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Biomedical subjects

Gerry Shaw

Publications and source records attributed to Gerry Shaw.

18 recordsLinked to original sources
PubMed2006

Comparison of two ELISA methods for measuring levels of the phosphorylated neurofilament heavy chain.

BACKGROUND: Recent studies suggest that the quantification of neurofilament subunits in cerebrospinal fluid (CSF), blood and amniotic fluid may reflect neuroaxonal damage and be of high clinical value. The present study aims to cross-validate two different independently developed ELISA techniques for the quantification of the phosphorylated axonal forms of the neurofilament heavy chain (pNfH). METHODS: The London ELISA method is based on barbitone buffer and the commercially available SMI35 capture antibody. The Gainesville method uses Tris-buffered saline (TBS) and an affinity purified chicken polyclonal capture antibody (C-pNfH). Coded CSF from 50 patients with neurological diseases were analyzed in duplicate by both laboratories, each using both ELISA methods, but with each lab using their own detection antibody, tertiary antibody and chromogen. Methods were compared using Bland-Altman plots. Correlation and regression analyses were used to allow for transformation of values between both methods. RESULTS: The Bland-Altman plots demonstrated that 96% of all samples fell into the narrow 95% limits of agreement (0.04 units of OD). There was a high correlation (Spearman R=0.92, p<0.0001 and Pearson R=0.98, p<0.0001) between the Gainesville (Y) and the London (X) method with Y=0.132+1.104(*)X. The previously determined upper reference limit of 0.73 mg/l (London method) corresponds to 0.94 mg/l for the Gainesville method. CSF pNfH levels above the reference limit were observed in patients with encephalitis, encephalomyelitis, hydrocephalus, subarachnoid haemorrhage, spino-muscular atrophy, stroke and cancer with both methods agreeing in all cases. CONCLUSION: The two assays are in excellent agreement, suggesting that pNfH, which has a number of unusual protein chemical features, may be the biomarker of choice for the routine and robust detection of axonal injury and degeneration in both research and clinical contexts.

Adolescent↗
PubMed2006

Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum.

The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.

Administration, Inhalation↗
PubMed2006

Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods.

BACKGROUND: Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity. METHODS: Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions. RESULTS: At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7. CONCLUSIONS: Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.

14-3-3 Proteins↗
PubMed2005

Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury.

Several lines of reasoning suggest that the phosphorylated axonal form of the neurofilament subunit NF-H is likely to be released from damaged and diseased neurons in significant amounts. Detection of this protein in serum or CSF might therefore provide information about the presence and degree of neuronal loss. We therefore developed a sensitive NF-H ELISA capable of detecting picogram quantities of phosphorylated NF-H (pNF-H). This assay showed that soluble pNF-H immunoreactivity is readily detectable in the sera of adult rats following various types of experimental spinal cord injury (SCI) and traumatic brain injury (TBI), but is undetectable in the sera of normal animals. Here we describe details of the time course and extent of serum pNF-H expression following experimental SCI and TBI. Following SCI, serum pNF-H showed an initial peak of expression at 16h and a second, usually larger, peak at 3 days. Following TBI, lower levels of serum pNF-H were detected with a peak at 2 days post-injury. We also show that the higher levels of pNF-H released from injured spinal cord as compared to brain are in line with the approximately 20-fold higher levels of pNF-H present in spinal cord. These findings suggest that serum levels of pNF-H immunoreactivity may be used to conveniently monitor neuronal damage and degeneration in experimental and presumably clinical situations.

Animals↗
PubMed2005

Functional mechanisms of apoptosis-related proteins in neonatal rat cerebellum are differentially influenced by ethanol at postnatal days 4 and 7.

