Structural implications of a Val-->Glu mutation in transmembrane peptides from the EGF receptor.

Certain specific point mutations within the transmembrane domains of class I receptor tyrosine kinases are known to induce altered behavior in the host cell. An internally controlled pair of peptides containing the transmembrane portion of the human epidermal growth factor (EGF) receptor (ErbB-1) was examined in fluid, fully hydrated lipid bilayers by wide-line 2H-NMR for insight into the physical basis of this effect. One member of the pair encompassed the native transmembrane sequence from ErbB-1, while in the other the valine residue at position 627 was replaced by glutamic acid to mimic a substitution that produces a transformed phenotype in cells. Heteronuclear probes having a defined relationship to the peptide backbone were incorporated by deuteration of the methyl side chains of natural alanine residues. 2H-NMR spectra were recorded in the range 35 degrees C to 65 degrees C in membranes composed of 1-palmitoyl-2-oleoyl phosphatidylcholine. Narrowed spectral components arising from species rotating rapidly and symmetrically within the membrane persisted to very high temperature and appeared to represent monomeric peptide. Probes at positions 623 and 629 within the EGF receptor displayed changes in quadrupole splitting when Val(627) was replaced by Glu, while probes downstream at position 637 were relatively unaffected. The results demonstrate a measurable spatial reorientation in the region of the 5-amino acid motif (residues 624-628) often suggested to be involved in side-to-side interactions of the receptor transmembrane domain. Spectral changes induced by the Val-->Glu mutation in ErbB-1 were smaller than those induced by the analogous oncogenic mutation in the homologous human receptor, ErbB-2 (Sharpe, S., K. R. Barber, and C. W. M. Grant. 2000. Biochemistry. 39:6572-6580). Quadrupole splittings at probe sites examined were only modestly sensitive to temperature, suggesting that each transmembrane peptide behaved as a motionally ordered unit possessing considerable conformational stability.

Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail.

BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known. RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. CONCLUSIONS: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.

Reducing anxiety in parents before and during pediatric anesthesia induction.

Fear and anxiety in a child undergoing surgery are correlated positively with the parent's level of anxiety, and interventions to decrease the parent's anxiety are appropriate. The purpose of this study was to determine whether viewing a video of an actual pediatric inhalation induction would reduce the level of parental anxiety. Eighty patients requiring an inhalation anesthetic induction were selected and randomized to 2 groups. Parents in the experimental group (group 1; n = 40) viewed a video demonstrating pediatric mask induction. Parents in the control group (group 2; n = 40) received an information pamphlet only. Anxiety was measured perioperatively in the parents and their children. Mean arterial pressure for children in group 1 was significantly lower during preoperative holding and following induction (P < .05). The level of anxiety postoperatively of children and parents in group 1 was significantly lower than that of children and parents in group 2 (P < .05). Viewing a preoperative video seems to be beneficial. Decreasing the parent's level of anxiety preoperatively may have a positive effect on the child's level of anxiety expressed postoperatively.

Val(659)-->Glu mutation within the transmembrane domain of ErbB-2: effects measured by (2)H NMR in fluid phospholipid bilayers.

