Effects of bone density alterations on strain patterns in the pelvis: application of a finite element model.

Insufficiency fractures occur when physiological loads are applied to bone deficient in mechanical resistance. A better understanding of pelvic mechanics and the effect of bone density alterations could lead to improved diagnosis and treatment of insufficiency fractures. This study aimed to develop and validate a subject-specific three-dimensional (3D) finite element (FE) model of a pelvis, to analyse pelvic strains as a function of interior and cortical surface bone density, and to compare high strain regions with common insufficiency fracture sites. The FE model yielded strong agreement between experimental and model strains. By means of the response surface method, changes to cortical surface bone density using the FE model were found to have a 60 per cent greater influence compared with changes in interior bone density. A small interaction was also found to exist between surface and interior bone densities (< 3 per cent), and a non-linear effect of surface bone density on strain was observed. Areas with greater increases in average principal strains with reductions in density in the FE model corresponded to areas prone to insufficiency fracture. Owing to the influence of cortical surface bone density on strain, it may be considered a strong global (non-linear) indicator for insufficiency fracture risk.

The role of charged amphipathic helices in the structure and function of surfactant protein B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.

Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10.

A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

Membrane-perturbing domains of HIV type 1 glycoprotein 41.

Structural and functional studies were performed to assess the membrane actions of peptides based on HIV-1 glycoprotein 41,000 (gp41). Previous site-directed mutagenesis of gp41 has shown that amino acid changes in either the N-terminal fusion or N-leucine zipper region depressed viral infection and syncytium formation, while modifications in the C-leucine zipper domain both increased and decreased HIV fusion. Here, synthetic peptides were prepared corresponding to the N-terminal fusion region (FP-I; gp41 residues 519-541), the nearby N-leucine zipper domain (DP-107; gp41 residues 560-597), and the C-leucine zipper domain (DP-178; gp41 residues 645-680). With erythrocytes, FP-I or DP-107 induced dose-dependent hemolysis and promoted cell aggregation; FP-I was more hemolytic than DP-107, but each was equally effective in aggregating cells. DP-178 produced neither hemolysis nor aggregation, but blocked either FP-I- or DP-107-induced hemolysis and aggregation. Combined with previous nuclear magnetic resonance and Fourier transform infrared spectroscopic results, circular dichroism (CD) spectroscopy showed that the alpha-helicity for these peptides in solution decreased in the order: DP-107 >> DP-178 > FP-I. CD analysis also indicated binding of DP-178 to either DP-107 or FP-I. Consequently, DP-178 may inhibit the membrane actions mediated by either FP-I or DP-107 through direct peptide interactions in solution. These peptide results suggest that the corresponding N-terminal fusion and N-leucine zipper regions participate in HIV infection, by promoting membrane perturbations underlying the merging of the viral envelope with the cell surface. Further, the C-leucine zipper domain in "prefusion" HIV may inhibit these membrane activities by interacting with the N-terminal fusion and N-leucine zipper domains in unactivated gp41. Last, exogenous DP-178 may bind to the N-terminal and N-leucine zipper domains of gp41 that become exposed on HIV stimulation, thereby preventing the fusogenic actions of these gp41 regions leading to infection.

Surfactant protein B and C analogues.

Mammalian lung surfactant is a mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D). Its major function is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film highly enriched in dipalmitoyl phosphatidylcholine (DPPC), thereby preventing alveolar collapse during expiration. SP-A and SP-D are large hydrophilic proteins, which play an important role in host defense, whereas the small hydrophobic peptides SP-B and SP-C interact with DPPC to generate and maintain a surface-active film. Surfactant replacement therapy with bovine and porcine lung surfactant extracts, which contain only polar lipids and SP-B and SP-C, has revolutionized the clinical management of premature infants with respiratory distress syndrome. Newer surfactant preparations will probably be based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. The development of peptide analogues of SP-B and SP-C offers the possibility to study their molecular mechanism of action and will allow the design of surfactant formulations for specific pulmonary diseases and better quality control. This review describes the hydrophobic peptide analogues developed thus far and their potential for use in a new generation of synthetic surfactant preparations.

Conformational mapping of the N-terminal segment of surfactant protein B in lipid using 13C-enhanced Fourier transform infrared spectroscopy.

Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs.

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.

Conformational mapping of a viral fusion peptide in structure-promoting solvents using circular dichroism and electrospray mass spectrometry.

