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L A Allen

Publications and source records attributed to L A Allen.

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

Transformation of C3H 10T1/2 cells by low doses of ionising radiation: a collaborative study by six European laboratories strongly supporting a linear dose-response relationship.

For the assessment of radiation risk at low doses, it is presumed that the shape of the low-dose-response curve in humans for cancer induction is linear. Epidemiological data alone are unlikely to ever have the statistical power needed to confirm this assumption. Another approach is to use oncogenic transformation in vitro as a surrogate for carcinogenesis in vivo. In mid-1990, six European laboratories initiated such an approach using C3H 10T1/2 mouse cells. Rigid standardisation procedures were established followed by collaborative measurements of transformation down to absorbed doses of 0.25 Gy of x-radiation resulting in a total of 759 transformed foci. The results clearly support a linear dose-response relationship for cell transformation in vitro with no evidence for a threshold dose or for an enhanced, supralinear response at doses approximately 200-300 mGy. For radiological protection this represents a large dose, and the limitations of this approach are apparent. Only by understanding the fundamental mechanisms involved in radiation carcinogenesis will further knowledge concerning the effects of low doses become available. These results will, however, help validate new biologically based models of radiation cancer risk thus providing increased confidence in the estimation of cancer risk at low doses.

Animals↗
PubMed1997

MARCKS regulates membrane ruffling and cell spreading.

The dynamic rearrangement of the actin cytoskeleton is fundamental to most biological processes including embryogenesis, morphogenesis, cell movement, wound healing and metastasis [1]. Membrane ruffling and reversible cell-substratum interactions underlie actin-driven cell movement. Protein kinase C (PKC) stimulates membrane ruffling and adhesion [2], but the mechanism by which this occurs is unknown. Myristoylated alaninerich C kinase substrate (MARCKS) is a PKC substrate that cycles on and off membranes by a mechanism termed the myristoyl-electrostatic switch [3-6]. While at the membrane, MARCKS binds to and sequesters acidic phospholipids including phosphatidyl-inositol-4,5-bisphosphate (PIP2) [7]. MARCKS also binds and cross-links filamentous actin, an activity which is regulated by PKC-dependent phosphorylation and calcium-calmodulin [3]. In this report, we demonstrate that expression, in fibroblasts, of MARCKS containing a mutation which abrogates the myristoyl-electrostatic switch prevents cell spreading. The MARCKS mutant arrests the cell during an early stage of spreading, characterized by profuse membrane blebbing, and prevents the formation of membrane ruffles and lamellae usually found at the leading edge of spreading cells. This defect in the regulation of the actin cytoskeleton is accompanied by a decrease in cell-substratum adhesion. Our results provide direct evidence that MARCKS and PKC regulate actin-dependent membrane ruffling and cell adhesion, perhaps via a PIP2-dependent mechanism.

Actins↗
PubMed1997

Effects of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast.

Oxygen is an important environmental regulator for the transcription of several genes in Saccharomyces cerevisiae, but it is not yet clear how this yeast or other eukaryotes actually sense oxygen. To begin to address this we have examined the effects of oxygen concentration on the expression of several nuclear genes (CYC1, CYC7, COX4, COX5a, COX5b, COX6, COX7, COX8, and COX9) for proteins of the terminal portion of the respiratory chain. COX5b and CYC7 are hypoxic genes; the rest are aerobic genes. We have found that the level of expression of these genes is determined by oxygen concentration per se and not merely the presence or absence of oxygen and that each of these genes has a low oxygen threshold (0. 5-1 microM O2) for expression. For some aerobic genes (COX4, COX5a, COX7, COX8, and COX9) there is a gradual decline in expression between 200 microM O2 (air) and their oxygen threshold. Below this threshold expression drops precipitously. For others (COX5a and CYC1) the level of expression is nearly constant between 200 microM O2 and their threshold and then drops off. The hypoxic genes COX5b and CYC7 are not expressed until the oxygen concentration is below 0.5 microM O2. These studies have also revealed that COX5a and CYC1, the genes for the aerobic isoforms of cytochrome c oxidase subunit V and cytochrome c, and COX5b and CYC7, the genes for the hypoxic isoforms of cytochrome c oxidase subunit V and cytochrome c, are coexpressed at a variety of oxygen concentrations and switch on or off at extremely low oxygen concentrations. By shifting cells from one oxygen concentration to another we have found that aerobic genes are induced faster than hypoxic genes and that transcripts from both types of gene are turned over quickly. These findings have important implications for cytochrome c oxidase function and biogenesis and for models of oxygen sensing in yeast.

