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Biomedical subjects

S C Feinstein

Publications and source records attributed to S C Feinstein.

At least 37 records · Page 2Linked to original sources
PubMed1995

Imaging real-time neurite outgrowth and cytoskeletal reorganization with an atomic force microscope.

An atomic force microscope was used to image the morphology and structural reorganization of rat NIH/3T3 fibroblasts and PC-12 cells growing in petri dishes. NIH/3T3 fibroblasts had a uniform morphology and an extensive cytoskeletal network. Cell thickness varied from approximately 2-3 microns above the nucleus to approximately 20-30 nm over the distal processes, and cytoskeletal fibers as small as 30 nm wide were observed. Imaging over an extended period of time showed a limited degree of cytoskeletal reorganization. Localized force dissection did not induce significant retraction of cellular processes and immediate cell death. Differentiating PC-12 cells with a neuronal phenotype had a nonuniform morphology, abundant cytoskeletal elements, neuritic processes, and growth cones. The cell thickness varied from approximately 5-8 microns over the nucleus to approximately 100-500 nm over the neuritic processes; growth cones approximately 50-700 nm wide and end structures approximately 30-150 nm wide were visible. Repeated imaging showed reorganization of the growth cone, especially the appearance and disappearance of beadlike features and fibrous organization. Thus an atomic force microscope can be used for high-resolution real-time studies of the dynamic subcellular mechanisms that drive cell behavior.

3T3 Cells↗
PubMed1994

Distribution of intracerebral ventricularly administered neurotrophins in rat brain and its correlation with trk receptor expression.

To assess the potential effectiveness by which injected neurotrophins can diffuse throughout the brain, we used autoradiographic and immunohistochemical techniques to examine the brain distributions of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) after a single injection into the lateral cerebral ventricle (ICV) in rats. As described previously, ICV-injected NGF labeled cholinergic neurons in the basal forebrain. Injection of BDNF resulted in few or no labeled neurons in the basal forebrain or in the substantia nigra. However, very intense labeling was associated with the ventricular walls and immediate parenchyma. The distribution of NT-3 after ICV injection was intermediate between that of NGF and BDNF. In the basal forebrain, similar neurotrophin distributions were observed in neonate versus adult animals. Our in situ hybridization analysis has shown that mRNA encoding the BDNF receptor(s) (trkB) is highly expressed by ependymal cells as well as by many neurons and glia. On the other hand, expression of the high-affinity NGF receptor (trkA) is restricted to cholinergic neurons in basal forebrain and striatum. In addition, staining with antisera specific for the trkA or trkB receptors demonstrated that their expression patterns closely reflect their mRNA distributions. Taken together, these data suggest that the presence of the trkB receptor on the ependymal layer of the ventricle and its expression throughout the brain parenchyma represents a significant impediment to the adequate diffusion of ICV-injected BDNF into the brain for delivery to target neurons.

Animals↗
PubMed1994

Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau.

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

Aging↗
PubMed1994

Sense and antisense transfection analysis of tau function: tau influences net microtubule assembly, neurite outgrowth and neuritic stability.

Microtubules are fundamental elements participating in many aspects of cell behavior and maintenance, yet the factors regulating microtubule behavior in vivo remain poorly understood. Employing the nerve growth factor (NGF)-responsive cell line, PC12, we have used sense and antisense DNA transfection strategies to examine the role of the microtubule-associated protein (MAP) tau in several aspects of neuronal cell behavior. Stable transfectants over-expressing tau accumulate more microtubule mass and extend neurites more rapidly than control cells, while transfectants under-expressing tau exhibit reduced microtubule levels and slower neurite outgrowth. Further, tau over-expressing cells are markedly more resistant to nocodazole-induced neuritic degeneration when compared to wild-type or tau under-expressing cells. These observations provide direct support for the model that tau is capable of influencing: (i) net microtubule assembly, (ii) the rate of neurite elongation and (iii) neuritic stability. These capabilities suggest that tau plays crucial roles in the development and maintenance of neuronal cells.

Animals↗
PubMed1993

Molecular analysis of the nerve growth factor inducible ornithine decarboxylase gene in PC12 cells.

