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Biomedical subjects

N Marks

Publications and source records attributed to N Marks.

At least 19 recordsLinked to original sources
PubMed2001

Opposite effects of lithium on proximal and distal caspases of immature and mature primary neurons correlate with earlier paradoxical actions on viability.

To provide an explanation for earlier paradoxical findings of lithium on survival of mature and immature neurons, this study monitors changes in cytosolic caspases in rat cerebellar granule cells (CGC) grown 2-7 days in vitro (DIV), or in murine E-17 cortical neurons. Data show Li+ protects mature 7-DIV CGC parallel to a decrease in proximal and distal caspases but increases levels for immature 2-DIV-CGC or E-17 cortical neurons. Caspases mirror viability based on morphological analyses (dye uptake, phase-contrast, DNA fragmentation), and suggest protection occurs by suppressing activation of a cascade resulting in distal effectors that destroy proteins essential for neuronal survival. Protection was dose-dependent with EC50 3.0 mM and extended to 64 h in K+-serum deprived apoptotic media. Neuronal extracts contain a spectrum of proximal (-2, -8, -9) and distal (-3, -6) caspases sensitive to Li+ on assay with preferred peptide substrates and by immunoblotting. The lack of direct effect on activated cytosols indicates Li+ acts upstream only on intact cells, at sites for recruitment of pivotal procaspases. Alterations of procaspase-9 p46 and membrane-bound cytochrome c (Apaf-1) point to interaction with an intrinsic Mt-mediated pathway as one of the targets. The opposite effects on caspases and viability of immature or embryological neurons point to existence of alternative pathways that alter during neurite outgrowth suggesting the use of Li+ as a probe to unravel events relevant to neurogenesis.

Animals↗
PubMed2001

Differential expression of carnosine, homocarnosine and N-acetyl-L-histidine hydrolytic activities in cultured rat macroglial cells.

N-acetyl-L-histidine (NAH) and N-acetyl-L-aspartate (NAA) are representatives of two series of substances that are synthesized by neurons and other cells in the vertebrate central nervous system (CNS). Histidine containing homologs of NAH are beta-alanyl-L-histidine or carnosine (Carn) and gamma-aminobutyrl-L-histidine or homocarnosine (Hcarn). A homolog of NAA is N-acetylaspartylglutamate (NAAG). These substances belong to a unique group of osmolytes in that they are synthesized in cells that may not to be able to hydrolyze them, and are released in a regulated fashion to a second compartment where they can be rapidly hydrolyzed. In this investigation, the catabolic activities for NAH, Carn, and Hcarn in cultured macroglial cells and neurons have been measured, and the second compartment for NAH and Hcarn has been identified only with astrocytes. In addition, oligodendrocytes can only hydrolyze Carn, although Carn can also be hydrolyzed by astrocytes. Thus, astrocytes express hydrolytic activity against all three substrates, but oligodendrocytes can only act on Carn. The cellular separation of these hydrolytic enzyme activities, and the possible nature of the enzymes involved are discussed.

Amidohydrolases↗
PubMed1999

Recent advances on neuronal caspases in development and neurodegeneration.

In view of a large and growing literature, this overview emphasizes recent advances in neuronal caspases and their role in cell death. To provide historical perspective, morphology and methods are surveyed with emphasis on early studies on interleukin converting enzyme (ICE) as a prototype for identifying zymogen subunits. The unexpected homology of ICE (caspase-1) to Caenorhabditis elegans death gene CED-3 provided early clues linking caspases to programmed cell death, and led later to discovery of bcl-2 proteins (CED-9 homologs) and 'apoptosis associated factors' (Apafs). Availability of substrates, inhibitors, and cDNAs led to identification of up to 16 caspases as a new superfamily of unique cysteine proteinases targeting Asp groups. Those acting as putative death effectors dismantle neurons by catabolism of proteins essential for survival. Caspases degrade amyloid precursor protein (APP), presenilins (PS1, PS2), tau, and huntingtin, raising questions on their role in neurodegeneration. Brain contains 'inhibitors of apoptosis proteins' (IAPs) survivin and NAIP associated also with some neuronal disorders. Apoptotic stress in neurons initiates a chain of events leading to activation of distal caspases by pathways that remain to be fully mapped. Neuronal caspases play multiple roles for initiation and execution of cell death, for morphogenesis, and in non-mitotic neurons for homeostasis. Recent studies focus on cytochrome c as pivotal in mediating conversion of procaspase-9 as a major initiator for apoptosis. Identifying signaling pathways and related events paves the way to design useful therapeutic remedies to prevent neuronal loss in disease or aging.

