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Ventriculoperitoneal shunts (VPS) and gastrostomies are frequently provided in daily practice. This study investigated the incidence of VPS infection and the survival rate among adult patients who underwent gastrostomy at least 1 month after VPS placement. This single-center retrospective cohort study was conducted among patients with a VPS, who underwent a gastrostomy. This procedure was performed on a standby basis after a period of at least 1 month had elapsed since VPS placement. Subsequent VPS infection and survival rates were assessed over a period of at least 6 months. We reviewed 31 patients who had a VPS at the time of gastrostomy. Gastrostomy was performed endoscopically in 29 cases and via open surgery in 2 cases. The average interval between VPS insertion and gastrostomy was 1135.5 ± 1717.1 days. A single case of VPS infection (3.2%) was diagnosed during the study. This infection rate was not significantly different than that among 230 patients who underwent their first VPS placement (without gastrostomy) at our institution during the same time period (P = .57); there was also no significant difference in the survival rate, compared to 38 age-matched patients (with cerebrovascular disease, but without a VPS) who underwent gastrostomy (P = .73). Gastrostomy performed after an interval of at least 1 month after VPS placement was extremely safe in adult patients, and their prognosis was excellent. Additional studies are required to develop appropriate nutritional interventions for patients with a VPS.

Introduction: In 2010, a large-scale multi-institutional study in Japan showed a good prognosis for percutaneous endoscopic gastrostomy (PEG). However, the function and efficacy of PEG are not fully understood by patients, families, and health-care professionals; thus, the number of PEG treatments in Japan has declined. Therefore, we aimed to investigate the safety of the PEG procedure and subsequent survival after PEG. Methods: In total, 249 PEGs were performed at Juzenkai Hospital from 2005 to 2017. PEG was originally performed using the pull method and then by a modified introducer method from mid-2011. We examined procedure-related complications and survival rates after PEG. Results: Fifty-one (20.5%) procedure-related complications occurred; emergency surgery was required in 4 cases. Infections accounted for 76.5% (39/51) of complications. More infections occurred with the pull method than with the modified introducer method. The 1-year survival rate was 66.8%; the median survival time was 678 days. Nine patients (3.6%) died within 30 days; no deaths were directly related to PEG. Sex, age, and albumin level before surgery significantly influenced the prognosis. Conclusion: Due to changes in the PEG insertion method and other factors, PEG has become a safer treatment method. Additionally, PEG-based nutritional supplementation is associated with adequate survival.

Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto MMC resin was developed as a capture step. Binding of murine mAb occurred at neutral pH. The bound mAb was eluted with a gradient from 0.3 M NaCl to 0.3 M arginine/0.3 M NaCl at pH 7.0. The Capto MMC-purified murine mAb was further purified by hydroxyl apatite chromatography. Similarly, rabbit mAb was processed with some modifications. Binding of rabbit mAb to Capto MMC required a lower pH. Elution of the bound rabbit mAb was achieved by a gradient to 0.3 M NaCl, pH 7.0.

Arginine is widely used in such applications as protein refolding, solubilization of proteins and small molecules, protein and small molecule formulation, column chromatography and viral inactivation as summarized in this review. What makes arginine effective in these applications is largely based on its ability to suppress protein-protein interactions and protein-surface interactions. The mechanism of these widespread effects of arginine on proteins can be explained at least in part from its unique interactions with the protein surface. Here we describe the modes of the interactions of arginine with model compounds and proteins and also water molecules, and then attempt to explain the mechanism of its effect on proteins by comparing with the interactions that occur between protein and protein denaturants or stabilizers.

Humanin (HN), a short amino acid peptide, protects neurons as well as other cells from amyloid β-induced toxicities and other stresses. A number of HN binding proteins have been identified and their involvements in HN-mediated neuroprotection have been suggested in some cases. However, the way HN binds to the target molecules has never been clarified. Here we will review the structures of HN and HN analogs in solution as a function of solvent conditions and attempt to relate their structural characteristics to the functional properties.

A variety of excipients are used to stabilize proteins, suppress protein aggregation, reduce surface adsorption, or to simply provide physiological osmolality. The stabilizers encompass a wide variety of molecules including sugars, salts, polymers, surfactants, and amino acids, in particular arginine. The effects of these excipients on protein stability in solution are mainly caused by their interaction with the protein and the container surface, and most importantly with water. Some excipients stabilize proteins in solution by direct binding, while others use a number of fundamentally different mechanisms that involve indirect interactions. In the dry state, any effects that the excipients confer to proteins through their interactions with water are irrelevant, as water is no longer present. Rather, the excipients stabilize proteins through direct binding and their effects on the physical properties of the dried powder. This review will describe a number of mechanisms by which the excipients interact with proteins in solution and with various interfaces, and their effects on the physical properties of the dried protein structure, and explain how the various interaction forces are related to their observed effects on protein stability.

