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Biomedical subjects

I Fischer

Publications and source records attributed to I Fischer.

At least 127 records · Page 7Linked to original sources
PubMed1992

Isolation and characterization of periaxolemmal and axolemmal enriched membrane fractions from the rat central nervous system.

In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.0M sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K+ ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.0M fraction was also highly enriched in Na+, K+ ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage-dependent Na+ channel, axon-specific fodrin, and MAP-1B. Based on these characteristics we concluded that the 0.8M and 1.0M subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipin10 was unique with equally high levels in myelin and in the 0.8M and 1.0M subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GA/1. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K+ and Cl- channels, which based on channel sizes and open times, are largely distinct from one another.

Animals↗
PubMed1991

Differential hormonal modulation of brain antigens recognized by the AB-2 monoclonal antibody.

The expression of monoclonal antibody AB-2 immunoreactivity is age- and sex-dependent in radial glia of developing rat hypothalamus and is regulated by prenatal exposure to gonadal steroids. In the present study, several proteins were recognized by AB-2 and were distributed selectively in subcellular fractions from neonatal hypothalamus (HYP), remaining forebrain (FB), and brainstem regions. Immunoblots revealed polypeptide bands in 3 major molecular weight classes: one at approximately 195 kDa in the cytosolic compartment; and two doublets at 220 kDa and 340 kDa in both microsomal and crude mitochondrial membrane fractions. The 220 kDa and 340 kDa doublets were also Triton-insoluble, suggesting a cytoskeletal association. The 195 kDa-AB-2-immunoreactive band was present in both Triton-soluble and insoluble fractions. AB-2 also recognized several acidic glycolipids extracted from postnatal rat brain regions on immunoblots following high performance thin layer chromatography. One of the bands from postnatal rat brain extracts migrated similarly to purified bovine brain sulfatide, which was also immunoreactive with AB-2. AB-2 immunoreactivity with proteins, polar lipids, and sulfatide suggests that the epitope is a carbohydrate present in multiple cellular compartments. AB-2 recognized the same molecular bands in males and females. Testosterone treatment selectively decreased the level of the 195 kDa AB-2-immunoreactive polypeptide. The 195 kDa AB-2-immunoreactive polypeptide possibly acts in radial glia in the determination of sexually dimorphic neurons in the preoptic area/hypothalamus.

Animals↗
PubMed1991

Expression of plasmolipin in oligodendrocytes.

Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602-610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligodendrocyte cultures. Double-label immunofluorescence showed that plasmolipin was expressed by galactocerebroside (GC)-positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC-positive cells. During the following 3 weeks in culture plasmolipin staining of oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmolipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenates from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by oligodendrocytes.

Animals↗
PubMed1991

Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain.

Microtubule associated proteins MAP1B and MAP2 are important components of the neuronal cytoskeleton. During early development of the brain, MAP1B (340 kDa) is present as two isoforms that differ in their level of phosphorylation, while MAP2 is expressed as a single high molecular weight isoform (MAP2B, 280 kDa) and a low molecular weight form (MAP2C, 70 kDa). In this study we examined and compared the sensitivities of MAP1B and MAP2, obtained from MT preparations and brain homogenates of young rats, to degradation by calcium-activated neutral protease, calpain II. We found that in MAPs prepared from microtubules the two isoforms of MAP1B had comparable sensitivity to calpain-mediated proteolysis. Similarly, the high and low molecular weight forms of MAP2 were equally sensitive to digestion by calpain. However, although both MAPs were very susceptible to calpain-mediated proteolysis, MAP1B was more resistant to degradation by calpain than MAP2. Furthermore, the endogenous degradation of MAPs in neonate brain homogenates was calcium-dependent and inhibited by leupeptin, and the pattern of degradation products for MAP1B and MAP2 was similar to that of calpain-mediated proteolysis. These data suggest that calpain can play a role in the regulation of MAPs levels during brain development, in relation to normal neuronal differentiation and disorders associated with neurodegeneration.

Animals↗
PubMed1991

The phylogenic expression of plasmolipin in the vertebrate nervous system.

Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.

Amphibians↗
PubMed1991

Regulation of microtubule associated protein 2 (MAP2) expression by nerve growth factor in PC12 cells.

In the presence of nerve growth factor (NGF), PC12 cells cease to divide and differentiate, extending long microtubule-containing neurites. We showed by immunoblot analysis that MAP2 was detectable in PC12 after 4 days of NGF treatment and that its levels increased five- to sevenfold after 12 days of NGF treatment. The apparent molecular weight of MAP2 in PC12 cells was similar to that of rat brain MAP2 (280,000), with a doublet representing the MAP2 isoforms. However, the relative levels of MAP2 in differentiated PC12 cells were 5-10% of those found in rat brain. Immunofluorescence analysis of NGF-treated PC12 cells revealed that MAP2 co-localized with tubulin and was present in cell bodies and neurites. Northern blot analysis showed that the levels of MAP2 mRNA increased in PC12 cells during NGF-treatment in a pattern that paralleled the protein levels, suggesting that MAP2 expression is transcriptionally regulated.

Adrenal Gland Neoplasms↗
PubMed1991

Early in vitro genesis and differentiation of axons and dendrites by hippocampal neurons analyzed quantitatively with neurofilament-H and microtubule-associated protein 2 antibodies.

Differentiating neurons initially extend neurites that are the precursors of axons and dendrites. The temporal pattern of neurite outgrowth has been studied extensively, but mostly qualitative analyses have been used to study this phenomenon. We have examined neurite outgrowth of hippocampal neurons in primary cultures using a polyclonal antibody against microtubule-associated protein 2 (MAP2) and a novel monoclonal antibody against the phosphorylated form of high neurofilament subunit (NF-H). These antibodies serve as markers for dendrites and axons, respectively. The neurite staining patterns were quantified during the first 10 days in culture and the analysis revealed that primary processes undergo three phases of differentiation: (i) in the first 24 h, the majority of primary neurites express MAP2 only and a small percentage express both MAP2 and NF-H; (ii) between 24 and 48 h, NF-H expression increases and it is coexpressed with MAP2 in many neurites as they begin to lengthen; and (iii) between 48 h and 4 days, MAP2 and NF-H protein expression occurs in separate populations of neurites. While most of the earliest forming primary neurites appear to be dendritic (MAP2 only), the coexpression of dendritic and axonal protein markers in a group of early forming processes suggests that these neurites may not be predetermined to become a dendrite or an axon. Our data also indicate that NF-H is detectable early in primary neurite development and that, based on in vivo localization and morphology of cultured neurites, the phosphorylated form of NF-H is concentrated in axons.

Animals↗
PubMed1991

Differential appearance of extensively phosphorylated forms of the high molecular weight neurofilament protein in regions of mouse brain during postnatal development.

The appearance and accumulation of extensively phosphorylated forms of the high molecular weight neurofilament protein (H-phos) was studied in six regions of mouse brain during postnatal development by quantitative immunoblot analyses. H-phos (migrating at 200 kDa) was detected in brainstem, cerebellum, cortex and hippocampus as early as postnatal day 1. While NF-H levels increased dramatically during subsequent postnatal development in these regions, and reached levels similar to those observed in adult brain by postnatal day 14, quantitative differences were observed in both the rate and the extent of increase among individual regions. The most rapid accumulation of H-phos was observed in brainstem and cortex, where H-phos increased within the first postnatal week to levels comparable to those of adult brain. However, H-phos exhibited a slower developmental change in cerebellum, where the levels increased uniformly over the first two postnatal weeks. In hippocampus, the major increase in H-phos levels was delayed until the second postnatal week. In contrast to its early detection in the above regions, H-phos was not detected in immunoblot analyses of olfactory bulb or hypothalamus cytoskeletons at postnatal day 1, indicating that in these regions the accumulated levels of posttranslationally modified forms of this protein appeared relatively late. Furthermore, H-phos levels in hippocampus did not level off at postnatal day 14 and continued to increase until at least postnatal day 21. Immunoblot analyses of whole embryonic brain revealed the presence of H-phos as early as embryonic day 17, demonstrating that some mouse brain regions carry out extensive phosphorylation of NF-H during embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Turnover of cytoskeletal proteins in vivo.