Animals↗
PubMed1999

Laryngocele: an anatomical variant.

It is commonly believed that external laryngoceles always penetrate the thyrohyoid membrane at the site of penetration of the neurovascular bundle. We present a case where the site of penetration was posterosuperior to this. Careful dissection of the neck of a laryngocele sac is important to prevent damage to the neurovascular bundle.

Aged↗
PubMed1999

Gangliosides attenuate ethanol-induced apoptosis in rat cerebellar granule neurons.

Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 microM MK-801 (an NMDA receptor antagonist), and 20-200 mM ethanol for 1-4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 microM MK-801 or 100 microM glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 microM gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 microM LIGA20 (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.

Animals↗
PubMed1999

Women & HIV.

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CD4 Lymphocyte Count↗
PubMed1998

The semisynthetic glycosphingolipid LIGA20 potently protects neurons against apoptosis.

LIGA20 (a semisynthetic GM1 with n-dichloroacetyl sphingosine) having antinecrotic properties was evaluated as an antiapoptotic agent using cerebellar granule cells as a neuronal model. Neurons exposed to medium containing 5 mM K+ in absence of serum (apoptotic medium) for up to 22 h resulted in 65% cell death, accompanied by oligonucleosomal laddering, and a 15-fold-increase in caspase-3 activity. The relationships between times required for enzyme activation and cell death suggests a linkage between these two events. Pretreatment with 10 microM LIGA20 for 10 min significantly attenuated the aforementioned apoptotic changes. LIGA20 also attenuated apoptosis induced by Pb2+, ethanol, and C6-ceramide. Data show that LIGA20 is a versatile neuroprotective agent protecting cells from multiple pathways of cell death.

Animals↗
PubMed1998

Activation of caspase-3 and apoptosis in cerebellar granule cells.

Caspase-3 activity increased dramatically in cytosolic extracts of rat cerebellar granule cells exposed to apoptotic conditions (basal medium Eagle (BME) containing 5 mM K+ without serum) when assayed with Ac-DEVD-amc, but not with Ac-YVAD-afc, a preferred substrate for caspase-1. This provided a basis to examine relationships between enzyme activity and cell viability for purposes of selecting an optimal time for comparing neuroprotective agents or strategies. Exposure of neurons to an apoptotic medium containing 5 mM K+ in absence of serum led to a rapid 5- to 10-fold increase in caspase-3 within 2-4 hr but without significant cell loss, or morphological alterations. Exposure to apoptotic medium followed by replacement with maintenance medium containing 25 mM K+ and serum led to a rapid fall in caspase-3 and prevention of cell death. This strategy was not effective after 13 hr exposure despite a large fall in enzyme activity. These temporal changes infer systems for rapid enzyme turnover and/or activation of cytoplasmic components linked to later DNA degradation. The effects of cycloheximide point to requirements for protein synthesis, and those of Glu exclude a caspase-3 dependent pathway for necrotic cell damage. Brief treatment with 10 microM LIGA20, an anti-necrotic agent, also attenuated cell loss and caspase-3 activity, indicating a broad spectrum of neuroprotection. Rapid and long-lasting effects, together with its biophysical properties, suggest that this semisynthetic ganglioside acted upstream at or near a membrane site. As such, gangliosides provide useful agents to further probe pathways relevant to neuronal death in culture.