Alzheimer's disease (AD) is a complex disease, involving multiple factors such as the production of aggregation-prone amyloid beta (Abeta) peptides, the formation of fibrillarly tangles of microtubule-associating proteins, Tau, and the polymorphism of cholesterol binding protein, APOE4. While understanding the mechanism of AD and the involvement of key players should lead to rational drug discovery against this disease, a traditional screening approach should also work for identifying drugs using AD models. We have used a cellular AD model, in which a cell death was induced by AD-causing neurotoxicities, and then screened the genes, which rescued the cells from the cell death. This resulted in isolation of a gene encoding a novel 24-amino acid long peptide, termed Humanin (HN), which protected neuronal cells at approximately microM level. Surprisingly, these gene products and the synthetic peptides not only protected neurons from cell death induced by Abeta-related neurotoxicities, but also Abeta-unrelated neurotoxicities. While a broad range of activities of HN against AD-related insults is discovered, the detailed mechanism of its action is still obscure. Structure analysis of HN showed that it is largely disordered and flexible at low peptide concentrations and heavily aggregates at high concentrations. Interestingly, one of the HN analogs, which is 10000-times more active than the parent HN molecule (i.e. active below nM range), was found to be monomeric. Based on findings of structural analyses, we propose here that membrane environment may enable HN to achieve high affinity for target protein(s) with multiple-transmembrane domains, such as G-protein coupled receptors.

We have examined the structure of two Humanin (HN) analog peptides, HNG and AGA-(C8R)HNG17, in the presence of sodium dodecylsulfate (SDS) and trifluoroethanol (TFE) using CD and sedimentation velocity. Both HNG and AGA-(C8R)HNG17 underwent complex conformational changes with increasing concentrations of SDS and TFE, in contrast to general trend of increasing alpha-helix with their concentration. To our surprise, both peptides appear to converge into a similar structure in SDS and TFE at higher concentrations; e.g., above 0.05 % SDS or 30-40 % TFE. Sedimentation velocity analysis showed extensive aggregation of HNG at 0.1 mg/ml in PBS in the absence of SDS, but a highly homogeneous solution in 0.1 % SDS, indicating formation of a uniform structure by SDS. These two peptides also formed an intermediate structure both in SDS and TFE at lower concentrations, which appeared to be associated with extensive aggregation. It is interesting that the structure changes of these peptides occur well below the critical micelle concentration of SDS, suggesting that conformational changes are mediated through molecular, not micellar, interactions with SDS.

The structure of a highly potent Ser14Gly analog of antiAlzheimer peptide, Humanin, was examined by circular dichroism (CD). The secondary structure is more disordered in water than in phosphate-buffered saline (PBS). The peptide structure in water is little dependent on both peptide concentration and temperature. On the contrary, the peptide structure was significantly different in PBS from the structure in water, which is more apparent at a higher peptide concentration and temperature. The observed different structure in PBS appears to be due to self-association of the peptide, which is enhanced by elevated temperature and, hence, via hydrophobic interactions. The wild-type Humanin also behaved similarly, i.e., it assumed a disordered structure in water but underwent conformational changes in PBS. Although high peptide concentrations for CD measurements are not encountered in vivo, the results suggest the tendency of the peptide to interact hydrophobically with other structures as well as with itself. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.

Human brain serine racemase (hSR) was expressed in large amounts in E. coli with N-terminal His-tag (His-hSR). His-hSR expressed in inclusion body was solubilized and purified to homogeneity by Ni-NTA affinity column. Purified His-hSR was refolded in Tween 20/cycloamylose with approximately 50% efficiency, and refolded His-hSR was isolated by Q Sepharose column chromatography. The refolding conditions are described in detail. His-hSR catalyzed the elimination of L-Ser as well as L-Ser-O-sulfate to form pyruvate.