Exposure of the developing nervous system to ethanol (EtOH) produces neurological aberrations associated with fetal alcohol syndrome. During a well-defined period, cerebellar neurons are highly susceptible to EtOH-induced death, primarily through apoptosis. Neonatal rodent cerebellum is exquisitely sensitive to EtOH on postnatal days 4-6 (P4-6); however, at slightly later developmental ages (P7 and later), EtOH effects are minimal. We have previously shown that EtOH differentially influences expression of apoptosis-related proteins of the Bcl-2 survival-regulatory gene family in P4 and P7 cerebellum. In the present study, the effects of EtOH on multiple functional mechanisms of Bcl-2, Bcl-xL, and Bax were investigated to characterize further the processes underlying these disparate EtOH sensitivities. For these analyses, we addressed the following questions, by using P4 and P7 cerebellar tissue following in vivo exposure: 1) Are there differential patterns of expression of antiapoptotic Bcl-2 or proapoptotic Bax in EtOH-vulnerable Purkinje cells that could contribute to the different degrees of temporal EtOH vulnerability? 2) How does EtOH affect intracellular localization of apoptosis-related proteins? 3) Does cleavage of Bax contribute to EtOH sensitivity? 4) Does EtOH differentially modulate cerebellar protein-protein interactions of Bcl-2, Bcl-xL, and Bax at the vulnerable vs. the resistant ages? Overall, we show that, at P4, the EtOH-mediated effects on Bcl-2, Bcl-xL, and Bax favor a prodeath response, whereas most of the intracellular responses to EtOH exposure at P7 promote survival. Such differential responsiveness likely plays a major role in the disparate ethanol vulnerability at these two postnatal ages.

Age Factors↗
PubMed2005

Extending the clinicopathological spectrum of neurofilament inclusion disease.

We describe features of a patient that broadens the clinical and pathological spectrum of neurofilament inclusion disease (NFID). The patient was a 52-year-old man with a 5--6 year history of progressive, asymmetrical spastic weakness of the upper and lower extremities; L-DOPA-unresponsive parkinsonism; and SPECT evidence of asymmetrical frontoparietal and basal ganglia hypoperfusion. The brain had marked frontoparietal parasagittal cortical atrophy, including the motor cortex, with histopathological evidence of neurofilament- and alpha-internexin-immunoreactive neuronal inclusions. The corticospinal tract had degeneration, but there was minimal lower motor neuron pathology. There was also severe neuronal loss and gliosis in the posterolateral putamen and the substantia nigra, mimicking multiple system atrophy; however, glial cytoplasmic inclusions were not detected with alpha-synuclein immunohistochemistry. This case extends the clinical and pathological spectrum of NFID to include cases with predominant parkinsonian and pyramidal features.

Carrier Proteins↗
PubMed2005

MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity.

Actins↗
PubMed2004

Characterization of the bovine neurofilament NF-M protein and cDNA sequence, and identification of in vitro and in vivo calpain cleavage sites.

Neurofilaments are the major components of the neuronal cytoskeleton, and accumulations of these proteins are associated with several important human diseases. Here we report the cloning and sequencing of bovine NF-M, the first NF-M cloned from a large domestic mammal. The bovine sequence proves to be generally more similar to that of human NF-M than the previously described mammalian sequences, suggesting that bovine neurofilaments are a useful model for biochemical studies of application to humans. However, we noted some unusual features within the 16 lys-ser-pro (KSP2) type phosphopeptide repeats and also note that the number of these repeats correlates well with the size of animal. We also characterized two in vitro calpain cleavage sites by direct peptide sequencing, finding that both are located in the glutamic acid rich E segment. Finally, we show biochemically that the more abundant and stable of these calpain fragments can also be detected in vivo.

Amino Acid Sequence↗
PubMed2004

Pycnogenol and vitamin E inhibit ethanol-induced apoptosis in rat cerebellar granule cells.

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol-induced activation of caspase-3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure.

Animals↗
PubMed2003

Ethanol effects on neonatal rat cortex: comparative analyses of neurotrophic factors, apoptosis-related proteins, and oxidative processes during vulnerable and resistant periods.

