Efficacy and safety of alpha lipoic acid-capped silver nanoparticles for oral applications.

Silver nanoparticles (AgNPs) are widely studied for their broad-spectrum antimicrobial effects, and can be utilised readily in biomaterials, however the cellular safety of specific AgNP formulations should be profiled prior to clinical usage. This study determined the cytotoxic effect of small sized (6 nm) alpha lipoic acid capped-AgNPs on human gingival fibroblasts (HGF), as compared to ionic silver and clinical antiseptics. The metabolic pathway was investigated to determine the cellular effects on HGF cells. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) was established for a range of oral related bacteria. Results showed that cell viability decreased with increasing AgNP concentration, whereas lower concentrations of AgNPs, (≤5 µg ml-1) caused a significant increase in cell proliferation at 24 and 72 hour time points. The cytotoxicity profile of AgNPs exhibited significantly lower concentrations, relative to the dose of clinical efficacy, when compared to clinical antiseptics. Caspase 3/7 was not significantly altered when HGF cells were treated with 0.225 µg ml-1 AgNPs, indicating cell necrosis rather than apoptosis. Quantitative RT2-PCR detected an upregulation of genes associated with oxidative stress and the G2M cell cycle checkpoint at ≤4 hours, but expression levels returned to levels consistent with control cells at 24-96 hours. An MIC range of 2.5-12.5 µg ml-1 (min. Escherichia coli, Streptococcus mutans, S. mitis; max. Staphylococcus aureus) was determined across the bacterial species tested and an MBC range of 5-100 µg ml-1 (min. E. coli, max. S. mutans). The antimicrobial profile was similar to that of AgNO3 which suggested that the antimicrobial effect may be influenced by free Ag+ release. It was concluded that alpha lipoic acid capped AgNPs possess limited cytotoxic activity to HGF cells when compared to clinically utilised oral antiseptics, observed via the cellular recovery after initial AgNP treatment and a lack of cumulative cytotoxic effect, whilst maintaining a broad range antimicrobial effect of the AgNPs.

Fluorescent monitoring of kinase activity in real time: development of a robust fluorescence-based assay for Abl tyrosine kinase activity.

Fluorescent biosensors hold great promise for drug discovery. Using a solid-phase version of protein semi-synthesis, we incorporated two fluorophores at specific sites within a truncated version of the c-Crk-II protein. The resulting fluorescent protein biosensor permits the real-time monitoring of Abl kinase activity and provides a robust and rapid method for assaying Abl kinase inhibitors.

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II.

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.

Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation.

BACKGROUND: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence. As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation. RESULTS: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process. CONCLUSIONS: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.

Peptide ligation and its application to protein engineering.

The ability to assemble a target protein from a series of peptide fragments, either synthetic or biosynthetic in origin, enables the covalent structure of a protein to be modified in an unprecedented fashion. The present technologies available for performing such peptide ligations are discussed, with an emphasis on how these methodologies have been utilized in protein engineering to investigate biological processes.

A functional, discontinuous HIV-1 gp120 C3/C4 domain-derived, branched, synthetic peptide that binds to CD4 and inhibits MIP-1alpha chemokine binding.

This paper describes a branched synthetic peptide [3.7] that incorporates sequence discontinuous residues of HIV-1 gp120 constant regions. The approach was to bring together residues of gp120 known to interact with human cell membranes such that the peptide could fold to mimic the native molecule. The peptide incorporates elements of both the conserved CD4 and CCR5 binding sites. The 3.7 peptide, which cannot be produced by conventional genetic engineering methods, is recognized by antiserum raised to native gp120. The peptide also binds to CD4 and competitively inhibits binding of QS4120 an antibody directed against the CDR2 region of CD4. When preincubated with the CD4+ve MM6 macrophage cell line, which expresses mRNA for the CCR3 and CCR5 chemokine receptors, both 3.7 and gp120 inhibit binding of the chemokine MIP-1alpha. The peptide also inhibits infection of primary macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate peptide for a vaccine against HIV-1 and represents a novel approach to the rational design of peptides that can mimic complex sequence discontinuous ligand binding sites of clinically relevant proteins.

Introduction of unnatural amino acids into proteins using expressed protein ligation.

Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.

Synthetic peptides representing discontinuous CD4 binding epitopes of HIV-1 gp120 that induce T cell apoptosis and block cell death induced by gp120.