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease. It has been generally accepted that the proapoptotic property of the familial ALS (FALS)-linked mutant SOD1 genes plays an important role in the pathogenesis of some FALS cases. We found here that expression of N19S-SOD1, a novel SOD1 mutant originally found in a sporadic ALS patient, induces lower grade death in NSC34 cells than FALS-linked mutant SOD1. In agreement, intracytoplasmic aggregate formation and SOD1 polymerization are less prominently induced by ectopic expression of N19S-SOD1 than FALS-linked mutant SOD1. We further found that additional cell stresses, such as inhibition of proteasomal activity or up-regulation of intracellular oxidative stress, enhance N19S-SOD1-induced aggregate formation and polymerization of N19S-SOD1. Such analysis of the intracellular polymerization and the ubiquitination of N19S-SOD1 have further suggested that it is recognized as a misfolded protein, like FALS-linked mutant SOD1, whereas wild-type SOD1 is not. Altogether, it is speculated that the N19S mutation of SOD1 in cooperation with associated cell stresses contributes to the onset of ALS as a risk factor.

A novel factor, termed Humanin (HN), antagonizes against neurotoxicity by various types of familial Alzheimer's disease (AD) genes [V642I and K595N/M596L (NL) mutants of amyloid precursor protein (APP), M146L-presenilin (PS) 1, and N141I-PS2] and by Abeta1-43 with clear action specificity ineffective on neurotoxicity by polyglutamine repeat Q79 or superoxide dismutase 1 mutants. Here we report that HN can also inhibit neurotoxicity by other AD-relevant insults: other familial AD genes (A617G-APP, L648P-APP, A246E-PS1, L286V-PS1, C410Y-PS1, and H163R-PS1), APP stimulation by anti-APP antibody, and other Abeta peptides (Abeta1-42 and Abeta25-35). The action specificity was further indicated by the finding that HN could not suppress neurotoxicity by glutamate or prion fragment. Against the AD-relevant insults, essential roles of Cys(8) and Ser(14) were commonly indicated, and the domain from Pro(3) to Pro(19) was responsible for the rescue action of HN, in which seven residues turned out to be essential. We also compared the neuroprotective action of S14G HN (HNG) with that of activity-dependent neurotrophic factor, IGF-I, or basic FGF for the antagonism against various AD-relevant insults (V642I-APP, NL-APP, M146L-PS1, N141I-PS2, and Abeta1-43). Although all of these factors could abolish neurotoxicity by Abeta1-43, only HNG could abolish cytotoxicities by all of them. HN and HN derivative peptides may provide a new insight into the study of AD pathophysiology and allow new avenues for the development of therapeutic interventions for various forms of AD.

It has been found that insulin-like growth factor I (IGF-I) exerts cytoprotection against Abeta amyloid-induced neuronal cell death. Deposits of Abeta amyloid are one of the pathological hallmarks of Alzheimer's disease (AD). Here, we examined whether IGF-I exerts protective activity against cell death induced by a familial AD (FAD)-linked mutant of amyloid precursor protein (APP), and we found that IGF-I protected cells from toxicity of FAD-associated V642I mutant of APP in multiple cell systems. IGFBP-3 blocked this action of IGF-I, but not of des(1-3)IGF-I, which was as active as IGF-I in the presence of IGFBP-3. The data also demonstrated that the IGF-I receptor (IGF-IR) mediates the protective activity of IGF-I. The antagonizing function of the IGF-I/IGF-IR system against V642I-APP, which is further antagonized by IGFBP-3, provides a molecular clue to the understanding of AD pathophysiology and to the establishment of potential therapy for AD.

Since an apolipoprotein E4 (ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [ApoE4(141–149) 2 or ApoE(141–155) 2 ] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial whether these effects are mediated by LRP. Here, we examined whether ApoE4(141–149) 2 -induced toxicity is mediated by LRP in a neuronal cell system where ApoE4 toxicity is mediated by LRP: serum-deprived F11 neuronal cells. In these cells, where ApoE4 exerted toxicity by apoptosis in a manner sensitive to both caspase inhibitors and pertussis toxin (PTX), ApoE4(141–149) 2 also caused cell death by apoptosis but in a caspase-inhibitor-resistant, PTX-resistant manner. ApoE4(141–149) 2 -induced death was not inhibited by antisense oligonucleotides to LRP. Therefore, we conclude that ApoE4(141–149) 2 is able to exert neurotoxicity without involving LRP.

A single mutation has resulted in large differences in neuroprotective activity of a 24 amino acid Humanin (HN). A mutation of Ser7Ala (S7A-HN) resulted in loss of activity, while a mutation of Ser14Gly (S14G-HN) resulted in about 1000-fold increase. The mechanism of the effects conferred by these mutations have been totally unclear, although our recent structure analysis suggested a possibility of the effect of mutation on the structure stability. Here, we have studied the effects of buffer and temperature on the structure of these three HN peptides. These peptides showed a similar disordered structure at 10 °C in 10 mM phosphate, pH 6.0. They were also similar in phosphate-buffered saline (PBS) as long as the temperature was kept low at 10 °C. However, a large difference was observed in both phosphate buffer and PBS between the peptides, when the temperature was raised to a physiological temperature of 37 °C. While S14G-HN showed small changes in both solutions at 37 °C, the less active HN and inactive S7A-HN showed much larger changes under the identical conditions. In addition, it appeared that structure change at 37 °C was faster for S7A-HN than HN. These results show that the structure stability at 37 °C increases in the order of S7A-HN, HN and S14G-HN, in correlation with their neuroprotective activities.