Certain point mutations within the hydrophobic transmembrane domains of class I receptor tyrosine kinases have been associated with oncogenic transformation in vitro and in vivo [Gullick, J., and Srinivasan, R. (1998) Breast Cancer Res. Treat. 52, 43-53]. An important example is the replacement of a single (hydrophobic) valine by (charged) glutamate in the rat protein, Neu, and in the homologous human protein, ErbB-2. It has been suggested that the oncogenic nature of this Val-->Glu substitution may derive from alteration of the transmembrane domain's ability to take part in direct side-to-side associations. In the present work, we examined the basis of this phenomenon by studying transmembrane portions of ErbB-2 in fluid bilayer membranes. An expression system was designed to produce such peptides from the wild-type ErbB-2, and from an identical region of the transforming mutant in which Val(659) is replaced by Glu. All peptides were 50-mers, containing the appropriate transmembrane domain plus contiguous stretches of amino acids from the cytoplasmic and extracellular domains. Deuterium heteronuclear probes were incorporated into alanine side chains (thus, each alanine -CH(3) side chain became -CD(3)). Given the presence of natural alanine residues at positions 648 and 657 within ErbB-2, this approach afforded heteronuclear probes within the motif Ser(656)AlaValValGlu(660), thought to be important for homodimer formation, and nine residues upstream of this site. Further peptides were produced, by site-directed mutagenesis, to confirm spectral assignments and to provide an additional probe location at position 670 (11 residues downstream of the motif region). On SDS-polyacrylamide gels, the transmembrane peptides migrated as predominant monomers in equilibrium with smaller populations of homodimers/-oligomers. CD spectra of both wild-type and transforming mutant peptides were consistent with the transmembrane portions being basically alpha-helical. (2)H NMR spectra of each transmembrane peptide were obtained in fluid phospholipid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) from 35 to 65 degrees C. Results were consistent with the concept that the glutamic acid residue characterizing the mutant is uncharged at neutral pH. Narrowed spectral components from species rotating rapidly and symmetrically within the membrane appeared to represent monomeric peptide. Mutation of Val(659) to Glu within the hydrophobic domain induced changes in side chain angulation of at least 6-8 degrees at Ala(657) (i.e., within the five amino acid motif thought to be involved in homodimer formation), and downstream of this site to residue 670. There was little evidence of effect at the upstream site (Ala(648)) at the membrane surface. This result argues that the transforming mutation is associated with significant intramolecular rearrangement of the monomeric transmembrane helix-extending over some four helix turns-which could influence its lateral associations. In addition, temperature effects on spectral quadrupole splittings suggested that there is greater peptide backbone flexibility for the wild-type transmembrane region.

Expression and membrane assembly of a transmembrane region from Neu.

Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways. The present work addresses problems surrounding expression, isolation, secondary structure recovery, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes. A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli. The sequence also contained 11-12 amino acids from each side of the transmembrane domain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product. This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption losses. Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior. Dissolution in certain acidic organic solvents led to material with increased alpha-helix content, which behaved in detergent as mixtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes. For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression. The same acidic organic solvents used to dissolve and dissociate expressed transmembrane peptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.

Sequence specific analysis of the heterogeneous glycan chain from peanut peroxidase by 1H-NMR spectroscopy.

The cationic peanut peroxidase is a complex enzyme consisting of a heme group, two calcium ions and three complex carbohydrate chains at positions Asn60, 144 and 185. Details of the heme and calcium ligation, necessary for oxidation, have recently been revealed from the three-dimensional structure of the peroxidase. However, the three glycans that may be important for the stability of the enzyme as well as its activity were not resolved. In order to determine the configuration of one of these glycans, PNGase A was used to cleave the glycan from the enzyme at Asn-144. This glycan was studied by two dimensional 1H-NMR spectroscopy to identify the sugar linkages. The results indicated a glycan structure comprising a Man alpha1-6(Xyl beta1-2)Man beta1-4GlcNAc beta1-4(Fuc alpha1-3)GlcNAc beta core but with an additional Man alpha1-3 appendage linked to Man3. The glycan also appeared to be heterogeneous as was noted from a single terminating galactose being linked to approximately 20-25% glycan.

Ranitidine-induced acute dystonia.

Acute dystonia is a dramatic form of extrapyramidal side effects of antipsychotic medications. Although extrapyramidal reactions have been noted in related drugs, there are no existing reports associated with ranitidine. This report describes a case of an acute dystonic reaction secondary to a commonly prescribed, currently approved over-the-counter drug, ranitidine.

Specificity and Zn2+ enhancement of the S100B binding epitope TRTK-12.