The developing central nervous system (CNS) is highly susceptible to ethanol, with acute or chronic exposure producing an array of anomalies and cell loss. Certain periods of vulnerability have been defined for various CNS regions, and are often followed by periods of relative ethanol resistance. In the present study, neonatal rats were acutely exposed to ethanol during a time when peak cell death is found in developing cerebral cortex (postnatal day 7; P7), and during a later neonatal period of ethanol resistance (P21). Comparisons at the two ages were made of basal levels of neurotrophic factors (NTFs), and in addition, ethanol-mediated changes in NTFs, apoptosis-related proteins, antioxidant activities, and generation of reactive oxygen species (ROS) were quantified at 0, 2, and 12 h following termination of exposure. It was found that at P21, basal levels of NTF nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were considerably higher than at P7, possibly affording protection against ethanol neurotoxicity at this age. Following ethanol treatment at P7, approximately equal numbers of pro-apoptotic and pro-survival changes were produced, although most of the pro-apoptotic alterations occurred rapidly following termination of treatment, a critical period for initiation of apoptosis. At P21, however, the large majority of ethanol-mediated changes were adaptive, favoring survival. We speculate that the capacity of the older CNS to upregulate a number of protective elements within the cellular milieu serves to greatly mitigate ethanol neurotoxicity, while in younger animals, such adjustments are minimal, thus enhancing ethanol vulnerability within this developing region.

Adaptation, Physiological↗
PubMed2003

A neurotoxic peripherin splice variant in a mouse model of ALS.

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.

Alternative Splicing↗
PubMed2003

The role of neurotrophic factors, apoptosis-related proteins, and endogenous antioxidants in the differential temporal vulnerability of neonatal cerebellum to ethanol.

BACKGROUND: Ethanol produces abnormalities in the developing nervous system, with certain regions being vulnerable during well-defined periods. Neonatal rodent cerebellum is particularly susceptible to ethanol during the early postnatal period [on postnatal days 4-5 (P4-5)], while this region is resistant to ethanol at a slightly later time (P7-9). We assessed basal levels of several substances which may be involved in differential temporal ethanol vulnerability in neonatal cerebellum, and analyzed alterations in these substances after early ethanol exposure. METHODS: Assessments were made of neurotrophic factors nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4; apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Bcl-xs, Bad, phosphorylated-Bad, phosphorylated-Akt, and phosphorylated-c-Jun N-terminal kinase; and the antioxidants superoxide dismutase, glutathione reductase, and catalase. These analyses quantified basal levels (in controls), and sequential changes following acute ethanol exposure at the vulnerable and resistant cerebellar periods (P4, P7). RESULTS: Comparisons of basal levels of the molecules assessed between P4 and P7 revealed higher levels of total proapoptotic Bad at p4, higher levels of the protective pAkt kinase at P7, and lower levels of proapoptotic pJNK at P7. Other basal levels did not differ. While ethanol-mediated alterations were found at both ages favoring both apoptosis and survival, the apoptosis-promoting changes produced on P4 exceeded those on P7, and most occurred within the first 2 hr after exposure, a critical survival/death period. The number of alterations favoring survival were similar at the two ages, but at P7 most occurred within the first 2 hr after exposure, possibly acting in a protective manner. CONCLUSIONS: Differential temporal vulnerability to ethanol in the neonatal cerebellum appears to be paralleled by cellular alterations in neurotrophic factors, apoptosis-regulatory proteins, and/or antioxidant activities which generally favor apoptosis at the most sensitive age and survival at the resistant age.

Age Factors↗
PubMed2002

Axonally transported peripheral signals regulate alpha-internexin expression in regenerating motoneurons.