Aerobiosis↗
PubMed1996

Molecular determinants of the myristoyl-electrostatic switch of MARCKS.

MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmodulin and actin, and which has been implicated in cell motility, phagocytosis, membrane traffic, and mitogenesis. MARCKS cycles on and off the membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A.(1995) Trends Biochem. Sci. 20, 272-276). Here we define the molecular determinants of the myristoyl-electrostatic switch. Mutation of the N-terminal glycine results in a nonmyristoylated form of MARCKS which does not bind membranes and is poorly phosphorylated. This indicates that myristic acid targets MARCKS to the membrane, where it is efficiently phosphorylated by PKC. A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmitoylated, is phosphorylated by PKC but not released from the membrane. Thus two palmitic acid moieties confer sufficient membrane binding energy to render the second, electrostatic membrane binding site superfluous. Mutation of the PKC phosphorylation sites results in a mutant which does not translocate from the membrane to the cytosol. A mutant in which the intervening sequence between the myristoyl moiety and the basic effector domain is deleted, is not displaced from the membrane by PKC dependent phosphorylation, fulfilling a theoretical prediction of the model. In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluorescence microscopy demonstrates that specific protein-protein interactions also specify the intracellular localization of MARCKS.

Animals↗
PubMed1996

Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages.

It has long been known from the results of ultrastructural studies that complement- and immunoglobulin G (IgG)-opsonized particles are phagocytosed differently by macrophages (Kaplan. G. 1977. Scand. J. Immunol. 6:797-807). Complement-opsonized particles sink into the cell, whereas IgG-coated particles are engulfed by lamellipodia, which project from the cell surface. The molecular basis for these differences is unknown. We used indirect immunofluorescence and confocal microscopy to examine how cytoskeletal proteins associate with phagosomes containing complement-opsonized zymosan (COZ) particles or IgG beads in phorbol-myristateacetate-treated peritoneal macrophages. During ingestion of COZ, punctate structures rich in F-actin, vinculin, alpha-actinin, paxillin, and phosphotyrosine-containing proteins are distributed over the phagosome surface. These foci are detected beneath bound COZ within 30 s of warming the cells to 37 degrees C, and their formation requires active protein kinase C. By contrast, during Fc receptor-mediated phagocytosis, all proteins examined were uniformly distributed on or near the phagosome surface. Moreover, ingestion of IgG beads was blocked by tyrosine kinase inhibitors, whereas phagocytosis of COZ was not. Thus, the signals required for particle ingestion, and the arrangement of cytoskeletal proteins on the phagosome surface, vary depending upon which phagocytic receptor is engaged. Moreover, complement receptor (CR)-mediated internalization required intact microtubules and was accompanied by the accumulation of vesicles beneath the forming phagosome, suggesting that membrane trafficking plays a key role in CR-mediated phagocytosis.

Actinin↗
PubMed1996

Mechanisms of phagocytosis.

Recent advances in research on phagocytosis include a better appreciation of the cross-talk between phagocytic receptors, the definition of multiple signaling domains within these receptors, and a deeper understanding of the downstream effector pathways leading to actin polymerization and particle internalization. Phagosome maturation in macrophages proceeds via a series of membrane fusion and fission events, which modify the phagosome in small increments, and appears to be regulated, in part, by GTP-binding proteins and perhaps by protein kinase C. The isolation of dysphagic mutants of Dictyostelium discoideum presages the identification of new genes required for phagocytosis.

Actins↗
PubMed1996

Possible selves in major depression.

Ratings of possible selves and resultant self-concept discrepancies were examined in 25 patients diagnosed with major depression and 25 control subjects. Self-concept discrepancies significantly discriminated patients from controls. The presence of negative features in the self-schema was a stronger indicator of depressive symptomatology than was the absence of positive self-appraisal. Depressives' future projections of self were less pessimistic than predicted by cognitive theories of depression.

Adult↗
PubMed1995

Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.

MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immuno-fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos-) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos- mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.

Alkaloids↗
PubMed1995

Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c.

