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Biomedical subjects

D Alford

Publications and source records attributed to D Alford.

At least 19 recordsLinked to original sources
PubMed2000

The irrelevant sound effect: does speech play a special role?

Memory for order is markedly impaired by the presence of irrelevant sound, even though participants are instructed to ignore the sound. Although a great deal of research has disclosed some features of the task and of the sound that augment or reduce the degree of interference, one important issue of the irrelevant sound effect not yet resolved is whether speech has a special status. This study revealed, within a design of adequate power, that the same physical stimulus (sine wave speech), whether perceived as speech or as nonspeech sound, produces similar degrees of disruption and is less disruptive of serial recall than natural speech. This outcome suggests that the acoustic constituents of sound rather than its source are most influential in determining the impact of irrelevant material.

Adolescent↗
PubMed2000

Interference from degraded auditory stimuli: linear effects of changing-state in the irrelevant sequence.

Cognitive performance, particularly on a number of tasks involving short-term memory for order, is impaired by the mere presence of irrelevant background sound. The current study examines the features of the irrelevant sound that determine its disruptive potency. Previous research suggests that the amount of variability in an irrelevant stream is related to the degree of disruption of memory. The present experiments used a parametric approach to manipulate degree of change more precisely. Increasing levels of degradation, effected either by low-pass filtering (speech) or by digital manipulation (speech and nonspeech), monotonically decreased the degree of interference. The findings support the following propositions: (i) the degree of physical change within an auditory stream is the primary determinant of the degree of disruption; and, (ii) the effects of irrelevant speech and irrelevant nonspeech sounds are functionally similar.

Auditory Perception↗
PubMed2000

Morphogenetic and proliferative responses to heregulin of mammary epithelial cells in vitro are dependent on HER2 and HER3 and differ from the responses to HER2 homodimerisation or hepatocyte growth factor.

The effects of heregulin on the cell line HB2, derived from immortalised human luminal mammary epithelial cells, have been examined. HB2 cells, which normally form smooth spherical colonies in collagen gels, exhibited a striking heregulin-induced morphological change to colonies projecting a large number of spiky branches. A mitogenic effect of heregulin on HB2 cells was also seen, which was more pronounced on collagen than on plastic, whereas cell motility was unaffected. HB2 cells were found to express the heregulin receptor subunits HER2 and HER3, but not HER4. Treatment of HB2 cells with heregulin also induced tyrosine phosphorylation of a band shown by immunoprecipitation to contain HER3. Using specific receptor-blocking antibodies, it was found that both the morphogenetic and proliferative responses of heregulin in HB2 cells were mediated by HER2 and HER3. To compare the effects of HER2 in heregulin signaling to heregulin-independent HER2 homodimerisation (thought to be a carcinoma-associated event), HB2 cells were transfected with the trk-neu hybrid receptor which could be induced to form homodimers by NGF. Although activated HER2 homodimers induced proliferation in the HB2 transfectants in collagen, a morphological response in collagen was not seen, suggesting that HER3 signaling is important for morphogenesis in this cell type.

Breast↗
PubMed1998

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.

Animals↗
PubMed1998

Integrin-matrix interactions affect the form of the structures developing from human mammary epithelial cells in collagen or fibrin gels.

The HB2 cell line, developed from luminal epithelial cells cultured from milk, forms ball-like structures in collagen gels which show a uniform branching response to hepatocyte growth factor. The alpha2beta1 integrin is the major integrin expressed by luminal epithelial cells, and the role of this integrin in mammary morphogenesis has been analysed using HB2 cells cultured in collagen gels and antibodies which affect integrin function. Selectivity of response was followed by comparing effects on morphogenesis in fibrin, where the alphavbeta1 integrin interacts with the matrix. In the presence of hepatocyte growth factor, using alpha2 and beta1 antibodies in collagen and alphav and beta1 antibodies in fibrin, complete blocking of the cell-matrix interaction inhibits cell survival. With partial blocking of the integrin-ligand interaction, the cells proliferate but form dissociated colonies. Activating antibodies to the beta1 integrin subunit which enhance the matrix interaction dramatically inhibit the branching and motility responses to hepatocyte growth factor. A series of non-blocking alpha2 reactive antibodies also inhibit these responses specifically in or on collagen. Studies with ras-transfected HB2 cells emphasise the importance of the alpha2beta1 collagen interaction in the development of form since HB2ras cells, which express reduced levels of the alpha2beta1 integrin, form dissociated colonies in collagen but not in fibrin. Treatment of HB2ras cells with a beta1 activating antibody, however, induces the formation of compact colonies. Even though the ras-transformants form colonies in agar, complete blocking of the alpha2beta1/collagen interaction does not allow survival in collagen. The results indicate that in mammary morphogenesis, the strength of the interaction of integrins with the extracellular matrix modulates the response to motogenic factors and contributes to the definition of form.

