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M Fahnestock

Publications and source records attributed to M Fahnestock.

At least 37 records · Page 2Linked to original sources
PubMed1997

Long-term potentiation trains induce mossy fiber sprouting.

It has been shown that both amygdaloid and hippocampal kindling induce sprouting of the mossy fibers in the dentate gyrus. In this study, we investigated whether non-epileptogenic stimulation could also induce mossy fiber sprouting. Long-term potentiation (LTP) was induced in the dentate gyrus by the application of brief, high frequency trains to the perforant path. The potentiating stimulation was applied each day for 10 days, and the tissue was prepared for Timm labelling 7 days later. Sprouting was significantly increased in the LTP group compared to the implanted control rats. These results suggest that mossy fiber sprouting is not damage-induced and is dependent on neuronal activation.

Animals↗
PubMed1997

Nerve growth factor accelerates seizure development, enhances mossy fiber sprouting, and attenuates seizure-induced decreases in neuronal density in the kindling model of epilepsy.

Recurrent seizure activity induced during kindling has been reported to produce a functional synaptic reorganization of the mossy fibers in the hippocampus. To date, it is unclear whether this kindling-induced growth is secondary to decreases in hilar neuron density, which are presumed to reflect hilar neuronal cell loss, or whether it is related specifically to an activation-dependent plasticity. We recently demonstrated that blocking nerve growth factor (NGF) biological activity retards seizure development and inhibits the sprouting of mossy fibers. We now demonstrate that intraventricular administration of NGF itself accelerates the progression of kindling epileptogenesis, increases mossy fiber sprouting in the CA3 region and in the inner molecular layer (IML), but reduces seizure-induced decreases in hilar cell density. These findings provide support for a role of NGF in kindling and kindling-induced mossy fiber sprouting. In addition, the results dissociate this form of epileptogenesis from hilar cell loss or decreases in hilar cell density attributable to increases in hilar area, thereby supporting seizure-induced mossy fiber sprouting as being primarily attributable to the combined effects of neuronal activation and the activation-induced upregulation of growth factors.

Animals↗
PubMed1996

Stimulatory G-protein alpha-subunit mRNA levels are not increased in autopsied cerebral cortex from patients with bipolar disorder.

Increased alpha-subunit (alpha s) levels of both the 45- and 52-kDa isoforms of the stimulatory guanine nucleotide binding protein (G-protein), have been found in postmortem brain and mononuclear leukocytes from patients with bipolar disorder (BD). The pathophysiological mechanism responsible for increased alpha s protein levels is unknown, however, it may involve increased expression of the gene encoding this protein. To assess this possibility, alpha s mRNA levels were determined by RT-PCR in postmortem brain from 10 subjects with an antemortem diagnosis of BD and age- and sex-matched control subjects in whom we had previously reported increased alpha s protein levels. There were no significant differences in alpha s mRNA levels in frontal, temporal, or occipital cortex between BD and control subjects. Cerebral cortex alpha s mRNA levels did not correlate with age or postmortem interval. These findings do not support the notion that higher alpha s levels found in BD postmortem brain are a result of increased gene expression.

Adult↗
PubMed1996

Nerve growth factor mRNA and protein levels measured in the same tissue from normal and Alzheimer's disease parietal cortex.

Nerve growth factor (NGF) mRNA and protein levels were determined in parietal cortex samples from both normal and Alzheimer's disease (AD) patients. NGF protein levels were slightly elevated in AD patients compared to controls, but NGF mRNA levels were unchanged in the same tissue samples. Thus, small but reproducible increases in NGF protein reported in AD cortex do not result from increases in NGF mRNA.

Aged↗
PubMed1996

Method for quantitation of low-abundance nerve growth factor mRNA expression in human nervous tissue using competitive reverse transcription polymerase chain reaction.