The N-terminal domain of human immunodeficiency virus (HIV)-1 glycoprotein 41,000 (FP; residues 1-23; NH2-AVGIGALFLGFLGAAGSTMGARS-CONH2) is involved in the fusion and cytolytic processes underlying viral-cell infection. Here, we use circular dichroism (CD) spectroscopy, along with electrospray ionization (ESI) mass spectrometry and tandem (MS/MS) mass spectrometry during the course of hydrogen/deuterium exchange, to probe the local conformations of this synthetic peptide in two membrane mimics. Since amino acids that participate in defined secondary structure (i.e., alpha-helix or beta-sheet) exchange amido hydrogens more slowly than residues in random structures, deuterium exchange was combined with CD spectroscopy to map conformations to specific residues. For FP suspended in the highly structure-promoting solvent hexafluoroisopropanol (HFIP), CD spectra indicated high alpha-helix and disordered structures, whereas ESI and MS/MS mass spectrometry indicated that residues 5-15 were alpha-helical and 16-23 were disordered. For FP suspended in the less structure-promoting solvent trifluoroethanol (TFE), CD spectra showed lower alpha-helix, with ESI and MS/MS mass spectrometry indicating that only residues 9-15 participated in the alpha-helix. These results compare favorably with previous two-dimensional nuclear magnetic resonance studies on the same peptide.

Fine trabecularized carbon: ideal material and texture for percutaneous device system of permanent left ventricular assist device.

The development of a percutaneous artificial internal organ system requires a reliable biocompatible connection between the external environment and the inside of the human body. Such is necessary for the success of a permanent left ventricular assist device. However, the search for a satisfactory interface at the epidermal level has proven to be difficult. Carbon has been proposed for this application, but its texture does not typically promote ingrowth from surrounding tissue. We have therefore employed a new processing method to produce a fine trabecularized carbon implant. The method for preparing the implant involves infiltrating low temperature pyrolytic carbon into the surface of a carbon core which is wrapped with carbon fabric. This results in a tightly woven porous structure of carbon (carbon fiber diameter: 35-50 microm, maximal pore size >200 microm) with gradually increasing porosity from 15-75%. We implanted test samples percutaneously in a calf for in vivo histological evaluation. Thirty days after implantation epidermal downgrowth was minimal. Microscopic analysis revealed that a thin fibrous capsule surrounded the implant, and mature connective tissue with accompanying blood vessels filled the pores of the fine trabecularized carbon layer. From these results we suggest that fine trabecularized carbon is ideally suited for a percutaneous device system in a permanent left ventricular assist device.

Effect of pressure-flow relationship of centrifugal pump on in vivo hemodynamics: a consideration for design.

We have been developing centrifugal pumps for an implantable left ventricular assist device. We manufactured 2 prototype centrifugal pumps (PI, PII). These two have similar designs except for the PII having a volute casing and a large output port. To determine the differences in the hydraulic characteristics between the PI and PII, we carried out in vitro and in vivo experiments. In vitro study showed that the PII had a shallower H-Q curve than that of the PI, and the PII required a pump speed faster than the PI for the same flow rate and pressure head. On the other hand, in vivo study showed that the PII demonstrated a flow pulsatility greater than that of the PI at 1,900 rpm and 8 L/min although no significant change was observed at low pump speeds (< or = 1,500 rpm). This greater pulsatility consisted of a large discharge according to the small differential pressure during the systolic phase and a small discharge according to the large differential pressure during the diastolic phase. In contrast, the PI, having the steeper H-Q curve, showed a small discharge in the systolic phase and a large discharge in the diastolic phase. These results showed that pulsatility synchronized with the native heart beating depended on the slope of the H-Q curve. As a result, the slope of the H-Q curve is important to determine the component of pulsatility synchronized with native cardiac output. Regarding the slope of the H-Q curve, a pump having a volute casing and a large outlet port demonstrates a shallow slope in the H-Q curve. In conclusion, we suggest that a centrifugal pump for use in left ventricular aortic bypass should be designed considering the effect on the native heart pulsatility.

The vasoregulatory role of endothelium derived nitric oxide during pulsatile cardiopulmonary bypass.

