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M Pilon

Publications and source records attributed to M Pilon.

31 records · Page 2Linked to original sources
PubMed1992

Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor.

Time courses of the import into isolated chloroplasts of the purified ferredoxin precursor were measured at different protein concentrations. Analysis of the initial import rates indicates the presence of one saturable import system with an apparent Km value of approximately 100 nM and a Vmax corresponding to the uptake of approximately 2.5 x 10(4) precursor proteins per minute per chloroplast. We conclude that the in vitro observed activity of the chloroplast protein import machinery, functioning independently from cytosolic factors, would be enough to allow chloroplast development at physiological rate.

Biological Transport↗
PubMed1992

New insights into the import mechanism of the ferredoxin precursor into chloroplasts.

We have investigated the import pathway of the nuclear-encoded chloroplast protein ferredoxin. By using purified precursor protein and washed intact chloroplasts in a defined in vitro uptake system, we show that preferredoxin is fully import-competent by itself. In addition, we show also that the in vitro, in a wheat germ lysate, synthesized preferredoxin is not stably associated with another protein. Import is dependent only on ATP and does not require the presence of cytosolic proteins. Translocation could be largely stimulated by the thiol reducing agent dithiothreitol (DTT). To determine whether DTT acts on the precursor or on the chloroplast, we modified the 5 cysteines in the precursor by a reaction with iodoacetamide, thereby preventing the formation of disulfide bridges in the precursor. The import of this modified precursor was still stimulated by the addition of DTT, indicating that DTT had a stimulating effect on the chloroplast import machinery. In the case of the modified precursor, the import must have taken place without iron-sulfur cluster attachment in the stroma. The modified precursor could be imported with a similar efficiency as the parent precursor showing that import takes place independently from cofactor assembly.

Adenosine Triphosphate↗
PubMed1991

Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Expression in Escherichia coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin.

The precursor of the chloroplast protein ferredoxin from Silene pratensis was expressed in Escherichia coli. When a low copy number plasmid was used, the preferredoxin level was low, and the protein was soluble. The expression level was increased by using a high copy number plasmid. In protease-deficient cells transformed with the latter plasmid, the preferredoxin accumulated up to 1% of total protein, and it was found in insoluble aggregates. These aggregates were dissolved in 4 M urea, and the protein was purified to homogeneity. Amino-terminal sequencing confirmed the amino acid sequence as deduced from the copy DNA. However, the first methionine residue of the expected sequence was absent in E. coli. The purified precursor was readily imported by isolated chloroplasts and processed to the mature size.

Chloroplasts↗
PubMed1989

The importance of the amino terminus of the mitochondrial precursor protein apocytochrome c for translocation across model membranes.

The importance of the various regions of the apocytochrome c molecule and the effect of covalent coupling of the heme group to the protein for the translocation across a model-membrane was studied by using chemically and enzymatically prepared fragments of horse heart apo- and holocytochrome c and model-membranes composed of phosphatidylserine and phosphatidylcholine. Binding experiments showed that fragments of apocytochrome c with the highest net positive charge have the highest affinity for negatively charged large unilamellar phosphatidylserine vesicles. Monolayer experiments demonstrated that the amino-terminal fragments were only able to penetrate into a phosphatidylserine monolayer whereas the carboxyl-terminal fragments, in addition, penetrated into a phosphatidylcholine monolayer although with a lower efficiency. The covalent coupling of the heme group to both a small amino-terminal fragment residue numbers 1-38 and to the entire precursor protein resulted in a marked decrease in the ability to penetrate into a phosphatidylserine monolayer. Translocation experiments with trypsin enclosed in vesicles, showed that only the amino-terminal fragments of the precursor protein and not carboxyl-terminal peptides or the heme-containing fragments of the mature protein were able to cross the bilayer and become digestible by trypsin at the opposite side of the bilayer. Circular dichroism measurements with the various peptides both in an aqueous and lipidic environment were performed to investigate the conformation of apocytochrome c after interaction with model-membranes. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.

Amino Acid Sequence↗
PubMed1987

Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study.

(1) The interaction of apocytochrome c with different molecular species of phosphatidylserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 mN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidylserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidylserine is indicated by the low Kd of 0.017 microM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using 14C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interfacial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 A2/molecule in the absence of lipid to 700-900 A2/molecule after penetration of dioleoylphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidylserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.

Apoproteins↗
PubMed1987

Evidence for a nonprolactin, non-growth-hormone mammary mitogen in the human pituitary gland.