In an effort to understand molecular mechanisms by which nerve growth factor (NGF) regulates gene expression, we have isolated a full-length rat cDNA clone encoding ornithine decarboxylase (ODC) and utilized this probe to identify and examine the transcriptionally active, NGF inducible ODC gene in rat PC12 cells. This same gene is also responsive to epidermal growth factor, basic fibroblasts growth factor, and dibutyryl cAMP. Primer extension analysis demonstrates that both basal and NGF induced transcription of the ODC gene utilize the same major transcriptional start site, demonstrating that NGF acts to increase transcriptional activity at the basal start site as opposed to unmasking an alternative, stronger start site. Functional promoter analysis reveals the presence of a constitutive core promoter residing between positions -201 and +390, relative to the start site of transcription. Additional analyses reveal that sequences in the region -7800 to +2257 are insufficient to mediate NGF induced transcriptional activation, demonstrating that at least some of the regulatory sequences necessary for NGF mediated transcriptional induction of the ODC gene must reside at relatively enormous distances from the transcriptional start site. Such a long distance transcriptional regulatory mechanism is unique when compared with other NGF responsive genes that have been similarly analyzed.

Animals↗
PubMed1991

A general radiochemical-color method for quantitation of immunoblots.

Quantitative interpretation of protein immunoblotting procedures is hampered by a variety of technical liabilities inherent in the use of photographic and densitometric methods. In this paper, we present a novel, simple, and generally applicable alternative procedure to acquire quantitative data from immunoblots. Our strategy employs both the standard alkaline phosphatase color reaction and radiolabelled Protein A. The color reaction is used to localize the polypeptide of interest after transfer to a solid support. The colored bands are then excised and the radioactivity in the colocalized Protein A is quantitated in a gamma counter. In addition to avoiding the problems associated with photographic and densitometric procedures, our assay also overcomes common problems associated with variable gel lane width and individual band distortion. The resulting data is linear over a range of at least 50-fold (10-500 ng of specific protein, for the example used in this study) and is highly reproducible.

Alkaline Phosphatase↗
PubMed1991

Analytical purification of the slow, high affinity NGF receptor: identification of a novel 135 kd polypeptide.

Although nerve growth factor (NGF) action is mediated by the slow, high affinity NGF receptor, little is known regarding its molecular composition or mode of action. We have used reversible chemical cross-linkers and affinity chromatography strategies to purify the slow NGF receptor covalently cross-linked to its NGF ligand. Subsequent uncoupling of the cross-links reveals that the receptor-ligand complex is composed of only a novel 135 kd polypeptide interacting with NGF. The previously characterized 85 kd fast, low affinity NGF receptor is not a component of the cross-linked slow receptor-ligand complex. This newly identified 135 kd polypeptide is either the entire slow NGF receptor, or it might be one component of a larger, multisubunit slow NGF holo-receptor.

Animals↗
PubMed1990

Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis.

We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.

Bacteriophage lambda↗
PubMed1990

Efficient transfection and expression of heterologous genes in PC12 cells.

The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.

Animals↗
PubMed1985

Nerve growth factor rapidly induces ornithine decarboxylase mRNA in PC12 rat pheochromocytoma cells.

The mechanism by which nerve growth factor (NGF) stimulates ornithine decarboxylase (OrnDCase; EC 4.1.1.17) activity in the rat pheochromocytoma cell line PC12 was investigated. As demonstrated previously, NGF rapidly induces OrnDCase activity in a dose-dependent manner, with maximal enzymatic activity at 4-6 hr after exposure to NGF. Activity subsequently returns to near basal levels. A cloned OrnDCase cDNA was used to analyze the levels of OrnDCase RNA. In response to NGF administration, OrnDCase RNA levels were induced. The time course of the OrnDCase RNA induction paralleled that of the enzyme activity induction, and the magnitude of both inductions was quantitatively the same. Increased concentration of OrnDCase RNA was clearly detected at the earliest time point examined, 2 hr. No change was observed in the size of OrnDCase RNA. The dose-response curves for both RNA and enzyme activity inductions were also similar. Thus, increased OrnDCase RNA levels fully account for, and are responsible for, the induction of activity. Further, one-third of the OrnDCase RNA induction was unaffected by cycloheximide treatment but was fully blocked by actinomycin D treatment, suggesting that NGF acts through at least two mechanisms to mediate the OrnDCase induction. The first mechanism is cycloheximide insensitive and the second is mediated through an event requiring ongoing protein synthesis. Both mechanisms require ongoing transcription, as evidenced by the complete sensitivity of the induction process to actinomycin D.

Animals↗
PubMed1985

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors.

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.

Adrenal Gland Neoplasms↗