Synthesis of polyethylene glycol (PEG) derivatives and PEGylated-peptide biopolymer conjugates

We synthesized a library of 50 poly(ethylene glycol) (PEG) derivatives to expand the extent of conjugation with biologically active molecules (biopolymers, peptides, drugs, etc.) and biomaterial substrates. The formation of PEG derivatives was confirmed with HPLC, (1)H and (13)C NMR. PEG derivatives were polymerized into networks in order to study the role of PEG and terminal functional groups in modulating the hydrophilicity of biomaterials and cell-biomaterial interaction. The resulting surface hydrophilicity and the number of adhered fibroblasts were primarily dependent on the PEG concentration with the molecular weight and the terminal functional group of PEG derivatives being less important. One of PEG derivatives, PEG-bis-glutarate, was utilized to link peptide sequences to gelatin backbone in the formation of novel biomedical hydrogels. PEG-peptide conjugates were characterized by mass spectroscopy. PEG-peptide modified gelatins were characterized by gel permeation chromatography.     

Interaction of poly(ethylene-glycols) with air-water interfaces and lipid monolayers: investigations on surface pressure and surface potential.

We have characterized the surface activity of different-sized poly(ethylene-glycols) (PEG; M(r) 200-100,000 Da) in the presence or absence of lipid monolayers and over a wide range of bulk PEG concentrations (10(-8)-10% w/v). Measurements of the surface potential and surface pressure demonstrate that PEGs interact with the air-water and lipid-water interfaces. Without lipid, PEG added either to the subphase or to the air-water interface forms relatively stable monolayers. Except for very low molecular weight polymers (PEGs < 1000 Da), low concentrations of PEG in the subphase (between 10(-5) and 10(-4)% w/v) increase the surface potential from zero (with respect to the potential of a pure air-water interface) to a plateau value of approximately 440 mV. At much higher polymer concentrations, > 10(-1)% (w/v), depending on the molecular weight of the PEG and corresponding to the concentration at which the polymers in solution are likely to overlap, the surface potential decreases. High concentrations of PEG in the subphase cause a similar decrease in the surface potential of densely packed lipid monolayers spread from either diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (DPPC), or dioleoyl phosphatidylserine (DOPS). Adding PEG as a monolayer at the air-water interface also affects the surface activity of DPhPC or DPPC monolayers. At low lipid concentration, the surface pressure and potential are determined by the polymer. For intermediate lipid concentrations, the surface pressure-area and surface potential-area isotherms show that the effects due to lipid and PEG are not always additive and that the polymer's effect is distinct for the two lipids. When PEG-lipid-mixed monolayers are compressed to surface pressures greater than the collapse pressure for a PEG monolayer, the surface pressure-area and surface potential-area isotherms approach that of the lipid alone, suggesting that for this experimental condition PEG is expelled from the interface.     

The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.     

Insertion and folding of the amino-terminal amphiphilic signal sequences of the mannitol and glucitol permeases of Escherichia coli.

Peptides which correspond to the NH2-terminal 23 or 22 residues of the mannitol and glucitol permeases (enzymes IImtl and IIgut of the bacterial phosphotransferase system; mtl-23 and gut-22) and which are believed to function in envelope targeting were synthesized chemically, and their interactions with lipid model membranes were studied. Both wild-type peptides penetrated phospholipid monolayers up to high surface pressures, and partition constants of 8.0 x 10(4) M-1 and 4.2 x 10(4) M-1, respectively, were derived from the incorporation isotherms of mtl-23 and gut-22 with monolayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine at 32 mN/m or bilayers of the same lipid. The mtl-23 peptide was highly alpha-helical in trifluoroethanol, sodium dodecyl sulfate, lysolecithin, or vesicles of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol, with estimated percentages of alpha-helix ranging between 60 and 85%. The interactions with model membranes of several single site mutants (S3P, D4P, and D4K) of mtl-23 which were defective in properly assembling the mannitol permease in the cytoplasmic membrane of Escherichia coli were also studied. The contents of alpha-helix of these peptides in detergent micelles or phospholipid bilayers were not significantly changed compared with those of the wild type, suggesting that the amphiphilic NH2-terminal membrane-targeting domain could still be formed in these mutants. However, the mutants which contained a proline in positions 3 or 4, i.e. NH2-terminal to the proposed amphiphilic alpha-helix, partitioned into phospholipid monolayers with partition constants that were 2 or 4 times smaller than those of the wild type. Based on these data, a model of the amphiphilic structure of the NH2-terminal domain of the mannitol permease is discussed. This domain may interact physiologically with amphiphilic interfaces of lipids and/or proteins during membrane insertion.     