A 24-amino acid long peptide, Humanin, protects neurons from Alzheimer's disease (AD)-related cell toxicities at sub-nM-uM concentrations. Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and AD-related and amyotrophic lateral sclerosis-related insults at fM concentrations. An attempt was made to further improve the activity of Humanin by fusing this peptide to ADNF9, a 9-amino acid long core peptide of the ADNF. This fusion resulted in a novel molecule, termed Colivelin, with the neuroprotective activity at fM range, which is approximately 10(3)-10(7) fold higher than the activity of Humanin and Humanin analogs and follows the activity profile of fM-active ADNF9. We have characterized the structural properties of Colivelin and compared with those of ADNF9 and Humanin in water and phosphate-buffered saline (PBS). The secondary structure of Colivelin was similar to that of ADNF9, but not that of Humanin, and hence was not the average of the contributions of the two peptides fused. Colivelin was stable and monomeric in PBS, consistent with the monomeric property of ADNF9, while Humanin showed strong tendency to self-associate. Thus, it is evident that the structural properties of Colivelin resemble those of ADNF9, rather than those of Humanin.

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.

Humanin (HN) is a 24-amino acid peptide that protects neuronal cells from death caused by Alzheimer's disease (AD)-related genes and amyloid-β (Aβ). Multiple studies have revealed its biochemical and neuroprotective characteristics in vitro; however, little has been known regarding whether HN is effective in vivo in AD model systems. We examined the effect of S14G-HN, a 1,000-fold more potent derivative of HN in vitro, on amnesia induced by Aβ25–35 in mice. The Y-maze test revealed that at least 50 pmol of S14G-HN by intracerebroventricular injection prevented Aβ-induced impairment of short-term/spatial working memory; however, 5 nmol of S14A-HN, a neuroprotection-defective mutant in vitro, did not prevent Aβ-induced amnesia. These results are in agreement with the structure–function correlation shown previously in vitro. In the water-finding task, S14G-HN prevented prolongation of finding latency (the time to find water) observed in Aβ-amnesic mice, indicating that S14G-HN also blocked Aβ-induced impairment of latent learning. In accordance with these observations, immunohistochemical analysis showed that S14G-HN sustained the number of cholinergic neurons in the basal forebrain and the striata nearly to the normal level. Furthermore, genistein, a specific inhibitor of tyrosine kinases, blocked recovery from scopolamine-induced amnesia by S14G-HN, suggesting that certain tyrosine kinase(s) are involved in the inhibitory function of S14G-HN in vivo. Taking these findings together, we conclude that S14G-HN has rescue activity against memory impairment caused by AD-related insults in vivo by activating the same intracellular neuroprotective machinery as elucidated previously in vitro. © 2005 Wiley-Liss, Inc.

AlsinLF, the product of the ALS2 gene, inhibits Cu/Zn-superoxide dismutase (SOD1) mutant-induced neurotoxicity via its Rho guanine nucleotide-exchanging factor domain. We here identified Rac1, a Rho family small GTPase, as a target for the Rho guanine nucleotide-exchanging factor activity of alsinLF. Rac1 associates with alsinLF. The amount of the GTP form of Rac1 is up-regulated by enforced overexpression of alsinLF. We further found not only that constitutively active Rac1 suppresses motoneuronal cell death induced by SOD1 mutants but also that the neuroprotective activity of alsinLF was completely inhibited by knocking down the endogenous Rac1 expression with small interfering RNA for Rac1, indicating that Rac1 is the major effector for alsinLF-mediated neuroprotection. Such alsinLF/Rac1-mediated neuroprotection occurs specifically against the SOD1 mutant-induced cell death but not against the cell death induced by any other neurotoxic insults in motoneuronal NSC34 cells. We further demonstrated that the alsinLF/Rac1-mediated neuroprotective signal is transmitted to the phosphatidylinositol 3-kinase/Akt anti-apoptotic axis. Among three Akt family proteins, Akt3 is the major downstream mediator for alsinLF/Rac1-mediated neuroprotection, which is specifically effective against SOD1 mutant-induced neurotoxicity.