To determine whether the human pituitary contains a previously unidentified, nonprolactin (non-hPRL), non-growth-hormone (non-hGH) factor capable of stimulating mammary development, we tested the effects of whole human pituitary extract (hPE) and pituitary extracts depleted of hPRL and hGH ("stripped hPE") in hypophysectomized, castrated estradiol (E2)-treated male rats and rhesus monkeys. Both whole and stripped hPE significantly stimulated rat mammary development (mean scores = 3.3 and 2.0, respectively, on a scale ranging from 0 to 4) in comparison with controls (mean score = 1.0). Mammary development was not due to minute concentrations of hGH or hPRL remaining in stripped hPE because 30- to 100-fold higher concentrations of hGH (Genentech) and 1000-fold higher concentrations of hPRL were required to stimulate significant mammary development. Non-pituitary extracts of human ovary, muscle, and serum, and bovine serum albumin did not stimulate rat mammary gland growth. Trypsin destroyed the mammary mitogenic activity of whole hPE, indicating that the unidentified factor is likely a protein. Mammary growth and development were also stimulated in hypophysectomized, E2-treated monkeys by stripped hPE (mean histological score = 3.25 vs. 1.35 in control animals). Monkeys receiving stripped hPE had undetectable levels of hGH and hPRL in serum sampled over a 24-hr period. These findings suggest that the human pituitary contains a non-hPRL, non-hGH factor that stimulates mammary growth and may be important in normal mammary growth and development and perhaps in breast cancer.

Animals↗
PubMed1987

Kinetics of l-Valine Uptake in Suspension-Cultured Cells and Protoplast-Derived Cells of Tobacco: Comparison of Wild-Type and the Val-2 Mutant.

A kinetic analysis was made of l-valine uptake in protoplast-derived cells (mesophyll protoplasts cultured for 6 days) and in suspension-cultured cells of tobacco (Nicotiana tabacum L., cv Xanthi). Cells from wild-type and Val(r)-2 mutant plants were compared. A low-K(m) component was found in protoplast-derived cells (K(m) = 45 +/- 5 micromolar) as well as in suspension-cultured cells (K(m) = 84 +/- 21 micromolar). In the mutant cells the V(max) of this component was 12- to 14-fold less than in wild-type cells. A second component (K(m) = 2.4 +/- 0.7 millimolar) was found in suspension-cultured cells but not in protoplast-derived cells; its V(max) was the same in wild-type and mutant cells. A third component was apparently unsaturable (linear component). It was present in protoplast-derived cells but not in suspension-cultured cells, and had the same magnitude in wild-type and mutant cells. The results are discussed with reference to the uptake of l-valine in leaf tissue, in which the three kinetic components have been found simultaneously. The reduced V(max) of the low-K(m) component in the Val(r)-2 mutant, and the differential expression of the other two components in suspension-cultured cells and protoplast-derived cells indicate that the kinetically distinguishable components represent physically distinct transport systems.

Journal Article↗
PubMed1984

Placental site, parity, and date of quickening.

The influence of placental site and parity on the date of quickening was studied in 112 gravidas. With placental site on the anterior uterine wall, quickening occurred at a mean gestational age of 19 weeks in primigravidas, and 17 weeks, five days in multiparas. With placental site on the posterior uterine wall, quickening occurred at a mean gestational age of 18 weeks in primigravidas, and 16 weeks, one day in multiparas. It is concluded that placental site influences quickening independent of parity and that it is a major factor in determining the stage at which fetal movements are first felt.

Female↗
PubMedDate not supplied

Persistent vegetative state: which sensory-motor variables should the physiotherapist measure?

This study used a modified Delphi technique to establish a list of core items which should be included in the measurement, by the physical therapist, of sensory-motor capacities of persons in a persistent vegetative state. Twenty-eight physical therapists with a mean of 5.9 years of experience working with this clientele participated in the study and identified 105 items as being important in the evaluation of the PVS patient (Round I). The study planning committee reduced these to 20 categories and asked the therapists to rate each category of items (a 5-point scale) as to it's importance (Round II). Physiotherapists identified 14 variables as being 'extremely' or 'very important' to include in such an evaluation. These included: tonus, voluntary movement on request, postural reactions, passive range of motion, tolerance to verticalization and postural status. These results are being used to develop a quantitative assessment instrument to be used by the physical therapist working with the PVS patient.

Arousal↗