Animals↗
PubMed1998

Adhesion molecules in breast cancer: role of alpha 2 beta 1 integrin.

An early event in the development of breast carcinomas is the loss of normal tissue architecture. In benign lesions and in situ tumours both luminal and myoepithelial cells are present, but in most invasive cancers the malignant cell has the phenotype of the luminal cell, and proliferates without contacting the myoepithelial cells or the basement membrane. The reduction in cell contacts is clearly crucial for the initiation of metastatic growth, and is accompanied by a loss of expression or function of cell adhesion molecules. Immunohistochemical studies using tissue and tumour sections indicate that a decrease in the level of expression of the alpha 2 beta 1 integrin is observed in many breast cancers. A specific and crucial role for this molecule in the maintenance of normal morphological differentiation has also been demonstrated in in vitro studies. The evidence from these studies suggests that, in mammary epithelial cells, oncogenes may be upstream regulators of the expression of the alpha 2 beta 1 integrin and of other specific molecules important for epithelial differentiation. These findings implicate oncogenes in the initial events relating to the disruption of tissue architecture that is seen in invasive breast cancer.

Breast Neoplasms↗
PubMed1996

Influenza-virus-liposome lipid mixing is leaky and largely insensitive to the material properties of the target membrane.

Monolayer intrinsic curvature, void stabilization, and membrane rupture tension have been suggested as important factors determining the rate of membrane fusion. Here, we have studied the kinetics of fusion between influenza virus and target liposomes as a function of various target membrane material properties. In order to examine the fusion process directly, a simple prebinding step is used and proven to be adequate to achieve fusion-rate-limiting kinetics. To test the hypothesis about membrane curvature and void stabilization, we studied the lipid mixing kinetics with dioleoylphosphatidylcholine (DOPC)/ganglioside GD1a (GD1a) liposomes containing lysooleoylphosphatidylcholine (LPC, positive curvature), dioleoyglycerol (DOG, negative curvature), arachidonic acid (AA, negative curvature), and hexadecane (HD, void stabilization). DOG, AA, and HD (at 4 mol%) showed no significant effect on the fusion kinetics, while LPC reversibly inhibited influenza HA mediated fusion only at very high concentrations. Using target liposomes with different membrane rupture tension values, no obvious correlation between membrane rupture tension and the rate of lipid mixing was observed. Moreover, a reported potential antiviral compound, tert-butylhydroquinone (t-b-HQ) (Bodian et al., 1993), showed no significant effect on the kinetics of influenza fusion. Finally leakage of liposome contents was detected during lipid mixing. For encapsulated molecules smaller than 450 MW, the kinetics of leakage is very similar to the kinetics of lipid mixing. In fact, leakage was also detected for encapsulated molecules up to 10 000 MW, suggesting that HA mediated lipid mixing is a very leaky process. Since "nonleaky fusion" has been the foundation of influenza fusion models, our work suggests the need for a major revision in the modeling of this process.

Influenza A virus↗
PubMed1996

Cell adhesion molecules in the normal and cancerous mammary gland.

Loss of normal tissue morphology is one of the first changes seen in the development of solid malignant tumours, including breast cancers. Since cell adhesion molecules play a crucial role in maintaining normal tissue architecture, investigations into the molecular mechanisms involved in the morphological changes occurring in malignancy that focus on the expression and function of cell adhesion molecules in malignant progression are extremely relevant. This review discusses the cadherin and integrin families of adhesion molecules which have been shown to be extremely important in the cell-cell and cell-matrix interactions of epithelial cells. Immunohistochemical studies using tissue and tumour sections indicate that a reduction in levels of expression or function of both types of cell adhesion molecules is indeed observed in many breast cancers. A specific and crucial role for these molecules in the maintenance of normal morphological differentiation has been demonstrated in vitro, where the noninvasive differentiated phenotype correlates with the normal functioning of E-Cadherin and alpha 2 beta 1 integrin. Further in vitro evidence suggests that in mammary epithelial cells, oncogenes may be upstream regulators of both the expression and function of E-Cadherin, the alpha 2 beta 1 integrin, and other epithelial specific molecules important for maintaining epithelial differentiation. In this way they could manifest their effects on the tumorigeneic potential of epithelial cells.