Animals↗
PubMed1998

Squamous cell carcinoma associated with lupus vulgaris.

Squamous cell carcinomas are known to arise in certain chronic, scarring dermatoses and also to be associated with exposure to ultraviolet radiation. We now report a case arising in a plaque of lupus vulgaris, the patient having received radiation from a Finsen lamp as a child for a tuberculous abscess in that region.

Aged↗
PubMed1997

Composition of white matter bovine brain coated vesicles: evidence that several components influence beta-amyloid peptide to form oligomers and aggregates in vitro.

Clathrin coated vesicles (CVs) purified from white matter of human or bovine brain contain amyloid precursor protein (APP), several C-terminal fragments encompassing the beta-amyloid domain (betaA), the alpha-secretase 11-12 kDa intermediate, ApoE and tau. The convergence of these components implicates CVs as potential sites for their interaction, yielding products linked to fibrillogenesis in Alzheimer's disease (AD). Analysis of components co-reactive with both anti-ApoE and betaA suggested presence of stable intravesicular conjugates. To evaluate these interactions in vitro, mixtures containing betaA(1-40), ApoE4 or E3 isoforms with and without lipid added as dymyristoyl phosphatidylcholine liposomes were co-incubated from 5 h to 7 days at 37 degrees C and analyzed on Western blots using a panel of antibodies recognizing betaA and ApoE. Data showed ApoE4 plus lipid induced betaA to form oligomers, conjugates and high Mr aggregates. The rates of formation for these products varied significantly with the ApoE isoform. E3 formed conjugates more rapidly, but these levels were exceeded by those of E4 at 7 days. ApoE4 plus lipid facilitated more rapid formation of higher Mr betaA aggregates which appeared in parallel with betaA oligomers containing up to seven molecules of betaA. Data suggest that the native ApoE, as found in CVs which contain lipid, can be an effective agent for promoting formation of betaA oligomers or other complexes that may be linked to formation of abnormal deposits in AD.

Amyloid beta-Peptides↗
PubMed1996

Structure-activity relationships of KNEFIRFamide (AF1), a nematode FMRFamide-related peptide, on Ascaris suum muscle.

Analogues of KNEFIRFamide (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2; AF1), an FMRFamide-related peptide (FaRP) originally isolated from Ascaris suum, were characterized in an A. suum muscle tension assay. AF1 had biphasic effects on this preparation, inducing a brief relaxation followed by excitation and spastic paralysis. Activity of AF1 in this assay was eliminated by N-terminal deletions and by deamidation of the carboxy-terminus. The potency of AF1 was greatly reduced by alanine substitution for any residue. Peptides that retained activity did not show the biphasic response observed with AF1, suggesting that the inhibitory and excitatory phases seen with AF1 may be due to activation of distinct receptors. The basis for the marked differences in potency observed between AF1 and the structurally related nematode FaRP, AF2 (KHEYLRFamide) was also tested. AF2 is approximately 1000-fold more potent than AF1 in this assay, but has physiological effects that are otherwise indistinguishable. KNEYIRFamide and KNEFLRFamide induced characteristic AF1/AF2 responses, but were much less potent than the native peptides. In contrast, KHEYIRFamide resembled AF1 in potency and pattern of responses. These data suggest that AF1 and AF2 act at distinct receptors, and hypothesis supported by the observation that KNEFIAFamide antagonized the effects of AF1 but not of AF2.

Alanine↗
PubMed1996

Lead (Pb+2) promotes apoptosis in newborn rat cerebellar neurons: pathological implications.