Fabrication of Langmuir-Blodgett film of a fullerene derivative with a cyclic peptide as an anchor

A novel cyclic octapeptide carrying a fullerene unit and poly(ethylene glycol) at the side chain (cyclo8-C 60 + PEG) was synthesized, and its monolayer formation at the air/water interface and on a substrate was studied. Surface pressure-area per molecule isotherms indicated that cyclo8-C 60 + PEG formed a stable monolayer at the air/water interface. The cyclo8-C 60 + PEG monolayers prepared from various spreading volumes (i.e., from various initial areas per molecule) overlapped nicely on a single curve, suggesting that the molecules were uniformly dispersed on the surface without aggregation of the fullerene units. The uniform dispersibility is due to the scaffold effect of the cyclic peptide unit to keep the fullerene units away from each other. The formed monolayer could be quantitatively transferred onto a solid substrate. UV-vis absorption spectroscopy of the Langmuir-Blodgett (LB) monolayer showed that the electronic structure of the fullerene unit was not affected by the formation of the monolayer. Cyclic voltammetry of the LB monolayer in an aqueous solution containing redox species indicated that the LB monolayer was densely packed. Furthermore, reversible redox peaks attributed to the one-electron reduction of the fullerene unit were observed, showing that the redox property of the fullerene unit was also retained in the monolayer. It is thus concluded that the cyclic peptide is a good candidate as a scaffold for stable monolayer formation at the air/water interface and for intact immobilization of the fullerene moiety onto a substrate.     

Anomalous settlement behavior of Ulva linza zoospores on cationic oligopeptide surfaces

Identification of settlement cues for marine fouling organisms opens up new strategies and methods for biofouling prevention, and enables the development of more effective antifouling materials. To this end, the settlement behaviour of zoospores of the green alga Ulva linza onto cationic oligopeptide self-assembled monolayers (SAMs) has been investigated. The spores interact strongly with lysine- and arginine-rich SAMs, and their settlement appears to be stimulated by these surfaces. Of particular interest is an arginine-rich oligopeptide, which is effective in attracting spores to the surface, but in a way which leaves a large fraction of the settled spores attached to the surface in an anomalous fashion. These 'pseudo-settled' spores are relatively easily detached from the surface and do not undergo the full range of cellular responses associated with normal commitment to settlement. This is a hitherto undocumented mode of settlement, and surface dilution of the arginine-rich peptide with a neutral triglycine peptide demonstrates that both normal and anomalous settlement is proportional to the surface density of the arginine-rich peptide. The settlement experiments are complemented with physical studies of the oligopeptide SAMs, before and after extended immersion in artificial seawater, using infrared spectroscopy, null ellipsometry and contact angle measurements.     

Formation of stable polypeptide monolayers at interfaces: controlling molecular conformation and orientation.