The calcium-binding protein S100B (an S100 dimer composed of two S100beta monomers) is proposed to act as a calcium-sensory protein through interactions with a variety of proteins. While the nature of the exact targets for S100B has yet to be defined, random bacteriophage peptide mapping experiments have elucidated a calcium-sensitive "epitope" (TRTK-12) for S100B recognition. In this work, interactions of TRTK-12 with S100B have been shown to be calcium-sensitive. In addition, the interactions are enhanced by zinc binding to S100B, resulting in an approximate 5-fold decrease in the TRTK-12/S100B dissociation constant. Moreover, Zn2+ binding alone has little effect. TRTK-12 showed little evidence for binding to another S100 protein, S100A11 or to a peptide derived from the N terminus of S100B, indicating both a level of specificity for TRTK-12 recognition by S100B and that the N-terminal region of S100B is probably not involved in protein-protein interactions. NMR spectroscopy revealed residues most responsive to TRTK-12 binding that could be mapped to the surface of the three-dimensional structure of calcium-saturated S100B, revealing a common region indicative of a binding site.

Epidermal growth factor receptor transmembrane domain: 2H NMR implications for orientation and motion in a bilayer environment.

As part of a study of receptor tyrosine kinase behavior in membranes, we have collected extensive NMR data from three well-defined probe locations within the transmembrane region of the human EGF receptor. Spectra were obtained for selectively deuterated alanine residues in a series of peptides corresponding to the putative transmembrane domain (with short extramembranous extensions). Peptides were incorporated into fluid unsonicated liposomes of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and POPC containing 33 mol % cholesterol to mimic common lipid composition of cell plasma membranes. The peptide concentration was in the range of 1-6 mol % relative to that of phospholipid. Data acquired at 35 degreesC have been analyzed quantitatively to determine their implications to receptor spatial orientation and dynamics. If it is presumed that the single transmembrane portion approximates an alpha-helix of 3.6 residues per turn, this helix was found to be tilted away from the membrane perpendicular, about which there was rapid axial diffusion. However, rotation about the peptide long axis was static on the NMR time scale of 10(-)4 s, and the peptide appeared to have a preferred direction(s) of lean. The results for this peptide, whose hydrophobic length is greater than the membrane hydrophobic thickness, were very similar between membranes of POPC and membranes of POPC containing 33 mol % cholesterol, despite considerable host matrix differences in thickness and order. Allowed values of peptide tilt occupied a narrow range: between 10 and 14 degrees in POPC and between 10 and 12 degrees in POPC/cholesterol. Although the existence of some preferred direction(s) of lean was demanded by the results, the direction of lean was not uniquely determined. We have interpreted these results, which were essentially unchanged at 65 degreesC, as reflecting the behavior of peptide monomers undergoing rapidly reversible peptide-peptide interactions. For transmembrane monomers, interference with rotation about the peptide long axis might be understood to arise from an energy benefit (in a tilted peptide) to prevention of particular amino acid side chains near the membrane surfaces from moving in and out of hydrophobic or hydrophilic environments. It will be desirable to test the conclusion of preferential lean of a monomeric receptor since such behavior could provide a mechanism for modulating monomer association with other species (i.e., signal transduction).

Differences between African American and Caucasian men participating in a community-based prostate cancer screening program.

Prostate cancer is a major contributor to morbidity and mortality in the male population, but public awareness of the cancer has been reported as minimal. We evaluated the effectiveness of an educational prostate cancer screening program on 944 men in a midwest urban community. Digital rectal examinations and PSA blood tests were provided at no charge to participants with a grant from the Michigan Department of Community Health. An educational intervention that stressed the importance of prostate cancer early detection and treatment was conducted before screenings. A brief questionnaire administered before and after the videotape and screenings, targeted both knowledge and attitudes concerning prostate cancer. Pre-test results revealed that African American men were significantly (t = 3.7, P = .00) less likely then white men to correctly identify early symptoms of prostate cancer and the basic components of a prostate checkup. Following program involvement, scores significantly improved in all areas and differences were no longer significant between the races. Racial differences were also found for screening preferences and modes of reaching men to participate in screening. African American men were twice as likely as white men to choose private appointments over mass screening (OR = 2.2, P = .00). Radio reached the most African Americans (25%) while newspaper reached the most Caucasians (34%). The decreased level of knowledge among African Americans regarding prostate etiology and clinical factors highlights the need for educational programs to target minority populations. The need for discretion also applies by providing minority-favored access with screening through private appointments.