Despite mandated reduction in environmental lead (Pb++), such exposure still poses a public health hazard for children, with devastating effects on CNS development. To replicate aspects of this neurotoxicity, we used cultured granule cells from newborn rat cerebella to study whether apoptotic or necrotic death is the major consequence of exposure to low micromolar concentrations of Pb++. At a low dose, 1 microM (EC50 approximately equal to 10 microM), Pb++ does not affect glutamate-induced neuronal necrosis but promotes neuronal apoptosis, as characterized morphologically by cell shrinkage and chromatin condensation, biochemically by the typical internucleosomal DNA fragmentation and functionally by dependence on new synthesis of macromolecules (cycloheximide- and actinomycin D-sensitive). The low micromolar doses of Pb++ that promote apoptosis are well within the blood level range reported to impair CNS function in children and to alter synaptogenesis in the neonatal rat brain. Thus these in-vitro results suggest that the highly neurotoxic action of Pb++ in the developing CNS of children might depend on a facilitation of apoptosis. The Pb+2-elicited potentiation of neuronal apoptosis is attenuated by treatment with the voltage-sensitive Ca+2 channel agonist Bay K8644, which suggests the possible use of this agonist for treatment of the neurotoxic effects of Pb++.

Animals↗
PubMed1994

Amyloid precursor protein is enriched in axolemma and periaxolemmal-myelin and associated clathrin-coated vesicles.

The amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal-myelin from rat brain and have determined by Western blot that APPs, Mr 100-110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal-myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal-myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti-C-terminal APP antibodies but not with anti-N-terminal antibody, appears to be a membrane-associated C-terminal fragment. Western blots with antibodies to Alzheimer precursor-like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100-110 and the 10.5 kDa C-terminal peptide. The demonstration of APP in axolemma and periaxolemmal-myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin-coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal-myelin. In addition, these vesicles contain cysteinyl and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100-110 and Mr 10.5 when analyzed with antibodies to APP672-695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C-terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C-terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C-terminus and may be a useful tool for altering C-terminal APP function.

Amino Acid Sequence↗
PubMed1994

Hydrolysis of amyloid precursor protein-derived peptides by cysteine proteinases and extracts of rat brain clathrin-coated vesicles.

Amyloid precursor proteins (APPs) and C-terminal fragments were colocalized with cysteine proteinase-like enzymes in purified rat brain clathrin-coated vesicles. Vesicular extracts degraded beta A4(12-28), yielding a product profile similar to that of purified rat brain cathepsin B. Cathepsin B degraded this peptide sequentially, with initial cleavage occurring at Val18-Phe19 and Phe19-Phe20 followed by release of dipeptides. Enzyme also hydrolyzed beta A4(1-40) at Phe19-Phe20 bond but at lower rates, likely due to aggregate formation. An octapeptide analogue of the domain adjacent to beta A4 (N-Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2) was also hydrolyzed by brain cathepsins B and L, and metalloendopeptidase 24.11. Enzymes acted at multiple sites, but only 24.11 cleaved the Met-Asp bond, thus resembling a proposed beta-secretase. Data imply that clathrin-coated vesicles contain cysteine-like proteinases capable of initiating the processing of APP or its fragments.

Amino Acid Sequence↗
PubMed1994

Primary structure of the human platelet serotonin 5-HT2A receptor: identify with frontal cortex serotonin 5-HT2A receptor.

Previous radioligand binding studies have demonstrated human platelet serotonin2A (5-HT2A) receptor binding sites. Pharmacological similarities between platelet and frontal cortex 5-HT2A receptor binding parameters have been demonstrated. However, it is not clear whether the platelet 5-HT2A receptor primary structure is identical to that of the brain receptor. Three overlapping cDNAs were obtained to span completely the coding region of the 5-HT2A receptor. These clones were sequenced with external and internal primers. The nucleotide sequence of human platelet 5-HT2A cDNA was identical to that reported for the human frontal cortex 5-HT2A receptor, except for nucleotide 102 (T-->C), which has been reported to represent a normal DNA polymorphism that does not alter the amino acid sequence. This finding may have implications in the study of neuropsychiatric disorders for which altered platelet 5-HT2A receptor binding has been demonstrated.

Adult↗