Subunit V, one of the nuclear-coded subunits of yeast cytochrome c oxidase, has two isoforms, Va and Vb. These alter the in vivo intramolecular rates of electron transfer within the holoenzyme (Waterland, R. A., Basu, A., Chance, B., and Poyton, R. O. (1991) J. Biol. Chem. 266, 4180-4186). The isozyme with Vb has a higher turnover rate and a higher intramolecular transfer rate than the isozyme with Va. To determine how these isoforms affect catalysis, we have examined their effects on the binuclear reaction center and on the interaction between cytochrome c oxidase and the two isoforms, iso-1 and iso-2, of yeast cytochrome c. Infrared spectroscopy of carbon monoxide liganded to heme a3 has revealed a single conformer for the binuclear reaction center in the isozyme with Vb but two discrete conformers in the isozyme with Va. The kinetics of interaction for all four pairwise combinations of isozymes with each subunit V isoform and the two cytochrome c isoforms are biphasic, with high and low affinity electron transfer reactions. In general, the isoforms of cytochrome c and subunit V do not alter the Km but do affect the TNmax. The TNmax for isozymes carrying Vb are higher at both high and low affinity sites for each cytochrome c isoform. Iso-1-cytochrome c supports a higher TNmax than Iso-2-cytochrome c. Surprisingly, the combinatorial effect of both sets of isoforms on TNmax is minimized with the pairs of isoforms (iso-1-cytochrome c and subunit Va or iso-2 and subunit Vb) that are co-expressed in cells. Together, these findings support the conclusion that the subunit V isoforms modulate catalysis and suggest that they do so by affecting the environment or structure of the binuclear reaction center. They also suggest that the coexpression of the two cytochrome c isoforms with two subunit V isoforms serves to minimize differences in electron transfer rates brought about by the subunit V isoforms.

Cytochrome c Group↗
PubMed1995

Self-complexity, self-evaluation, and depression: an examination of form and content within the self-schema.

Six studies examined the relationship between self-complexity and variables related to self-evaluation. Self-complexity was found to comprise two components: positive self-complexity and negative self-complexity. Positive self-complexity was sensitive to methodological factors, namely, variations in stimulus materials used for self-ratings. Negative self-complexity was relatively stable in the face of different rating stimuli and tasks and was related to trait measures of self-evaluation, psychic distress, and psychopathology. These findings were observed and replicated. Higher negative self-complexity was associated with increases in depression symptoms over time. Higher negative self-complexity also predicted a poorer prognosis and less complete recovery from depression in a clinical sample. Results are discussed in light of related research and possible social-cognitive mechanisms.

Adaptation, Psychological↗
PubMed1995

Dimensions of self-concept: a comparison of heroin and cocaine addicts.

Two groups of drug users on an inner-city inpatient drug detoxification unit were studied: 42 heroin addicts and 47 cocaine addicts. The two groups were compared on personality disorder diagnoses, personality traits, and demographic variables. Cocaine and heroin addicts scored similarly on: 1) number and kind of personality disorder diagnoses, with the exception of antisocial personality; 2) all personality traits measured; 3) positive and negative temperament; 4) description of self-concepts; and 5) positivity and negativity of self-concepts. It was also found that heroin addicts showed significantly higher levels of social deviance than the cocaine group, with significantly more antisocial personality disorder diagnoses, higher levels of social deviance, and lower scores on a Propriety Scale. Character pathology was more heterogeneous among cocaine users. Heroin addicts had used significantly longer and showed less educational and occupational achievement than the cocaine group. Our results also suggest that personality psychopathology in drug addicts is associated with lower self-esteem, more negative self-valuation, and longer duration of use.

Adult↗
PubMed1994

Genetic evidence supporting the role of peroxisome assembly factor (PAF)-1 in peroxisome biogenesis. Polymerase chain reaction detection of a missense mutation in PAF-1 of Chinese hamster ovary cells.

The peroxisome/plasmalogen-deficient Chinese hamster ovary (CHO) mutant cell line ZR-78.1 contains a missense mutation in its cDNA-encoding peroxisome assembly factor-1 (PAF-1). Using a rapid polymerase chain reaction assay, we now demonstrate that the genome of ZR-78.1 contains only the mutant allele. When mutant ZR-78.1 is fused with wild-type karyoplasts, occasional "negative nuclear hybrids" are observed that lack peroxisomes (Allen, L.-A. H., Morand, O. H., and Raetz, C. R. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7012-7016). Despite the fact that negative nuclear hybrids are tetraploid, they do not contain the wild-type PAF-1 gene, suggesting that a chromosome fragment bearing the wild-type copy of PAF-1 was lost. Negative nuclear hybrids reconstituted with wild-type cytoplasts do contain a wild-type PAF-1 gene, indicating that the cytoplasts somehow reintroduced the wild-type PAF-1 allele without increasing ploidy. These findings support the role of PAF-1 and exclude the hypothesis of an additional cytoplasmic requirement for reinitiation of peroxisome biogenesis in peroxisome-deficient CHO cells. The plasmalogen deficiency and some other biochemical properties of ZR-78.1 are partially corrected in 5-azacytidine-treated subclones. However, such pseudo-revertants do not contain peroxisomes, consistent with the fact that there is no wild-type PAF-1 gene to reactivate by demethylation.