Sequence-related behaviour of transmembrane domains from class I receptor tyrosine kinases.

2H NMR spectroscopy and freeze-fracture electron microscopy were used to compare the transmembrane domains of two Class I protein receptor tyrosine kinases (the EGF receptor and Neu/erbB-2) regarding overall behaviour in fluid lipid bilayer membranes. The 34-residue peptide, EGFRtm, was synthesised to contain the 23 amino acid hydrophobic stretch (Ile622 to Met644) thought to span the membrane of the human EGF receptor, plus the first 10 amino acids (Arg645 to Thr654) of the cytoplasmic domain. Deuterium probes replaced selected 1H nuclei at sites corresponding to Ala623, Met644, and Val650. The 38-residue peptide, Neutm, was synthesised having the 21 residue hydrophobic stretch (Ile660 to Ile680) calculated to span the membrane in rat Neu/erbB-2, plus residues Lys681 to Thr691 of the contiguous cytoplasmic domain. Deuterium probes replaced selected 1H nuclei at Ala661, Leu667, and Val676. A third peptide, Neutm*, was also prepared, corresponding to the transmembrane domain of a constitutively-activating Neu/erbB-2 transformant in which Val664 is replaced by Glu: it was deuterated in a manner identical to Neutm. Peptides were studied by 2H NMR spectroscopy at 1 mol% and 6 mol% in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in POPC containing 33 mol% cholesterol, over the range 12 degrees to 65 degreesC. Overall motion was found to be different for each of the three peptides under a given set of conditions. EGFRtm spectra were characteristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35 degreesC and above. In contrast, spectra of the transmembrane peptides, Neutm and Neutm*, were characteristic of significantly axially asymmetric motion under all conditions studied (and regardless of sample preparation method). Addition of 33% cholesterol to membranes was accompanied by spectral changes consistent with increased formation of peptide dimers/oligomers in all cases. The transformant peptide, Neutm*, showed greater spectral evidence of immobilisation than did the wild type - probably reflecting a greater tendency to form large oligomers. Sequence-related details within the transmembrane domains of Class I receptor tyrosine kinases appear to exert important control over their associations within membranes. Freeze-fracture electron microscopy of the NMR samples demonstrated their liposomal nature. Peptide-related intramembranous particles (IMPs) were present which likely represent oligomers of the transmembrane peptide. IMP size and distribution were similar under a given set of conditions for all three peptides, suggesting that the differences seen by NMR spectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accuracy of identifying lateral position.

The EGF receptor transmembrane domain: 2H NMR study of peptide phosphorylation effects in a bilayer environment.

Phosphorylation events are considered to be key control points in receptor tyrosine kinase function. We have used wide-line 2H NMR spectroscopy to look for physical effects of phosphorylating a threonine residue within the cytoplasmic domain of the human EGF receptor, as sensed at a distant site in the transmembrane portion. Modifications were made to Thr654 (a cytoplasmic residue suggested to be involved in regulation of EGF binding and of cytoplasmic domain function), and effects were sought at Ala623 (near the extracellular membrane surface but within the membrane-spanning region). The study was carried out on synthetic peptides corresponding to the EGF receptor transmembrane domain plus 10 or 11 residues of the cytoplasmic domain, assembled into lipid bilayer membranes. Three peptides were compared that differed only at Thr654. This residue was alternately: nonphosphorylated but left as a (-)-charged C-terminus (-Thr654COO-), nonphosphorylated and with a neighboring amidated glycine residue as the C-terminus (-Thr654GlyCONH2), or phosphorylated and with a neighboring amidated glycine residue as the C-terminus (-Thr654PO4-GlyCONH2). Bilayer membranes were composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or 2:1 POPC/cholesterol, containing 6 mol % peptide relative to phospholipid. The deuterated site, Ala623, was intrinsically conformationally sensitive; yet spatial orientation and motional order of the probe location were found not to be obviously influenced by phosphorylation.

Oligomerization of the EGF receptor transmembrane domain: a 2H NMR study in lipid bilayers.