The molecular self-organization and structural properties of peptide assemblies at different interfaces, using either amphipathic or hydrophobic polypeptide helices, is described. The two peptides under investigation form stable monolayers on the water surface under the conservation of their molecular conformation, as studied by circular dichroism and polarization-modulation Fourier transform infrared (FTIR) spectroscopy. Using surface plasmon resonance and reflection-absorption FTIR, we show that such molecular layers can be transferred unaltered to solid substrates. Most importantly, the molecular orientation of the hydrophobic helices on solid supports such as gold can be controlled by choosing a particular procedure for the layer formation. The helices were oriented parallel to the interface in Langmuir-Blodgett monolayers, and perpendicular to the interface in self-assembled monolayers. Our reflection-absorption FTIR measurements have delivered for the first time direct experimental evidence for the molecular conformation and orientation of pure peptide monolayers. Suitable reference spectra of polypeptides with defined conformation and orientation are necessary to use this technique for the determination of the molecular orientation of peptides in monomolecular films. We have solved the problem for alpha-helical polypeptides by using bacteriorhodopsin as a reference in combination with synthetic alpha-helices of defined interfacial orientation. The present study shows the possibility of constructing immobilized peptide monolayers with predefined macroscopic properties and molecular structure by choosing the proper polypeptide amino acid sequence, the technique used for layer formation, and the supporting surface properties.     

A neutron reflectivity study of polymer-modified phospholipid monolayers at the solid-solution interface: polyethylene glycol-lipids on silane-modified substrates.

The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously hydrophobized by chemically grafting a robust monolayer of octadecyltrichlorosilane (OTS). We show that this method results in homogeneous and continuous phospholipid monolayers on the silanated substrates and determine that the grafted PEG chains extend away from the monolayers into the solvent phase as a function of their density, as expected from scaling theories. In addition, ligands were coupled to the end of the PEG chains and selective binding was demonstrated using fluorescence microscopy. Our results demonstrate that these constructs are ideal for further characterization and studies with well-defined monomolecular films.     

Isolation and partial characterization of canine epidermal growth factor/urogastrone.

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.     

Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase

ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin-related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions.     

The solution conformation of tubulin-beta(422-434)-NH2 and its Nac-DATADEQG-NH2 fragment based on NMR

The solution conformation of tubulin-beta(422-434)-NH2 (YQQYQDATADEQG-NH2) and its Nac-DATADEQG-NH2 fragment has been studied by two-dimensional 1H-nmr spectroscopy in CD3OH/H2O (90/10 v/v) at neutral and low pH. The 13 amino acid peptide is a segment of the C-terminal region of tubulin, and is directly involved in the selective binding site with microtubule-associated proteins MAP-2 and the tau protein. Based on correlated spectroscopy, total correlation spectroscopy, and rotating frame nuclear Overhauser effect spectroscopy experiments, a complete assignment of all proton resonances was achieved, and the conformation of the backbone could be deduced from coupling constants, NH temperature coefficients, and nuclear Overhauser effects. The spectroscopic evidence indicates that the T8-Q12 section of both molecules forms one complete alpha-helical turn, stabilized by a NH (Q12)-C = O (T8) hydrogen bond. Furthermore, strong pH-dependent backfolding of the E11 side chain to its own NH proton was found. In addition, close proximity between the aromatic side chains of Y1, Y4, and the alpha-helical part, resulting in some substantial chemical shift changes when comparing the entire 13-mer with the octamer, could be explained in terms of a nonclassical kink in the DATA section. The conformational space is dominated by extended structures and the nonextended conformers are only a minor, yet spectroscopically clearly discernible entity. The presence of the alpha-helical region at the C-terminus of the 13-mer is important because binding studies of this peptide with MAP-2 indicate that the D10-E11-Q12-G13 fragment is critical for the binding interaction.     

FCS Analysis of Protein Mobility on Lipid Monolayers

In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.     