Nerve growth factor (NGF) is a polypeptide hormone important for the development, function, and survival of neurons. NGF is an important target for investigation because of its potential therapeutic importance in neurological diseases and injury, yet its low level of expression in nervous tissue makes it difficult to measure. We report here the development of a quantitative assay for human NGF mRNA, using competitive reverse transcription polymerase chain reaction (cRT-PCR), with mouse submandibular gland mRNA as an internal standard. Exhibiting high homology to the human transcript, mouse NGF mRNA is co-amplified with the human NGF transcript through both the reverse transcription and PCR steps using identical primers and conditions. Products are distinguished by a convenient unique restriction site in the mouse transcript. Phosphorimage detection and quantitation of radiolabeled product provides femtogram sensitivity. Generation of a standard curve from a dilution series of mouse mRNA allows the amount of co-amplified human NGF mRNA transcripts to be determined. We demonstrate the amplification and quantitation of human NGF mRNA from 175-350 nanograms of poly(A)+mRNA from human frontal and parietal cortex. This technique is ideal for rapid detection and quantitation of low-abundance NGF mRNA from limited amounts of tissue.

Animals↗
PubMed1995

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.

Animals↗
PubMed1995

Intraventricular administration of antibodies to nerve growth factor retards kindling and blocks mossy fiber sprouting in adult rats.

Repeated subconvulsive electrical stimulation of certain areas of the forebrain leads to kindling, a progressive and permanent amplification of evoked epileptiform activity, which is a model for human temporal lobe epilepsy. Recent studies have shown that kindling induces synthesis of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) but not neurotrophin-3 (NT-3) in the hippocampus and cortex. Kindling also elicits mossy fiber sprouting and functional synaptogenesis in the supragranular layer, the hilus, and the CA3 region of the hippocampus. Intraventricular administration of antibodies to NGF has been shown to effectively block septohippocampal sprouting in the adult rat, and has been reported to retard amygdaloid kindling. In the present study, we have investigated the possible role of NGF in both kindling and kindling-associated sprouting. We have confirmed a kindling-induced sprouting of the mossy fibers into the stratum oriens of the CA3 region of the hippocampus, utilizing a new semiquantitative method of analysis based on Timm staining. Previous studies found no overt signs of hippocampal damage with this kindling paradigm, indicating that the increased Timm staining likely reflects a purely activity-induced sprouting. Intraventricular infusion of affinity-purified anti-NGF IgGs (which cross-react with NT-3 but not BDNF) resulted in both significant retardation of kindling and inhibition of the kindling-induced mossy fiber sprouting. The findings suggest a role for NGF in both these phenomena.

Animals↗
PubMed1994

NGF mRNA is not decreased in frontal cortex from Alzheimer's disease patients.

Alzheimer's disease (AD) is characterized by neuronal dysfunction and degeneration in certain brain regions such as cortex, hippocampus and basal forebrain. Specific neurochemical defects such as decreases in cholinergic enzymes and in the amounts of mRNA in AD brain have also been reported. Nerve growth factor (NGF), a protein necessary for the development, regulation and survival of basal forebrain cholinergic neurons (BFCN), is synthesized in target areas of BFCN (cortex, hippocampus) and is supplied to BFCN by retrograde transport. Thus, NGF is under investigation both as a potential therapeutic agent and for its possible involvement in the pathogenesis of AD. In this study, postmortem brain tissues from both control and AD cases were investigated for amounts of poly (A)+ mRNA and NGF mRNA in the frontal cortex, a region rich in cholinergic afferents. Yields of poly(A)+ mRNA were similar from normal and AD tissues. Human NGF mRNA comigrated with murine NGF mRNA on Northern blots. Additionally, dot blot quantitation demonstrated that NGF mRNA levels do not differ in the inferior frontal gyrus of normal and AD patients. Thus, we conclude that levels of mRNA in general, and of NGF mRNA in particular, are unchanged in the frontal cortex of individuals affected by AD.

Aged↗
PubMed1994

Mouse NGF promoter upstream sequences do not affect gene expression in mouse fibroblasts.