Acyltransferases↗
PubMed1993

Measurement of vanadium, nickel, and arsenic in seawater and urine reference materials by inductively coupled plasma mass spectrometry with cryogenic desolvation.

Addition of a small dose (2%) of H2 to the aerosol gas flow enhanced analyte signals by a factor of 2-3, which compensated for the loss of analyte signal that accompanied earlier efforts at cryogenic desolvation with inductively coupled plasma mass spectrometry (ICP-MS). Vanadium, nickel, and arsenic at microgram per liter levels in urine, river, and seawater reference materials were determined. The polyatomic ions ClO+, CaO+, and ArCl+, which normally cause severe overlap interferences for these elements, were attenuated to manageable levels by cryogenic desolvation. The samples were simply diluted with 1% HNO3 so that the chloride could be removed as HCl. The analytical results obtained for these standard reference materials agreed closely with the certified or recommended values. The detection limit ranges (3 sigma) obtained were 10-1000 ng L-1 for V, 0.03-20 micrograms L-1 for Ni, and 4-7000 ng L-1 for As in the original samples. The samples were introduced by flow injection to minimize clogging of the sampling orifice.

Arsenic↗
PubMed1993

Perception of self and other in major depression.

Previous research on the nature of person perception in depression has been inconclusive. This investigation differs from earlier studies in that extensive free-response descriptions of other people and self were collected from patients with major depression and from nonpsychiatric control Ss. In comparison with control Ss, depressed patients described fewer positive aspects not only of self but also of parents and significant others and reported more negative aspects of these people. Cluster analysis (HICLAS) also showed that more cognitive differentiation of negative self-perceptions (negative self-complexity) was characteristic of clinical depression. In both control Ss and patients, a positive (or negative) view of self was highly correlated (.85 or more) with a positive (or negative) view of parents and significant others. These correlations were significantly stronger than those between self and less important others.

Adult↗
PubMed1992

Partial phenotypic suppression of a peroxisome-deficient animal cell mutant treated with aminoglycoside G418.

Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.

Acyltransferases↗
PubMed1992

Direct comparison between protons and alpha-particles of the same LET: I. Irradiation methods and inactivation of asynchronous V79, HeLa and C3H 10T1/2 cells.

A direct comparison was carried out of the biological effectiveness of protons and alpha-particles of the same linear energy transfer (LET) under identical conditions with a variety of in vitro biological systems. Monolayers of mammalian cells were irradiated with accelerated beams of protons (1.2 and 1.4 MeV) and alpha-particles (30 and 35 MeV) corresponding to LETs of 23 and 20 keV microns-1 for each particle type. For V79-4 cells it was observed that the linear term of the dose-response for cell inactivation by protons was significantly greater than that for alpha-particles of the same LET. For HeLa and HeLa S3 cells, also, the linear term appeared to be greater for protons, but this was not observed with more limited data for C3H 10T1/2 cells. The result for V79 cells is in agreement with the report of Belli et al. (1989) who observed that the biological effectiveness of protons rose sharply between 17 and 30 keV microns-1 in strong contrast to alpha-particles which reached a peak effectiveness at greater than 100 keV microns-1. These results place new constraints on the biologically relevant features of the microscopic structure of radiation tracks, and have implications for the mechanistic and practical comparison between radiations.

Alpha Particles↗
PubMed1990

A rapid selection for animal cell mutants with defective peroxisomes.

Chinese hamster ovary (CHO) cells take up and incorporate 9-(1'-pyrene)nonanol (P9OH) into phospholipids and neutral lipids. Exposure of P9OH-labeled cells to long wavelength ultraviolet (UV) light causes cell death, because excitation of the pyrene moiety generates reactive oxygen species. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A. and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. USA 83, 5170-5174) are much more resistant to P9OH/UV treatment than are wild-type cells. This phenotype is explained by a 7.5-fold reduction of P9OH incorporation into the ethanolamine-linked phospholipids in the mutant cells and 2.4- to 6-fold reduction of P9OH incorporation into all other phospholipids and triglycerides, suggesting a general defect in fatty alcohol metabolism. [U-14C]Hexadecanol incorporation into the phospholipids of the mutant cells is also impaired. In contrast, the fatty acid analog, 9-(1'-pyrene)nonanoic acid, is incorporated into cells two times more rapidly by the mutants than by the wild type. Resistance to P9OH/UV treatment affords a simple, new method for the selection of animal cell mutants defective in peroxisome biogenesis.

Animals↗