The turnover of the microtubule-associated proteins 1B and 2 (MAP1B and MAP2), tubulin, high molecular weight neurofilament protein (NF-H), and spectrin were studied by in vivo labeling. Radiolabeled [35S]methionine was injected intracranially to 10-day-old rats and the rate of turnover was measured for total and specific brain proteins. The turnover of total brain proteins was biphasic and consisted of a fast and a slow component with half lives of 6.5 +/- 0.4 and 14.2 +/- 0.7 (mean +/- S.E.M.) days, respectively. The turnover of individual cytoskeletal brain proteins was also biphasic. The fast decay rates of MAP1B, MAP2, tubulin and spectrin were 5.8 +/- 0.7, 6.9 +/- 0.3, 4.8 +/- 0.5 and 4.9 +/- 0.4 days, respectively, while the slow decay rates of these proteins were 12.0 +/- 1.3, 12.4 +/- 1.7, 15.0 +/- 0.5 and 16.0 +/- 1.2 days, respectively. In addition, the Triton X-100 insoluble fraction of MAP1B, tubulin, spectrin and NF-H showed monophasic decay rates of 29.0 +/- 2.3, 15.0 +/- 1.4, 16.0 +/- 0.9 and 18.5 +/- 1.5 days, respectively, which were similar to their slow decay rates in whole brain homogenates, suggesting that incorporation of these proteins into the cytoskeletal lattice increases their stability.

Animals↗
PubMed1990

Microtubule associated protein (MAP1B) is present in cultured oligodendrocytes and co-localizes with tubulin.

Differentiation of oligodendrocytes is accompanied by the extension of processes and the assembly of the myelin membrane. It is likely that the cytoskeleton plays an important role in this process in terms of changes in cell shape, transport of myelin components, and organization of the myelin membrane. Oligodendrocytes contain microtubules (MT) which associate with other components of the cytoskeleton, and microtubule associated proteins (MAPs) may mediate some of these interactions. In this study we have shown the presence of MAP1B in oligodendrocytes grown in primary glial cultures by double-label immunofluorescence using antibodies to galactocerebroside (GC) and MAP1B. The staining of the cultures showed that GC-positive oligodendrocytes were also stained with MAP1B antibodies. However, MAP1B stain was limited to cell bodies and processes, whereas GC stain was also seen in flattened membrane sheets and punctate staining in processes. MAP1B staining was also compared with that of myelin proteolipid (PLP), myelin basic protein (MBP) and beta-tubulin in secondary glial cultures that were enriched for oligodendrocytes. The results showed a typical staining of cell bodies and membranous profiles using PLP antibodies, and the staining of cell bodies and flattened regions of membranous sheets by MBP antibodies. In contrast, both polyclonal and monoclonal antibodies to MAP1B showed a uniform diffuse staining of cell bodies, major processes, and fine interconnected processes. Double-labeling of the cells showed that MAP1B was co-localized with tubulin, but was not present in glial fibrillary acidic protein (GFAP)-positive astrocytes. Western and Northern blot analyses of primary glial cultures showed that MAP1B had a molecular mass of 320 kDa and a mRNA of 10 kb. These values are identical to those previously reported for brain MAP1B (Safaei and Fischer, 1989) and demonstrate the presence of MAP1B in oligodendrocytes.

Animals↗
PubMed1990

Synthesis, axonal transport, and turnover of the high molecular weight microtubule-associated protein MAP 1A in mouse retinal ganglion cells: tubulin and MAP 1A display distinct transport kinetics.

Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton.

Animals↗
PubMed1990

Presence of the plasma membrane proteolipid (plasmolipin) in myelin.

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.

Animals↗
PubMed1990

Changes in microtubule-associated protein MAP1B phosphorylation during rat brain development.