The expression of nerve growth factor (NGF) is tightly controlled in a tissue-specific manner during development and in response to injury. In fibroblasts and in other cell types, expression of NGF is regulated at the transcriptional level. In order to elucidate the mechanism of this regulation, we have undertaken the analysis of the mouse NGF promoter in a mouse fibroblast cell line (LTA), using transient transfection of NGF promoter-human growth hormone (hGH) reporter gene plasmids. We find that sequences between +8bp and +120bp, containing an AP-1 site, confer increased levels of expression from the full length and truncated NGF promoters. When this region is deleted, a significant decrease in expression is observed from both the full length promoter and truncated versions thereof. A gradual increase in expression is observed with successive 5' deletions of both the AP-1 containing and AP-1 deleted promoters; this effect results from the juxtapositioning of adjacent plasmid sequences closer to the transcription initiation site and not from deletion of promoter sequences as was previously reported. When the NGF promoter is analyzed using a luciferase reporter plasmid, these 5' promoter deletions have no significant effect on reporter gene expression in fibroblasts. Thus, sequences downstream of the transcription start site influence NGF promoter activity in fibroblasts, but sequences upstream of the TATA box fail to affect promoter activity in these cells.

Animals↗
PubMed1994

Characterization of kallikrein cDNAs from the African rodent Mastomys.

Kallikreins comprise a family of serine proteases that are required for the processing of hormone precursors, thereby controlling many physiological processes including blood flow, ion transport, and inflammation. In rodents such as mouse, rat, and Mastomys, many kallikreins are expressed in the submandibular gland (SMG), but only a limited number, notably true tissue (glandular) kallikrein, are expressed in the kidney. We report here the cloning and characterization of kallikrein cDNAs from the Mastomys SMG. Two of these are expressed in the kidney as well as in the SMG, and one may code for the true tissue kallikrein of Mastomys. A third kallikrein is expressed only in the SMG and bears some resemblance to a murine nerve growth factor-associated protein. The existence of a family of Mastomys SMG kallikreins suggests that these enzymes act as prohormone-processing enzymes in Mastomys. DNA sequence analysis and hybridization studies demonstrate that, although Mastomys kallikreins are very similar in structure to both mouse and rat kallikreins, their expression patterns differ. The expression of more than one Mastomys glandular kallikrein in the kidney is similar to that in the rat, but the sequence and nonsexually dimorphic expression of the putative tissue kallikrein most closely resembles mouse. Mastomys represents an interesting hybrid between mouse and rat, providing an important animal model for studies of kallikrein expression and regulation.

Amino Acid Sequence↗
PubMed1994

The role of kallikreins in growth factor processing: the kallikrein gene family from the African rodent Mastomys.

1. Kallikreins are trypsin-like serine proteases which have been implicated in the biosynthesis of a number of growth factors and hormones. Mastomys, an African rodent, is the only mammal other than mouse known to contain high levels of kallikreins and growth factors in its submandibular gland. Mastomys has a large kallikrein family similar to mouse and rat. Cloning and sequencing of several of these cDNAs demonstrates a high degree of homology with mouse and rat kallikreins, but careful analysis suggests that it will not be possible to determine the function of these kallikreins by sequence information alone. 2. It is not known whether a Mastomys kallikrein processes and binds to nerve growth factor (NGF) as in mouse submandibular gland. Mastomys NGF is bound to a protein similar in size, charge, and lack of esterase activity to alpha-NGF, a mouse kallikrein. However, the Mastomys NGF complex does not contain a proteolytically active kallikrein as does the mouse NGF complex. Thus, the identification of kallikreins as growth factor processing enzymes and the presence of a kallikrein family member in high-molecular-weight complexes in species other than mouse is still in question.

Animals↗
PubMed1993

Nerve growth factor synthesis by mouse submandibular gland cells in culture.

Mouse submandibular gland (SMG) cells in culture rarely retain functional properties of SMG cells in vivo. We demonstrate that both primary SMG cells and the mouse SMG cell line SCA-9 secrete biologically active nerve growth factor (NGF). However, primary cells secrete 40-fold more NGF than SCA-9 cells, demonstrating that SCA-9 cells cannot substitute for primary SMG cells for the study of SMG NGF in vitro.