The role of pulsatile flow as a physiologic stimulus for endothelium mediated vasoregulation is poorly understood. Furthermore, non pulsatile flow, which is associated with increased vascular resistance and end-organ failure, has been demonstrated to lead to a decrease in nitric oxide (NO) production in vitro. Anesthetized pigs (23.4 +/- 0.3 kg) were placed on cardiopulmonary bypass using either non pulsatile or pulsatile perfusion for 60 min. In both groups, animals were maintained with a constant mean aortic flow (1.0-1.3 L/min). Serum samples obtained during bypass were assayed for the stable end-products of NO (nitrate [NO3-] and nitrite [NO2-]) by a method based on the Greiss reaction. Systemic vascular resistance was higher after 60 min in the non pulsatile (3712.5 +/- 481.2 dyne sec cm(-5)) vs the pulsatile group (2672.6 +/- 427.0 dyne sec cm(-5)), but not statistically significant (p > .05). However, NO production was decreased in the non pulsatile flow group (27 +/- 6%) vs the pulsatile flow group (14 +/- 5%) at a statistically significant level (p < .005). The results suggest that non pulsatile flow is associated with diminished endothelial shear stress and a reduction in endothelial nitric oxide production. This may contribute to the detrimental physiologic effects observed in prolonged non pulsatile flow states.

Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments.

Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.

A synthetic segment of surfactant protein A: structure, in vitro surface activity, and in vivo efficacy.

Surfactant protein A (SP-A) is a 248-residue, water-soluble, lipid-associating protein found in lung surfactant. Analysis of the amino acid sequence using the Eisenberg hydrophobic moment algorithm predicts that the SP-A segment spanning residues 114-144 has high hydrophobic moments, typical of lipid-associating amphipathic domains. The secondary structure, in vitro surface activity and in vivo lung activity of this SP-A sequence were studied with a 31-residue synthetic peptide analog (A114-144). Analysis of the secondary structure using circular dichroism and Fourier transform infrared spectroscopy indicated association with lipid dispersions and a dominant helical content. Surface activity measurements of A114-144 with surfactant lipid dispersions and the hydrophobic surfactant proteins B and C (SP-B/C) showed that A114-144 enhances surface activity under conditions of dynamic compression and respreading on a Langmuir/Wilhelmy surface balance. Synthetic surfactant dispersions containing A114-144 improved lung compliance in spontaneously breathing, 28-d premature rabbits to a greater degree than surfactant dispersions with synthetic SP-B/C and synthetic surfactant lipids alone. These observations indicate that inclusion of A114-144 may improve synthetic preparations currently used for surfactant replacement therapy.

Traumatic epididymitis: evaluation with color Doppler sonography.

OBJECTIVE: We performed this study to determine whether scrotal trauma can cause hyperemia of the epididymis. This diagnosis is helpful because traumatic epididymitis can be treated conservatively. MATERIALS AND METHODS: We retrospectively reviewed color Doppler and gray-scale sonograms of five patients who had suffered trauma to the scrotum that resulted in epididymal hyperemia, which we called traumatic epididymitis. We also reviewed the presentation and management of each patient. RESULTS: Color Doppler sonography revealed focal (one patient) and diffuse (four patients) hyperemia. Gray-scale images revealed epididymal enlargement in all patients. These findings were indistinguishable from those of infectious epididymitis by sonography. One patient also had hyperemia of the testis. Four of the five patients were managed conservatively; the other underwent surgical exploration for a coexisting testicular rupture. CONCLUSION: Careful evaluation of the epididymis with both gray-scale and color Doppler sonography should be part of every sonographic survey of the scrotum for blunt trauma. Traumatic epididymitis, which may be noted on color Doppler images, should not be confused with infectious epididymitis. Surgery is not necessary unless another injury requires it.

Synthesis, secondary structure and folding of the bend region of lung surfactant protein B.

Previous theoretical analysis of the primary structure of lung surfactant protein SP-B indicates a disulfide-linked, hydrophobic midsequence that forms a hairpin-like motif. Here, we experimentally investigate the secondary structure of the disulfide-stabilized bend region by synthesizing two 12-residue analogs of the SP-B midsequence. The native peptide has the same sequence for residues 35 to 46 as native human SP-B, while, in the second mimic peptide, Leu40 and Val41 were replaced with D-Ser and L-His. Both peptides contain cysteine residues at the N- and C-terminus (Cys35 and Cys46, respectively). Oxidation/reduction experiments with fast atom bombardment mass spectrometry showed mass shifts of approximately 2 daltons, consistent with the oxidized peptides existing in solution as monomers, each with one internal disulfide bond (Cys35-Cys46). Since circular dichroism and Fourier-transform infrared measurements show that both peptides assume turn conformations in structure-promoting solvents such as trifluoroethanol (TFE), a structural model is proposed in which Cys35 and Cys46 are brought in close apposition through an internal bend in the peptide. Consistent with this model are electron spin resonance (ESR) results of the mimic peptide nitroxide spin-labeled at Cys35 and Cys46. For the double spin-labeled mimic peptide in TFE. ESR spectra indicated broadening characteristic of either radical interactions or decreased mobility, or both. Increases in radical interactions for the double spin-labeled mimic peptide would be expected for Cys35 and Cys46 approaching within 14 A in structure-promoting solvents, while decreases in spin-label mobility could be due to the formation of a loop. Based on these observations with peptide analogs, residues 35 to 46 probably form a similar bend in the full-length protein.