During the course of a previous study by wideline 2H NMR, we noted spectral features suggesting the possibility of monitoring homodimer/oligomer interactions between transmembrane domains of the EGF receptor in lipid bilayers [Rigby, A. R., Shaw, G. S., Barber, K. R., & Grant, C. W. M. (1996) Biochemistry 35, 12591-12601]. In the present work this possibility was explored using the 34-residue peptide EGFRtm. The peptide sequence included the 23 amino acid hydrophobic stretch thought to span the membrane (Ile622-Met644 of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg645-Thr654). Selective deuteration was carried out at sites corresponding to Ala623, Met644, and Val650. Samples were studied from 12 to 65 degrees C by 2H NMR in fluid membranes having low peptide concentration (1 mol %) or high peptide concentration (6 mol %). Methyl groups proved to be technically particularly attractive probe locations. Reversible homodimer/oligomer interactions were detected in membranes of the common natural phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), without cholesterol. Effects on the EGF receptor transmembrane domain included alterations in peptide backbone motional order and/or conformation at the site of Ala623 within the membrane, and alterations in motional properties of the Val650 side chain in the cytoplasmic domain. There was little spectral evidence of stable oligomer formation except at the lowest temperature studied. Addition of 33% cholesterol to these membranes was accompanied by spectral changes consistent with the formation of more stable peptide oligomers, and by evidence that peptide-peptide interactions were sensed at all three probe locations. Peptide-peptide interactions remained easily reversible, particularly at higher temperatures. Freeze-fracture electron microscopy of the NMR samples demonstrated peptide-related intramembranous particles traversing the membranes. To our knowledge, this is the first electron microscopy description of receptor tyrosine kinases or their fragments in model membranes. In the presence of cholesterol, the peptide-related particles were generally larger, more sharply demarcated, and showed a tendency to cluster. These observations relate to models of receptor lateral association as an aspect of signal transduction, and to forces that may determine protein sorting and organization in cell membranes. We suggest that the cholesterol effects reflect a general phenomenon rather than one specific to the EGF receptor.

Globoside as a membrane receptor: a consideration of oligosaccharide communication with the hydrophobic domain.

Recognition of macromolecules by glycosphingolipids is closely correlated with the nature of the glycolipid carbohydrate; however, it is also thought to be secondarily modulated by the structure of the single fatty acid. In the present work, we sought insight into what physical effect a change in this fatty acid has on the extramembranous portion of globosides at liposomal surfaces mimicking systems for which modulated receptor function has been recorded in the past. Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of globotriaosylceramide (Gb3) were replaced with deuterium. In this location, the nonperturbing probe nuclei sampled cumulative conformational and orientational characteristics of the oligosaccharide chain at a sugar residue that is critical in specific binding of verotoxins. Deuterated Gb3 having 18:1 fatty acid was compared to the same species having 22:1 fatty acid, at 6.3 mol % in unsonicated bilayers of dipalmitoylphosphatidylcholine/cholesterol. Both produced narrow, apparently axially asymmetric 2H NMR spectra over a wide temperature range. Motional properties of the terminal sugar were measurably influenced by the fluidity of the host matrix; however, evidence was not found for conformational or orientational variation in this sugar brought about by the fatty acid alteration. In related experiments, acetate protons on the terminal N-acetyl galactosamine (GalNAc) residue of globotetraosylceramide (Gb4) were substituted with deuterium, and the natural fatty acid was replaced with 18:0 or 24:0 species deuterated at C2. Once again, species with short vs long fatty acid were examined for evidence of headgroup differences. Spectra of Gb4 were compared at 10 mol % in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and at 5 mol % in membranes containing 33 mol% cholesterol. Spectral splittings reflecting cumulative effects on conformation and order at the terminal deuterated sugar remained unchanged between species having 18:0 vs 24:0 fatty acid in POPC/cholesterol. In a pure POPC host matrix, there was clear evidence of a motional difference between the two--the longer chain Gb4 demonstrating spectral asymmetry--but the spectral width was unchanged. Transverse relaxation times, T2, were measured. Our findings appear to help correlate the conclusions of a number of workers dealing with the molecular basis of crypticity. We suggest that changes in glycolipid receptor function based on ceramide fatty acid variation have a major origin in the fatty acid's ability to determine the thermodynamics of interaction with the host matrix, as reflected in such parameters as glycolipid motional properties, local membrane curvature, and likely glycolipid time-dependent lateral associations. The result at low concentrations of glycolipid may often be only a subtly altered collective surface epitope, best detected by a specific recognition event.