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease, affecting mostly middle-aged people. There are no curative therapies for ALS. Several lines of evidence have supported the notion that the proapoptotic property of familial ALS (FALS)-linked mutant Cu/Zn-superoxide dismutase-1 (SOD1) genes may play an important role in the pathogenesis of some FALS cases. Here we found that activity-dependent neurotrophic factor (ADNF), a neurotrophic factor originally identified to have the anti-Alzheimer's disease (AD) activity, protected against neuronal cell death caused by FALS-linked A4T-, G85R- and G93R-SOD1 in a dose-responsive fashion. Notably, ADNF-mediated complete suppression of SOD1 mutant-induced neuronal cell death occurs at concentrations as low as 100 fM. ADNF maintains the neuroprotective activity even at concentrations of more than 1 nM. This is in clear contrast to the previous finding that ADNF loses its protective activity against neurotoxicity induced by AD-relevant insults, including some familial AD genes and amyloid beta peptide at concentrations of more than 1 nM. Characterization of the neuroprotective activity of ADNF against cell death caused by SOD1 mutants revealed that CaMKIV and certain tyrosine kinases are involved in ADNF-mediated neuroprotection. Moreover, in vivo studies showed that intracerebroventricularly administered ADNF significantly improved motor performance of G93A-SOD1 transgenic mice, a widely used model of FALS, although survival was extended only marginally. Thus, the neuroprotective activity of ADNF provides a novel insight into the development of curative drugs for ALS.

An unbiased functional screening with brain cDNA library from an Alzheimer's disease (AD) brain identified a novel 24-residue peptide Humanin (HN), which suppresses AD-related neurotoxicity. As the 1567-base cDNA containing the open reading frame (ORF) of HN is 99% identical to mitochondrial 16S ribosomal RNA as well as registered human mRNA, it was elusive whether HN is produced in vivo. Here, we raised anti-HN antibody and found that long cDNAs containing the ORF of HN (HN-ORF) produced the HN peptide in mammalian cells, dependent on the presence of full-length HN-ORF. Immunoblot analysis detected a 3-kDa protein with HN immunoreactivity in the testis and the colon in 3-week-old mice and in the testis in 12-week-old mice. HN immunoreactivity was also detected in an AD brain, but little in normal brains. This study suggests that HN peptide could be produced in vivo, and would provide a novel insight into the pathophysiology of AD.

Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and aggregate to form intracellular inclusions. We report that both small ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant proteins at lysine 75 in a motoneuronal cell line, the cell type affected in ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9 of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather than by SUMO1, significantly increased the stability of the proteins and accelerated intracellular aggregate formation. These findings suggest the contribution of sumoylation, particularly by SUMO3, to the protein aggregation process underlying the pathogenesis of ALS.

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimer's disease genes and by Aβ amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimer's disease targeting neuroprotection.

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193–9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905–4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473–475; (1993) J. Biol. Chem. 268, 18407–18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3-transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125 I-labeled NDF and then chemically crosslinked. Immunoprecipitation with anti-Her3 revealed a dense high M W band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero-oligomerization.

The partial molar volumes of 1-heptanol and 1-octanol in red cell ghosts, in egg phosphatidylcholine bilayers, and in water and phosphate buffer have been measured to a precision of better than 4% by using a density meter. In every case, the partial molar volume was independent of concentration in the range studied. In both membranes, the partial molar volume of each alcohol was close to its molar volume whereas in aqueous solution it was considerably less. Comparison of the two membranes suggests that the major contribution to the partial molar volume arises from alcohol-lipid interactions of each case. Further comparison with partial molar volumes in bulk solvents suggests that on average the alcohols retain a hydrogen bond in the lipid bilayer. The magnitude of the volume change in ghosts is some 5 times less than the corresponding area changes previously reported by others [Roth, S. H., & Seeman, P. (1972) Biochim. Biophys. Acta 255, 190-198]. These two observations can only be resolved by assuming either that the bilayer expands anisotropically, experiencing a decrease in thickness with increasing volume, or that conformational changes in membrane-associated proteins can occur at constant volume to increase membrane area. Finally, these data are used to test the critical volume hypothesis of general anesthetic action. A volume change of 0.15% in red cell ghost membranes is found to be associated with anesthesia, which compares with a value of 0.2% predicted previously from pressure reversal of anesthesia studies. In egg phosphatidylcholine bilayers, a volume change of 0.36% is associated with anesthesia. The larger change in the lipid bilayer compared to the biomembrane originates solely in their different membrane/buffer partition coefficients.