The amino-terminal peptide of HIV-1 glycoprotein 41 fuses human erythrocytes.

The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41,000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519-541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.

Antivirals that target the amino-terminal domain of HIV type 1 glycoprotein 41.

Functional and structural studies were made to assess whether a class of antiviral agents targets the N-terminal domain of the glycoprotein 41,000 (gp41) of human immunodeficiency virus type 1 (HIV-1). Previous experiments have shown that the amino-terminal peptide (FP-I; 23 amino acids, residues 519-541) of HIV-1 gp41 is cytolytic to both human erythrocytes (non-CD4+ cells) and Hut-78 cells (CD4+ lymphocytes). Accordingly, FP-I-induced hemolysis may be used as a surrogate assay for evaluating the role of the N-terminal gp41 domain in HIV-cell interactions. Here, we studied the blocking of FP-I-induced lysis of erythrocytes by the following anti-HIV agents: (1) IgG [i.e., anti-(518-541) IgG] raised to an immunoconjugate of Arg-FP-I, (2) apolipoprotein A-1 (apo A-1) and a peptide based on apo A-1, (3) dextran sulfate, (4) gp41 peptide (residues 637-666), and (5) anionic human serum albumins. Dose-response curves indicated that their relative potency in inhibiting FP-I-induced hemolysis was approximately correlated with their previously reported anti-HIV activity. Electron spin resonance (ESR) studies showed that FP-I spin labeled at the N-terminal alanine binds to anti-(518-541) IgG, dextran sulfate, and anionic albumins. The high in vitro antiviral activity and low cytotoxicity of these agents suggest that blocking membrane-FP-I interactions offers a novel approach for AIDS therapy or prophylaxis.

Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.

We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

The amino-terminal peptide of HIV-1 gp41 interacts with human serum albumin.

Structural and functional studies were made to assess interactions between human serum albumin (HSA) and the amino-terminal peptide (FP-I; 23-residue peptide 519-541) of glycoprotein 41,000 (gp41) of human immunodeficiency virus type-1 (HIV-1). Circular dichroism (CD) spectroscopy indicated that the peptide binds to albumin with dominant alpha-helical character. Peptide binding to albumin was also examined using FP-I spin labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). Electron spin resonance (ESR) spectra of FP-II bound to HSA at 38 degrees C indicated that the spin label at the amino-terminal residue (Ala-519) was motionally restricted. The ESR spectrum of 12-nitroxide stearate (12-NS)-labeled HSA was identical to that obtained with FP-II, indicating that the reporter groups for the 12-NS and FP-II probes are similarly bound to albumin. Contrarily, ESR spectra of HSA labeled with FP-III indicated high mobility for the reporter group (Met-537) at the aqueous-protein interface. This suggests that the N-terminal gp41 peptide binds as an alpha helix (residues 519-536) to fatty acid sites on HSA, such that Ala-519 of the peptide residues in the interior of the protein while Met-537 lies outside the protein in aqueous solution. It is also of interest that addition of HSA to human red blood cells dramatically reduced the ability of FP-I to induce hemolysis, presumably through peptide-albumin binding that inhibited FP-I interactions with red cell membranes. The significance of these results focuses on the following three points. The first is that high serum levels of albumin may limit the efficacy of anti-HIV therapies using peptides based on the N-terminal gp41 domain. The second is that the elucidation of FP-I and HSA interactions with physical techniques may provide clues on the molecular features underlying viral FP-I combination with receptors on the target cell surface. Last, the affinity of albumin for the N-terminal gp41 peptide may play a subordinate role in the blocking of HIV infectivity in vitro that has been reported for chemically modified albumins.

The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4+ lymphocytes.

Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).