Identification and structural influence of a differentially modified N-terminal methionine in human S100b.

The calcium-binding protein S100b is a homodimer comprised of two identical 91-residue beta-subunits. Recombinant S100b is a heterogeneous protein, although the basis of this heterogeneity has not been established. We have used mass spectrometry and NMR spectroscopy to determine that heterogeneity in S100b arises from a mixture of formyl-S100b and desformyl-S100b when expressed in Escherichia coli. Reversed-phase HPLC purification of these two forms of S100b has allowed the differences in N-terminal composition to be used as a probe for tertiary contacts in the protein. The presence or absence of the N-terminal formyl group affected the chemical shifts of sequence neighboring residues and those in the linker of the protein (residues 40-43), indicating that these two regions are close in space.

Relationship between social support, self-esteem and codependency in the African American female.

Increasing numbers of minorities are seeking mental health assistance. Inclusion of cultural considerations is important for increasing sensitivity to those whose life experiences differ. For African Americans, therapy is better facilitated if one operates from a cultural specific frame of reference. African American women attending a women's support group were surveyed. Questionnaires querying dimensions on family relationships, self-esteem and dependency were utilized. Results indicated a relationship between social support, self-esteem and codependency in African American females. Social support and self-esteem were inversely associated with codependency. This study provides insight for mental health professionals in counseling African American females.

Pilin C-terminal peptide binds asialo-GM1 in liposomes: a 2H-NMR study.

Wideline 2H-NMR observations are described demonstrating the interaction of a synthetic peptide (PAK), representing residues 128-144 of the binding domain of pilin surface protein from Pseudomonas aeruginosa, with a complex glycosphingolipid thought to be its natural receptor. The receptor glycolipid (asialo-GM1) carried 2H probe nuclei on the terminal and next-to-terminal carbohydrate residues and was present as a minor component in fluid phosphatidylcholine liposomes. The peptide induced spectral changes that could be understood as arising from receptor motional changes, without receptor immobilization on the NMR time scale of 10(4) s-1. Spectral effects were reversed by reduction of the single peptide disulfide bond--a structural feature previously shown to be a determinant of PAK conformation (Campbell AP, McInnes C, Hodges RS, Sykes BD. 1995. Biochemistry 34:16255-16268). This is the first demonstration of PAK interaction with its epithelial cell receptor in liposomes.

Sphingolipid-derived signalling modulators: interaction with phosphatidylserine.

We previously described the synthesis of two deuterium-labelled sphingoid bases, which made it possible to perform NMR spectroscopy on this family of signalling modulators in membranes (Rigby, A.C, Barber, K.R and Grant, C.W.M. (1995) Biochim. Biophys. Acta 1240, 75-82). In the present work we sought to test the concept that such mediators may display altered physical behaviour through association with anionic lipids - as a possible mechanism of involvement in signal transduction. Lyso-dihydrogalactosylceramide with deuterium nuclei at C4 and C5 of the sphingosine backbone and at C'3 and C'4 of the galactose ring ([2H4]lyso-GalCer), and N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), were assembled as minor components into unsonicated fluid bilayer membranes of 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol. The effect of (anionic) phosphatidylserine was considered in this zwitterionic host matrix. The results present a picture of rapidly reversible interaction. The (-) charged phosphatidylserine exerted readily-measurable control over the orientation of the carbohydrate residue of [2H4]lyso-GalCer. In contrast, surrounding (-) charges exerted little spectral influence at the level of C4 and C5 of the lyso-GalCer, membrane-inserted, backbone; or at the level of the amino group of dimethylsphingosine. It would appear that packing alterations induced by the phosphatidylserine/sphingoid base association can translate into sizeable spatial constraints in the neighbouring aqueous domain.