2+ or protamine sulfate in microgram quantities triggered rapid, synchronous delivery of either solute. Delivery required a specific lipid composition: liposomes composed of 1:1 mole ratios of phosphatidylethanolamine:phosphatidylserine were able to deliver their contents, but not if phosphatidylcholine was substituted for phosphatidylethanolamine. Light microscopic observation of Kc cells incubated with free dye or antibody alone showed very little uptake, a result indicating that encapsulation within liposomes is a prerequisite for substantial delivery. Moreover, the stability of adhering vesicles in the absence of calcium or protamine sulfate, the lipid specificity, and the rapid onset of intracellular fluorescence after triggering suggest that vesicle-cell fusion is the predominant mode of solute uptake. Fusion of liposomes with the cell membrane was confirmed by freeze-fracture electron microscopy, which showed liposome vesicles first adhering to cell surfaces, then undergoing fusion when calcium or protamine sulfate was added.

Animals↗
PubMed1984

A monoclonal antibody that detects vimentin-related proteins in invertebrates.

Drosophila melanogaster contains a 46 000 MW cytoplasmic protein which is immunologically related to the intermediate filament protein vimentin of vertebrates. A monoclonal antibody raised against this protein was used to study its cross-reactivity with other vertebrate and invertebrate cells. Indirect immunofluorescence showed filamentous meshworks in all species tested. Protein blotting was used to determine the molecular weights of the proteins responsible for the wide range of cross-reactivity of this antibody. We present evidence that vimentin-like proteins are also present in invertebrates and form a cytoplasmic network in Paramecium. Furthermore, we demonstrate in vertebrates and invertebrates the presence of high molecular weight polypeptides which are immunologically related to vimentin.

Animals↗
PubMed1984

Intermediate-sized filaments in Drosophila tissue culture cells.

In using a monoclonal antibody against a major cytoplasmic protein of 46,000 mol wt, we have characterized an intermediate-sized (10 nm) filamentous cytoskeleton in Drosophila melanogaster tissue culture cells. Indirect immunofluorescence, immunoelectron microscopy, and protein blotting show that this cytoskeleton exhibits features typical of the vertebrate vimentin cytoskeleton, including the diameter and appearance of filaments, sensitivity to 10(-6) M colcemid, and insolubility in buffers containing 1% Triton X-100. The antibody cross-reacts with vimentin and desmin from baby hamster kidney cells and stains a vimentin cytoskeleton in the vertebrate Chinese hamster ovary cell line. We, therefore, conclude that the 46,000-mol wt Drosophila protein is homologous to vertebrate vimentin. Three minor, higher-molecular-weight polypeptides are also detected in the Drosophila cells that react with the antibody. At least two of these are members of a family of proteins with properties resembling those of the 46,000-mol wt intermediate filament protein.

Animals↗
PubMed1984

The Dunce gene of Drosophila: roles of Ca2+ and calmodulin in adenosine 3':5'-cyclic monophosphate-specific phosphodiesterase activity.

Two genetically distinct forms of cyclic nucleotide phosphodiesterases are present in adult Drosophila melanogaster. Form II, which specifically hydrolyzes adenosine 3':5'-cyclic monophosphate (cAMP), is controlled by the dunce+ gene. Mutants of this gene either eliminate this enzyme form entirely or alter its kinetic and thermal properties, suggesting that dunce+ is the structural gene for this enzyme. These mutants are defective in memory formation, habituation, and sensitization and exhibit elevated cAMP levels, implicating cAMP in these neurological processes. The other phosphodiesterase, Form I, which hydrolyzes both cAMP and guanosine 3':5'-cyclic monophosphate (cGMP), is not affected by dunce mutations. Because both cAMP and Ca2+ serve as intracellular second messengers in mediating the effects of neurotransmitters, the effects of Ca2+ on each form of phosphodiesterase have been investigated. Previous work has suggested that Form I is activated by calmodulin in a Ca2+-dependent manner. We confirm this activation and demonstrate that the activation involves the Ca2+-dependent association of two molecules of calmodulin with one Form I molecule. Under conditions permitting activation and association of Form I with calmodulin, we observe no interaction of Ca2+/calmodulin with Form II. Our studies suggest that the primary physiological defect, associated with a defective or absent Form II cAMP-specific phosphodiesterase and leading to the dunce neurological phenotype, is due to a direct failure to regulate the cAMP level in nerve cells rather than to a failure to mediate a signal resulting from a cAMP-induced Ca2+ influx, associated with presynaptic facilitation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Calcium receptor protein calmodulin isolated from cilia and cells of Paramecium tetraurelia.

A low molecular weight protein of about 17 000 as determined by SDS-polyacrylamide gel electrophoresis has been isolated from cilia and cell bodies, respectively, from Paramecium tetraurelia (wildtyp 51s). This protein has been identified as calmodulin by various properties previously ascribed to calmodulin from other vertebrate and invertebrate systems. These properties are heat stability, electrophoretic mobility and its ability to activate in the presence of calcium a calmodulin-dependent phosphodiesterase from pig brain. Calmodulin is present in rather high amounts in cell bodies (75 micrograms/g) and also in isolated cell-free cilia (up to 50 micrograms/g). Its presence in cilia suggests a role in the control of ciliary activity.

Animals↗