Biomedical subjects

Michael F Wilkemeyer

Publications and source records attributed to Michael F Wilkemeyer.

5 recordsLinked to original sources

PubMed2004

Ethanol antagonist peptides: structural specificity without stereospecificity.

Increasing evidence suggests that ethanol damages the developing nervous system partly by disrupting the L1 cell adhesion molecule. Ethanol inhibits L1-mediated cell adhesion, and compounds that antagonize this action also prevent ethanol-induced embryotoxicity. Two such compounds are the small peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL). We showed previously that NAP and SAL antagonize ethanol inhibition of L1 adhesion at femtomolar to picomolar concentrations. Here we demonstrate that, despite this extraordinary potency, both NAP and SAL lack stereospecificity. d-NAP, a peptide composed entirely of d-amino acids, was an effective ethanol antagonist in NIH/3T3 cells transfected with human L1 and in the NG108-15 neural cell line. Interestingly, Ala-substituted derivatives of d-NAP demonstrate the same structure-activity relation as the corresponding derivatives of l-NAP. The Ser-Ile-Pro motif was important for the ethanol antagonist activity of d-NAP, l-NAP, and l-SAL, with Ile being the most critical element in all three. Like l-NAP, d-NAP effectively reduced ethanol-induced growth retardation in mouse whole embryo culture. The potential resistance of d-peptides to proteases makes d-NAP a potentially attractive agent for the prevention of fetal alcohol syndrome.

3T3 Cells↗

PubMed2003

Differential effects of ethanol antagonism and neuroprotection in peptide fragment NAPVSIPQ prevention of ethanol-induced developmental toxicity.

NAPVSIPQ (NAP), an active fragment of the glial-derived activity-dependent neuroprotective protein, is protective at femtomolar concentrations against a wide array of neural insults and prevents ethanol-induced fetal wastage and growth retardation in mice. NAP also antagonizes ethanol inhibition of L1-mediated cell adhesion (ethanol antagonism). We performed an Ala scanning substitution of NAP to determine the role of ethanol antagonism and neuroprotection in NAP prevention of ethanol embryotoxicity. The Ser-Ile-Pro region of NAP was crucial for both ethanol antagonism and protection of cortical neurons from tetrodotoxin toxicity (neuroprotection). Ala replacement of either Ser-5 or Pro-7 (P7A-NAP) abolished NAP neuroprotection but minimally changed the efficacy of NAP ethanol antagonism. In contrast, Ala replacement of Ile-6 (I6A-NAP) caused a decrease in potency (>2 logarithmic orders) with only a small reduction (<10%) in the efficacy of NAP neuroprotection but markedly reduced the efficacy (50%) and the potency (5 logarithmic orders) of NAP ethanol antagonism. Ethanol significantly reduced the number of paired somites in mouse whole-embryo culture; this effect was prevented significantly by 100 pM NAP or by 100 pM P7A-NAP, but not by 100 pM I6A-NAP. The structure-activity relation for NAP prevention of ethanol embryotoxicity was similar to that for NAP ethanol antagonism and different from that for NAP neuroprotection. These findings support the hypothesis that NAP antagonism of ethanol inhibition of L1 adhesion plays a central role in NAP prevention of ethanol embryotoxicity and highlight the potential importance of ethanol effects on L1 in the pathophysiology of fetal alcohol syndrome.

Abnormalities, Drug-Induced↗

PubMed2002

Peptide antagonists of ethanol inhibition of l1-mediated cell-cell adhesion.

Ethanol inhibits cell-cell adhesion mediated by the L1 cell adhesion molecule. 1-Octanol potently antagonizes this cellular action of ethanol and also prevents ethanol-induced dysmorphology and cell death in mouse whole embryo culture. NAPVSIPQ (NAP) and SALLRSIPA (SAL) are active peptide fragments of two neuroprotective proteins: activity-dependent neuroprotective protein and activity-dependent neurotrophic factor. NAP and SAL are neuroprotective at femtomolar concentrations against a variety of neurotoxins and also prevent ethanol teratogenesis in mice. To explore the cellular basis for this action, we asked whether NAP and SAL antagonize ethanol inhibition of L1 adhesion. Aggregation assays were carried out in ethanol-sensitive, human L1-transfected NIH/3T3 cells in the absence and presence of NAP and SAL. Neither NAP nor SAL altered L1 adhesion or L1 expression; however, both peptides potently and completely antagonized the inhibition of L1 adhesion by 100 mM ethanol (EC(50): NAP, 6 x 10(-14) M; SAL, 4 x 10(-11) M). NAP also antagonized ethanol inhibition of cell-cell adhesion in bone morphogenetic protein-7-treated NG108-15 cells. In L1-expressing NIH/3T3 cells, SAL antagonism was reversible and could be overcome by increasing concentrations of ethanol. In contrast, NAP antagonism was irreversible and could not be overcome by increasing agonist concentration. Two scrambled NAP peptides (ASPNQPIV and PNIQVASP) were not antagonists at concentrations as high as 10(-7) M. Thus, two structurally unrelated classes of compounds, alcohols and small polypeptides, share two common actions: antagonism of ethanol inhibition of L1-mediated cell adhesion and prevention of ethanol teratogenesis. These findings support the hypothesis that ethanol inhibition of L1 adhesion contributes to ethanol teratogenesis.