Animals↗
PubMed1991

beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22.

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Structure and biosynthesis of nerve growth factor.

Most of our knowledge about NGF comes from extensive study of the mouse submaxillary gland protein. NGF from this source is isolated as a high molecular weight complex consisting of beta-NGF and two subunits, alpha and gamma, belonging to the kallikrein family of serine proteases. There are few other tissues where NGF is found in sufficient quantities for protein purification and study, although new molecular biological techniques have accelerated the study of NGFs from a variety of species and tissues. Mouse submaxillary gland NGF is synthesized as a large precursor that is cleaved at both N- and C-terminals to produce mature NGF. This biologically active molecule can be further cleaved by submaxillary gland proteases. The roles of the alpha and gamma subunits in the processing of the beta-NGF precursor, the modulation of the biological activity of beta-NGF, and the protection of mature beta-NGF from degradation have been well studied in the mouse. However, the apparent lack of alpha and gamma subunits in most other tissues and species and the existence of a large family of murine kallikreins, many of which are expressed in the submaxillary gland, challenge the relevance of murine high molecular weight NGF as a proper model for NGF biosynthesis and regulation. It is important therefore to identify and characterize other NGF complexes and to study their subunit interactions, biosynthesis, processing, and regulation. This review points out a number of other species and tissues in which the study of NGF has just begun. At this time, there exist many more questions than answers regarding the presence and the functions of NGF processing and regulatory proteins. By studying NGF in other species and tissues and comparing the processing and regulation of NGF from several sources, we will discover the unifying concepts governing the expression of NGF biological activity.

Animals↗
PubMed1989

Effects of ultrasound exposure in vitro on neuroblastoma cell membranes.

Mechanisms of action of ultrasound on cell membranes were studied on two murine C1300 neuroblastoma cell lines of minimum genetic diversity. Cavitation was established in rotating polystyrene centrifuge tubes by 1 MHz cw ultrasound; exposure time was 5 minutes. NS20Y and N2A cells exposed in suspension responded similarly by 86Rb+ transport and Na+-K+-ATPase activity assays, but differently by trypan blue dye exclusion and lysis assays. This indicates similar overall damage to the cell membranes, despite use of trypsin to release N2As only. Primary evidence of damage was lysis of NS20Ys and permeabilization of N2As. These results indicate that the same ultrasound exposure conditions can produce different effects in cells that differ in their membrane properties.

Animals↗
PubMed1988

Molecular cloning of a cDNA encoding the nerve growth factor precursor from Mastomys natalensis.

Mastomys natalensis is an African rat that has high levels of nerve growth factor (NGF) in its submaxillary glands. Like in the mouse, Mastomys NGF is found as a high-molecular-weight complex. However, the Mastomys complex differs from the mouse complex, in that the gamma-subunit is either missing or is less tightly bound in the Mastomys NGF complex. In the mouse, the gamma-subunit has been implicated in the processing of the beta-NGF precursor. The possible lack of gamma-subunits in the Mastomys NGF high-molecular-weight complex suggested that the Mastomys beta-NGF precursor might differ from the mouse beta-NGF precursor in some of its processing sites. In particular, Mastomys beta-NGF might lack the C-terminal dipeptide cleavage site implicated in beta-gamma subunit interactions in mouse NGF. In order to test this hypothesis, we isolated and sequenced a cDNA clone for Mastomys beta-NGF. We report here the cloning and sequencing of a cDNA coding for beta-NGF from Mastomys natalensis. The cDNA library was prepared from Mastomys submaxillary gland mRNA and the beta-NGF clone was isolated using a mouse cDNA as a probe. The nucleotide sequence of Mastomys beta-NGF is 95% homologous to that of mouse beta-NGF. In particular, the Mastomys beta-NGF precursor contains the same three C-terminal residues as the mouse, suggesting that the Mastomys beta-NGF precursor could interact with a gamma-like subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