Animals↗
PubMed1995

Membrane permeabilization by Listeria monocytogenes phosphatidylinositol-specific phospholipase C is independent of phospholipid hydrolysis and cooperative with listeriolysin O.

We have examined potential cooperative interactions of Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) and listeriolysin O (LLO), a pore-forming hemolysin, in a liposome lysis assay. Large unilamellar vesicles, approximately 0.1 micron in diameter, encapsulating the fluorescent probe calcein, were treated with PI-PLC or LLO at pH 6.0, and each was capable of causing dye release. With phosphatidylcholine/phosphatidylinositol/cholesterol liposomes at 0.1 microM lipid, minimal release of dye was observed on addition of 80 pM LLO or 7 nM PI-PLC. Addition of the two proteins together produced rapid dye release. Unexpectedly, essentially identical results were obtained with phosphatidylcholine/cholesterol liposomes. Thus, the effect of PI-PLC did not depend on lipid hydrolysis. Both proteins also released inulin (M(r) 5200) from liposomes. Membrane permeabilization was not accompanied by membrane fusion. Very little dye release from phosphatidylcholine/phosphatidylinositol/cholesterol liposomes was seen with PI-PLC from Bacillus thuringiensis, and addition of this enzyme to LLO produced no additional dye release; however PI-PLC from L. monocytogenes cooperated with perfringolysin O from Clostridium perfringens. PI-PLC from L. monocytogenes and LLO bind to phosphatidylcholine/cholesterol liposomes, and the rate of binding of each protein was not influenced by the presence of the other. These data support a postulated accessory role for PI-PLC with LLO in lysing the primary phagosome of a macrophage.

Bacterial Proteins↗
PubMed1994

Fusion of influenza virus with sialic acid-bearing target membranes.

We have monitored the fusion of intact A/PR/8/34 influenza virus with glycophorin-bearing liposomes and with ganglioside- (GD1a-) containing liposomes. The lipid bilayers of the glycophorin-bearing liposomes had several compositions, including pure dioleoylphosphatidylethanolamine (DOPE), pure egg phosphatidylethanolamine (EPE), and pure dioleoylphosphatidylcholine (DOPC). Examination of the temperature dependence of fusion for these and other compositions showed that even if the lipids are competent to form inverted hexagonal phases (HII), there is no enhancement of the fusion rate constant at the L alpha-HII phase transition temperature of the lipids, TH. Thus, the HII phase transition is not involved in the HA-mediated fusion mechanism. However, this mechanism is sensitive to lipid composition, in that PC bilayers fused more slowly than PE-containing bilayers above 20 degrees C. These results show that the HA-mediated fusion mechanism depends primarily upon specific lipid-protein interactions, although the fundamental parameters of lipid phase stability (interstice stabilization and monolayer spontaneous radius of curvature) may also be important. The fact that HII phase-component lipid bilayers in the glycophorin liposomes do not enhance the HA-mediated fusion rate strongly suggests that substantial bilayer-bilayer contact is not involved in HA-mediated fusion. Previously, we have shown that glycoprotein-bearing liposomes bind to HA-expressing cells specifically through HA-glycophorin interactions and that fusion is mediated by HAs not bound to glycophorin. Thus, with respect to the target membrane, the fusion site involves just the lipid bilayer. Our results with GD1a-containing liposomes strongly suggest that HAs bound to this sialic acid-bearing molecule are likewise incapable of participating in the fusion site. This could be due to a diminished lateral mobility of the HAs simultaneously bound to both closely apposed membranes. Finally, we find that the low-pH-induced viral inactivation is inhibited by binding to either glycophorin- or GD1a-containing target membranes.

Animals↗
PubMed1994

Branching morphogenesis of human mammary epithelial cells in collagen gels.