3T3 Cells↗

PubMed2002

Novel antagonists of alcohol inhibition of l1-mediated cell adhesion: multiple mechanisms of action.

1-Octanol antagonizes ethanol inhibition of L1-mediated cell adhesion and prevents ethanol teratogenesis in mouse whole embryo culture. Herein, we identify a new series of alcohol antagonists and study their mechanism of action. Cell aggregation assays were carried out in ethanol-sensitive, human L1-transfected NIH/3T3 cells in the absence and presence of 100 mM ethanol or 2 mM 1-butanol and candidate antagonists. Antagonist potency for 1-alcohols increased progressively over 5 log orders from 1-pentanol (C5) to 1-dodecanol (C12). Antagonist potency declined from 1-dodecanol (C12) to 1-tridecanol (C13), and 1-tetradecanol (C14) and 1-pentadecanol (C15) were inactive. The presence and position of a double bond in the 1-butanol molecule determined whether a compound was a full agonist (1-butanol), a mixed agonist-antagonist (2-buten-1-ol), or an antagonist (3-buten-1-ol). Increasing the concentration of agonist (1-butanol or ethanol) overcame the antagonism of 3-buten-1-ol, benzyl alcohol, cyclopentanol, and 3-pentanol, but not that of 4-methyl-1-pentanol, 2-methyl-2-pentanol, 1-pentanol, 2-pentanol, 1-octanol, and 2,6-di-isopropylphenol (propofol), suggesting that the mechanisms of antagonism may differ between these groups of compounds. These findings suggest that selective straight, branched, and cyclic alcohols may act at multiple, discrete sites to antagonize the actions of ethanol and 1-butanol on L1-mediated cell-cell adhesion.

1-Butanol↗

PubMedDate not supplied

Peptide-mediated protection from ethanol-induced neural tube defects.

Ethanol inhibition of L1-mediated cell adhesion may contribute to the spectrum of neurological, behavioral and morphological abnormalities associated with prenatal ethanol exposure. We showed previously that the neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) antagonize ethanol inhibition of L1 adhesion and prevent ethanol-induced growth retardation in mouse whole embryo culture. Here we ask whether NAP and SAL also prevent ethanol-induced major malformations of the nervous system. Gestational day 8.0 (3-5 somites) C57BL/6J mouse embryos were grown for 6 h in control medium, 100 mM ethanol and 10(-10) M peptides and then maintained for an additional 20 h in control medium. At the end of the culture period, only embryos having 18-19 somite pairs were examined and compared for the degree of neural tube closure. Ethanol exposure resulted in neural tube defects (NTDs) consistent with total dysraphia and anencephaly. Co-incubation with ethanol and L-NAP (all L-amino acids), D-NAP (all D-amino acids) or SAL significantly increased the percentage of embryos that had begun to close their neural folds at the level of the forebrain/midbrain junction or that had progressed beyond this stage of closure. P7A-NAP (NAPVSIAQ), which lacks neuroprotective activity, but retains activity as an antagonist of ethanol inhibition of L1 adhesion, was effective in preventing ethanol-induced NTDs. In contrast, I6A-NAP (NAPVSAPQ), which shows reduced efficacy as an ethanol antagonist but retains its neuroprotective efficacy, did not significantly diminish the induction of NTDs by ethanol. These findings demonstrate the ability of NAP and SAL to prevent ethanol-induced NTDs and support the hypothesis that ethanol teratogenesis is caused in part by ethanol inhibition of L1-mediated cell adhesion.

Animals↗