Microtubule-associated protein MAP1B from neonatal rat brain was separated on sodium dodecyl sulfate-containing polyacrylamide gels into two isoforms (high and low MAP1B), both of which were recognized by a panel of monoclonal and polyclonal antibodies against MAP1B. In addition, SMI31, a monoclonal antibody directed against phosphorylated epitopes of the neurofilament proteins, showed phosphatase-sensitive reactivity against the high isoform of MAP1B. The antigenic relationship between the phosphorylated isoform of MAP1B and neurofilaments was confirmed by the reactivity of SMI31 with the immunoprecipitated MAP1B protein. After dephosphorylation of MAP1B with alkaline phosphatase, the higher-molecular-weight isoform of MAP1B was no longer detectable with phosphate-insensitive anti-MAP1B antibodies, whereas there was a significant increase in the immunoreactivity of the lower-molecular-weight MAP1B isoform. These data suggest that the structural microheterogeneity of MAP1B is due to differences in phosphorylation. The two isoforms were present in all brain regions of the young rat. During brain development, the general decrease in MAP1B levels was accompanied by changes in the relative amount of the two isoforms. In particular, the phosphorylated isoform of MAP1B decreased dramatically to almost undetectable levels in adult brain. This conclusion was further supported by immunoblotting analysis that showed the disappearance of phosphorylated epitopes of MAP1B early during brain development. In addition, dephosphorylation experiments demonstrated the phosphatase sensitivity of the phosphorylated isoform throughout development.

Alkaline Phosphatase↗
PubMed1990

[Additional intra-abdominal injuries in multiple-traumatized patients with pelvic fractures. Difficulties in diagnosis and therapy].

Between 1.1. 1987 to 31.12. 1988 178 polytraumatized patients were treated with 62 patients demonstrating pelvic fractures. In 58% of patients injured by rapid falls pelvic fractures were diagnosed compared to only 25% of all patients with traffic accidents. 22% of all polytraumatized patients suffered from intraabdominal injuries, with raise to 47% in patients with pelvic fractures. To avoid unnecessary laparotomies caused by questionable positive lavage ultrasonography is recommended because of its high accuracy and lack of any diagnostic morbidity. 27 of 29 patients with intraabdominal injuries underwent laparotomy. One patient with an isolated kidney rupture was treated conservatively and one patient died before operation. The most common intraabdominal lesion was rupture of liver in 52% and splenic injury in 10 patients. Lesions of the intestine were diagnosed in 11 patients mostly in combination with parenchymal injuries. Concerning the therapeutical approach a conservative treatment is recommended in order to shorten time of operation in concern of the progressively increasing retroperitoneal hematoma. The overall mortality rate was 36%. Most common cause was direct injury of CNS in 14 patients of whom 6 patients suffocated during the first 24 hours. Unstable dislocated pelvic fractures were primary stabilized by external fixator which may help to avoid further progressive retroperitoneal hematoma.

Abdominal Injuries↗
PubMed1989

Regulation of microtubule-associated protein 2 (MAP2) mRNA expression during rat brain development.

The expression of MAP2 during rat brain development was studied by using specific antibodies and cDNA probes. MAP2 cDNAs were isolated from a rat brain lambda gt11 library, and their identity was confirmed by the reactivity of their fusion proteins with several independent monoclonal antibodies that recognize MAP2. Northern blot analyses of the RNA prepared from whole brains, cerebral cortex, hypothalamus, brain stem, olfactory bulbs, and cerebellum showed that the levels of MAP2 mRNA increase during the initial phase of development, reach a maximum between postnatal weeks 2 and 3, and then decrease in the adult. The time course and the kinetics of this change varied between different brain regions and appeared to reflect the pattern of morphological changes in these regions. RNA blots were also analyzed with beta-tubulin and beta-actin cDNA probes to ensure the quality and the quantity of the RNA. The levels of MAP2 mRNA and protein showed similar changes during the initial part of brain development and suggested a transcriptional control. However, while MAP2 protein levels remained high throughout development, MAP2 mRNA levels decreased in adulthood. We suggest that the increased stability of the MAP2 molecule may be a contributing factor in the developmental regulation of steady-state levels of MAP2.

Actins↗