To study the morphogenesis of human epithelial cells in vitro we have used a three-dimensional collagen matrix and a newly developed mammary epithelial cell line, 1-7 HB2. In standard medium 1-7 HB2 cells formed compact balls/spheres inside collagen type I gels, while cocultivation with various fibroblast cell lines or growth in fibroblast-conditioned media resulted in the appearance of branching structures. At least two different soluble factors secreted by fibroblasts were found to be implicated in the branching morphogenesis. Firstly, hepatocyte growth factor/scatter factor could induce branching in a concentration-dependent manner. Moreover, a polyclonal serum against hepatocyte growth factor/scatter factor completely inhibited the branching morphogenesis induced by medium conditioned by MRC-5 fibroblast cells. In contrast, a morphogenetic activity secreted by human foreskin fibroblasts was identified that appears to be different from hepatocyte growth factor/scatter factor and from a number of other well-characterized growth factors or cytokines. This model system has been used to examine the role of integrins in mammary morphogenesis. The expression of the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins was decreased when cells were plated on collagen gels. The addition of specific blocking monoclonal antibodies directed to the alpha 2- and beta 1-integrin subunits to growth media impaired cell-cell interactions and interfered with the formation of compact structures inside collagen gels, suggesting that the alpha 2 beta 1 integrin can control intercellular adhesion in mammary morphogenesis. In contrast one of the blocking monoclonal antibodies against the alpha 3-integrin subunit (P1B5) mimicked the effect of soluble 'morphogens'. Our results suggest that the modulation of alpha 3 beta 1 activity may represent an important event in the induction of branching morphogenesis of human mammary epithelial cells.

Animals↗
PubMed1992

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells.

The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma (S49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4.9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5.4, different from that of the virus preincubated at low pH without target membranes.

Cell Membrane↗
PubMed1990

An architecture for the fusion site of influenza hemagglutinin.

The recent finding that more than one Influenza hemagglutinin (HA) is required at the fusion site for HA-expressing fibroblasts, together with the crystal structure of HA at neutral pH, provide the basic elements of a plausible model for this fusion site. Within an aggregate of HA trimers at low pH, we propose fusion intermediates which are based upon a minimal alteration to the known neutral pH structure of HA and which should have reasonable activation energies. This is the first model of a glycoprotein-mediated fusion site which explicitly accounts for the disposition of the lipids within these intermediates. While the fusion site created by HA will not be the same as that of eukaryotic fusion complexes, general characteristics could be shared.

Animals↗
PubMed1989

Membrane fusion and inverted phases.

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.

Calorimetry, Differential Scanning↗
PubMed1989

Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases.

In the preceding paper (Ellens et al., 1989), it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, delta TI, below the L alpha/HII phase transition temperature, TH. TH and delta TI can be extremely sensitive to the lipid composition. The present work shows that 2 mol% of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower TH, delta TI, and the temperature for fast membrane fusion by 15-20 degrees C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo.

Chemical Phenomena↗
PubMed1988

Synexin enhances the aggregation rate but not the fusion rate of liposomes.

The effect of synexin on the calcium-induced fusion of large unilamellar liposomes was studied by using two assays for the mixing of aqueous contents. The results were analyzed in terms of the mass action kinetic model, which describes the overall fusion reaction as a two-step sequence consisting of a second-order process of liposome aggregation followed by a first-order fusion reaction. By using several different lipid compositions and varying the electrolyte composition, it was possible to select the rate-limiting step of the overall fusion process. When aggregation was the rate-limiting step, as in the case of Ca2+-induced fusion of phosphatidylserine (PS), phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3), and PS/PE (1:3) liposomes, synexin increased the overall fusion kinetics by increasing the aggregation rate constant (up to 100-fold). When aggregation was rapid compared to destabilization of apposed membranes, i.e., fusion was rate limiting, synexin either had no effect or reduced the overall fusion kinetics. In one such case involving liposomes composed of PA/PS/PE/phosphatidylcholine (PC) (10:15:65:10), synexin reduced the fusion rate constant by 50%. The effect of calcium-induced synexin polymerization was investigated by preincubation of synexin with calcium prior to addition of liposomes. Prepolymerization by Ca2+ always decreased the activity of synexin such that it was less than the activity of an equal amount of untreated monomers. However, it was found that the activity of synexin monomers polymerized to an average hexameric size was greater than that of one-sixth as many untreated monomers, with respect to the liposome aggregation rate constant. Neither polymers nor monomers increased the fusion rate constant.

Animals↗