The class IV neuronal intermediate filament (IF) family proteins includes the neurofilament (NF) triplet proteins NF-L, NF-M, and NF-H and also the more recently characterized alpha-internexin-NF66. It is well established that NF-L, -M, and -H protein and mRNA are downregulated after peripheral nerve injury. We examined alpha-internexin protein expression after three facial nerve lesion paradigms: crush, transection, and resection. Alpha-internexin immunoreactivity was absent in the perikarya of uninjured facial motoneurons but increased dramatically in all three injury paradigms, with maximum immunoreactivity observed at 7 d after injury. Twenty-eight days after nerve crush or transection, there was a dramatic decrease in the number of alpha-internexin-positive cells. In contrast, alpha-internexin remained elevated 28 d after nerve resection, an injury that hinders regeneration and target reinnervation. In situ hybridization studies showed an increase in alpha-internexin mRNA expression in the facial nucleus at 7 and 14 d after injury. Retrograde transport of fluorogold from the whisker pads to the facial nucleus was seen only in motoneurons that lacked alpha-internexin immunoreactivity, supporting the idea that target reinnervation and inhibitory signals from the periphery regulate the expression of alpha-internexin. Blockage of axonal transport through local colchicine application induced strong immunoreactivity in motoneurons. Alpha-internexin expression was also examined after central axotomy of rubrospinal neurons, which constitutively show alpha-internexin immunoreactivity. After rubrospinal tractotomy, alpha-internexin immunoreactivity transiently increased by 7 d after injury but returned to control levels by 14 d. We conclude that alpha-internexin upregulation in injured motoneurons suggests a role for this IF protein in neuronal regeneration.

Animals↗
PubMed2002

Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells.

The 293 cell line was derived by transformation of primary cultures of human embryonic kidney (HEK) cells with sheared adenovirus (Ad)5 DNA. A combination of immunostaining, immunoblot, and microarray analysis showed that 293 cells express the neurofilament (NF) subunits NF-L, NF-M, NF-H, and a-internexin as well as many other proteins typically found in neurons. Three other independently derived HEK lines, two transformed by Ad5 and one by Ad12, also expressed NFs, as did one human embryonic retinal cell line transformed with Ad5. Two rodent kidney lines transformed with Ad12 were also found to express NF proteins, although several rodent kidney cell lines transformed by Ad5 DNA and three HEK cell lines transformed by the SV40 early region did not express NFs. These results suggest that human Ads preferentially transform human neuronal lineage cells. We also demonstrate that the widely used HEK293 cells have an unexpected relationship to neurons, a finding that may require reinterpretation of many previous studies in which it was assumed that HEK293 cells resembled more typical kidney epithelial cells.

Adenoviruses, Human↗
PubMed2002

ANG II stimulation of neuritogenesis involves protein kinase B in brain neurons.

Stimulation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) signal transduction pathway has been linked to the neuromodulatory action of ANG II in the brain neurons of the spontaneously hypertensive rat (Yang H and Raizada MK. J Neurosci 19: 2413-2423, 1999). The cellular consequences of this signaling pathway, however, remain unknown in the brain neurons from the normotensive rat. The present study was designed to test the hypothesis that the PI3K-PKB signaling cascade activates an ANG II-mediated neuritogenic action by stimulating cellular growth-associated protein-43 (GAP-43) and neurite extension in Wistar-Kyoto rat brain neurons. ANG II activation of the ANG II type 1 receptor caused increases in PKB activity, cellular GAP-43 levels, and neurite extension in a time- and dose-dependent manner. Depletion of PKB by specific antisense oligonucleotides attenuated ANG II stimulation of both GAP-43 and neurite extension. PKB involvement in neuritogenic action is further supported by the observation that neurons that overexpress PKB develop extensive neuronal processes in the absence of ANG II. These observations demonstrate that PKB is directly involved in ANG II-mediated effects and may recruit both nuclear and cytoplasmic signaling systems for this action.

Angiotensin II↗
PubMed2002

Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem.

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker alpha-internexin, while the other stained with the astrocyte marker GFAP. The alpha-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These alpha-internexin positive cells coimmunostained for the neuronal markers MAP2, type III beta-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these alpha-internexin positive cells revealed small Ca(2+) currents with a peak current of -0.5 +/- 0.1 pA/pF at a membrane potential of -20 mV (n = 5) and half-maximal activation at -30 mV (n = 5). Na(+) currents with a peak current density of -154.5 +/- 49.8 pA/pF at a membrane potential of -15 mV (n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.

Age Factors↗