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Biomedical subjects

M Seeger

Publications and source records attributed to M Seeger.

53 records · Page 3Linked to original sources
PubMed1993

A simple method for negative and positive selection of murine and human IgM-bearing lymphocytes based on the use of antibody-coated silica microparticles.

The use of octadecyl silica microparticles is proposed as an alternative method for negative and positive selection of IgM-bearing lymphocytes. SiC18 microparticles coated with affinity purified IgG anti-IgM were evaluated in their efficiency to deplete IgM-bearing cells. 6 mg of SiC18-IgG depleted an average of 97% IgM-bearing cells from a murine spleen cell suspension and of 84% IgM-bearing cells from human spleen cells. The viability of the selected cells was not affected by this procedure. We also analyzed the ability of whole serum to release IgM-bearing cells from the complex SiC18-target cell. The IgM releasing efficiency was 98%, with a resulting viability of 85%. These results indicate that SiC18 microparticles, saturated with specific antibodies, may be used as an alternative method for depletion or purification of cells bearing the corresponding antigen, from complex cell suspensions. The method is simple, efficient and inexpensive, representing an interesting alternative to the immunomagnetic selection.

Animals↗
PubMed1993

Phosphate-starvation induced changes in Thiobacillus ferrooxidans.

We have analysed the response of the acidophilic chemolithotroph Thiobacillus ferrooxidans to phosphate starvation. Cultivation of the bacteria in the absence of added phosphate induced a remarkable filamentation of the cells. Polyacrylamide gel electrophoresis revealed several proteins whose levels increased upon phosphate limitation, as well as some polypeptides that were exclusively synthesized under this growth limitation. One of the proteins whose level increased by the lack of phosphate was apparently an acid phosphatase with a pH optimum of about 3.8, and a molecular mass of 26 kDa, which was located in the periplasm. The N-terminal sequence of a 26 kDa protein derepressed by starvation, which may correspond to the T. ferrooxidans starvation, which may correspond to the T. ferrooxidans phosphatase, showed 30% and 35% identity with the known sequence of Lysobacter enzymogenes and Escherichia coli alkaline phosphatases, respectively.

Acid Phosphatase↗
PubMed1993

Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline.

We performed a large-scale screen for mutations that affect the development of CNS axon pathways in the Drosophila embryo. We screened embryos from over 13,500 balanced lines and saved over 250 mutant lines whose phenotypes suggest possible defects in growth cone guidance. Here we focus on two new genes: commissureless (comm) and roundabout (robo). Mutations in comm lead to an absence of nearly all CNS axon commissures, such that growth cones that normally project across the midline instead now extend only on their own side. Mutations in robo lead to the opposite misrouting, such that some growth cones that normally extend only on their own side instead now project across the midline. The phenotypes of these two genes suggest that they may encode components of attractive and repulsive signaling systems at the midline that either guide growth cones across the midline or keep them on their own side.

Animals↗
PubMed1993

To cross or not to cross: a genetic analysis of guidance at the midline.

Previous studies have led to the suggestion that the midline of the developing Drosophila central nervous system might serve a dual function during growth cone guidance, providing attractive signals for those growth cones that project across the midline, and at the same time repulsive signals for those growth cones that stay on only their own side. In an attempt to discover some of the genes encoding key components of these two guidance systems, we performed a large-scale screen in Drosophila for mutants that affect the development of the axon commissures. Here we review the mutant phenotypes of two new genes in which either too few (commissureless) or too many (roundabout) growth cones cross the midline. These phenotypes suggest that these two genes encode important components for growth cone guidance at the midline.

Alleles↗
PubMed1992

Phosphate starvation affects the synthesis of outer membrane proteins in Thiobacillus ferrooxidans.

The outer membrane protein (omp40) component from the chemolithoautotrophic acidophilic Thiobacillus ferrooxidans is apparently regulated by the external pH and the concentration of phosphorus. Its amino-terminal sequence showed little identity with the Escherichia coli OmpC, OmpF or PhoE porins, but was 38.5% identical to the outer membrane channel-forming protein NosA from Pseudomonas stutzeri, whose expression is also regulated environmentally. In addition, the partial amino acid sequence of T. ferrooxidans omp40 showed between 34 and 38% identity with the amino-terminal end of the small outer membrane proteins Rck and PagC from Salmonella typhimurium and OmpX from Enterobacter cloacae.

Amino Acid Sequence↗
PubMed1992

A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast.

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.

Alkaline Phosphatase↗
PubMed1992

Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae.

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

Alleles↗
PubMed1991

Effect of external pH perturbations on in vivo protein synthesis by the acidophilic bacterium Thiobacillus ferrooxidans.

The response of the obligate acidophilic bacterium Thiobacillus ferrooxidans to external pH changes is reported. When T. ferrooxidans cells grown at pH 1.5 were shifted to pH 3.5, there were several changes in the general protein synthesis pattern, including a large stimulation of the synthesis of a 36-kDa protein (p36). The apparent low isoelectric point of p36, its location in the membrane fraction, and its cross-reaction with anti-OmpC from Salmonella typhi suggested that it may be a porin whose expression is regulated by extracellular pH.

Bacterial Proteins↗
PubMed1989

Antibiotic proteins of human polymorphonuclear leukocytes.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.

Amino Acid Sequence↗
PubMed1975

Characterization of erythrocyte membrane-associated enzymes (glyceraldehyde-3-phosphate dehydrogenase and phosphoglyceric kinase).

The purpose of our study was to determine why several of the glycolytic enzymes of the erythrocyte have the propensity for adhering to erythrocyte membranes while others do not. Two of these membrane-associated enzymes, glyceraldehyde phosphate dehydrogenase (GAPD) and phosphoglyceric kinase (PGK), have been shown to have controlling functions on intra-erythrocytic glycolysis and sodium-potassium transport. In order to test the hypothesis that the membrane-associated fraction of these enzymes consisted of isozymes with increased capacity to become membrane associated, the enzymes from cytosol or membrane sources were partially purified and characterized. Determination of molecular weight by gel filtration chromatography, measurement of certain kinetic parameters, and the curves relating to pH optimal activity indicated that there were no measurable differences between membrane and cytosol PGK and membrane and cytosol GAPD. It was possible to raise inhibitory antibodies to GAPD in certain species of mice, and these antibodies did not distinguish between GAPD isolated from either membrane or cytosol sources. GAPD was firmly bound to membranes and the membrane-associated fraction counted for approximately 60 per cent of total erythrocytic enzyme. Membrane-associated PGK was only loosely adherent, and accounted for only 1 per cent of the total erythrocytic enzyme. The reasons for membrane association have yet to be determined, but these inward facing membrane-associated enzymes appear to function by carrying the organization of the membrane into the cell interior.

Animals↗
PubMedDate not supplied

Presenilin 1 is actively degraded by the 26S proteasome.

The metabolic pathways governing the turnover of presenilin 1 (PS1) have been incompletely worked out. The PS1 holoprotein has low abundance in many cells and appears to undergo endoproteolytic cleavage near residue 298. We provide evidence that one mechanism by which the PS1 holoprotein is degraded is through the action of the 26S proteasome. We also show that the proteasome does not participate in the endoproteolytic cleavage.

Alzheimer Disease↗