RGD重组人IL-10的克隆、表达及其拮抗纤维化的初步研究

白细胞介素10(IL-10)是一种多效细胞因子,在炎症、免疫反应以及在疾病的发生过程中发挥着重要作用,RGD是能够特异与新生血管内皮细胞整合素结合的多肽序列。将RGD连接到IL-10的羧基端,期望构建新生血管内皮细胞特异导向结合型融合蛋白。以人外周血淋巴细胞cDNA为模板,扩增的PCR产物经克隆载体pMD18-T,连至原核表达载体pET-22b(+),转化大肠杆菌BL21(DE3),构建了pET-IL10-RGD表达载体的重组菌(pET-IL10-RGD/BL21)。SDS-PAGE分析表明:在19.3 kDa处有明显的新生蛋白带,符合理论预期。Western blot分析表明:诱导表达、分离纯化的目的蛋白能够与IL-10抗体特异结合,且纯化产物IL10-RGD具有与IL-10相同的生物学活性。利用培养的人皮肤成纤维细胞,观察了IL10-RGD对TGF-β1刺激成纤维细胞的I型胶原(Col1)、III型胶原(Col3)、α-平滑肌肌动蛋白(α-SMA)、结体组织生长因子(CTGF)蛋白水平及α-SMA免疫细胞化学的变化。结果表明:纯化产物IL10-RGD能够明显抑制TGF-β1刺激的成纤维细胞Col1、Col3、CTGF和α-SMA蛋白水平的升高;抑制TGF-β1诱导的成纤维细胞向肌成纤维细胞的转化。可见,成功克隆、表达并纯化了IL10-RGD融合蛋白,该融合蛋白能够明显拮抗TGF-β1诱导的纤维化,预示着该蛋白在瘢痕增生及皮肤纤维化治疗方面有着较好的应用前景。     

Protein kinase assay on peptide-conjugated gold nanoparticles

We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (alpha-pY) and Cy3-labeled secondary antibody (Cy3-alpha-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.     

A new technique for the visualization of the cytoskeleton in cultured fibroblasts with Coomassie blue R250

Staining of fibroblast monolayers with Coomassie brilliant blue R250 provides a relatively easy and fast method for visualization of cytoskeletal elements. Staining of detergent-extracted monolayers improves resolution and provides preparations comparable in definition to those obtained by specific immunofluorescence.     

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.     

The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.     

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.     

Monolayers of a model anesthetic-binding membrane protein: formation, characterization, and halothane-binding affinity

hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The alpha-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide.     

Injury-induced release of basic fibroblast growth factor from bovine aortic endothelium

Although the basic fibroblast growth factor (bFGF) gene lacks a traditional consensus signal peptide domain indicative for secretion, many cell types have receptors for bFGF. Since endothelium is a rich source of cell-associated bFGF, we asked under what conditions could bFGF be released or secreted from confluent cultures of bovine aortic endothelial (BAE) cells. The level of bFGF in BAE cell lysates was compared with the level of heparin-releasable bFGF in intact BAE cell monolayers, intact cells with exposed extracellular matrix (nonlytic matrices), and extracellular matrices prepared by cell lysis (lytic matrices). Less than 10% of total cell-associated bFGF was released from intact cell monolayers and nonlytic matrices. In contrast, the levels of bFGF released from lytic matrices depended upon the conditions used to prepare the matrices. Cell lysis at neutral pH generated matrices that released the highest bFGF levels (approximately 50% of total cell-associated bFGF). These matrices were heavily contaminated by histones, indicating the cellular release and adsorption of intracellular proteins to the matrix. Matrices prepared by BAE cell exposure to basic pH (100 mM NH4OH) contained low bFGF content and minor histone contamination. These latter matrices were chosen to study bFGF sequestration, under physiological conditions, into the extracellular matrix of confluent BAE cell cultures. Incubation with endotoxin, an agent acutely toxic to BAE cells, resulted in cellular release and adsorption of endogenous bFGF to cells and matrices, accompanied by histone deposition in the matrices. These results suggested that one mechanism for bFGF release from BAE cell monolayers was passive release induced by severe cell injury and/or cell lysis with secondary adsorption to the matrix.