A vaccine against HIV-1 virus would block initial infection and must target conserved residues. Since initial infection depends on binding of the viral envelope protein gp120 to CD4 on the cell surface, the CD4 binding site of gp120 is a target for vaccine design. To identify the optimal biologically active site, we synthesized a series of 32-mer peptides, based on conserved residues in the C3 and C4 regions of gp120. These included three of five sequence discontinuous residues known to be involved in CD4 binding, one or two of which were substituted with alanine. We also synthesized a 44-mer peptide with an additional branch to incorporate an extra C4 region sequence including a fourth CD4 binding residue. All these peptides used an oxidized Cys-X-Cys bridge to link the discontinuous sequence elements in a manner suggested by the known conserved disulfide bridges in gp120. Polyclonal sera raised to these peptides indicate that they all contain both B and T lymphocyte epitopes. Binding of the peptides to CD4-transfected HeLa cells reveals a hierarchy dependent on the number of relevant CD4 binding residues present. Furthermore, antibody cross-linking of peptides bound to the surface of human T cells results in apoptosis that is similar to the known properties of gp120. The peptide incorporating three CD4 binding residues competitively inhibited gp 120-induced T lymphocyte apoptosis. Thus, we have synthesized novel, branched peptides incorporating conserved discontinuous sequences from two different conserved domains of HIV-1 gp120 that contain T and B lymphocyte epitopes and mimic biological functions of the native protein. These synthetic peptides are candidates for future vaccine development.

Chemical ligation of unprotected peptides directly from a solid support.

In this article we describe a new, convenient procedure to carry out intramolecular (cyclization) and intermolecular native chemical ligations of unprotected peptides directly from a solid support. Our solid-phase ligation approach eliminates the need to manipulate peptide (alpha)thioacid and peptide (alpha)thioester intermediates in aqueous solution before the ligation step, thereby leading to a reduction in handling losses and significantly increasing the overall efficiency of the chemical ligation strategy. A key step in our ligation scheme is the ability to generate fully unprotected peptides tethered to a solid support through an (alpha)thioester linkage. This can be achieved efficiently using optimized Boc-solid-phase peptide synthesis on a 3-mercaptopropionamide-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer support (HS-PEGA). Once the synthesis is complete, the fully protected peptide (alpha)thioester resin is treated with HF to give the corresponding fully unprotected peptide (alpha)thioester resin. Using this procedure several polypeptides ranging from 15 to 47 residues were synthesized successfully. These peptide-resins were then used to perform both intramolecular (head-to-tail cyclizations) and intermolecular solid-phase ligations. The intramolecular solid-phase ligations proceeded much faster than their intermolecular counterparts, but in both cases the reactions were observed to be remarkably clean. The presence of aromatic thiol cofactors significantly accelerated the relatively slow intermolecular ligations. This novel methodology was then extended to provide a general method for performing sequential intermolecular ligations, allowing easy access to much larger polypeptide and protein systems.

Design and synthesis of a highly immunogenic, discontinuous epitope of HIV-1 gp120 which binds to CD4+ve transfected cells.

Here we report the design and synthesis of a novel 32-mer peptide, Lys364-378Val445-459.oxidized (named GC-1), which represents a discontinuous epitope from the C3 and C4 domains of gp120 from the HIV-1 IIIB isolate. This peptide induces high titre IgG antibody responses in mice, indicating that it has both B and T cell epitopes. Epitope mapping using reduced GC-1 and appropriate linear peptides demonstrated that a large proportion of the antibodies raised in mice were directed against discontinuous epitope(s). Furthermore, antibodies to GC-1 peptide cross-reacted with purified HIV-1 strain IIIB gp120, indicating the GC-1 mimicked at least one epitope of the native protein. The peptide, which incorporates three gp120 residues Asp 368, Glu 370 and Asp 457, previously shown to be critical for CD4 ligation, bound to the surface of a CD4 transfected human epithelial cell line HeLa, but not to the parent cell line and inhibited binding of recombinant HIV-1 gp120 to recombinant soluble CD4. We have synthesized the first of a series of discontinuous peptides which will be useful for the probing of interactions of HIV-1 gp120 with the CD4 molecule.

Laser-assisted angioplasty for arterial occlusion of the lower limb: initial results and follow-up.

A flashlamp-pumped pulsed dye laser operating at either 480 or 504 nm, coupled to an integral ball-tipped optical fibre, was used to recanalize occluded lower limb arteries. All channels created by the laser were augmented with balloon dilatation. We have treated 78 limbs in 71 patients; 46 limbs (59 per cent) had rest pain and 22 (48 per cent) of these had tissue loss. The median occlusion length was 18 (range 0.5-58) cm. Technical success was achieved in 58 limbs (74 per cent) with clinical success in 46 (59 per cent). Success rates fell with increasing length of occlusion. Two patients died in the perioperative period. A subgroup of 22 patients with marked discrete arterial calcification had a lower technical success rate than the subgroup without calcification (50 per cent versus 84 per cent, P less than 0.01). Both subgroups displayed a similar pattern of reclosure during follow-up. The cumulative patency rate after technical success was 67 per cent at 6 months and 45 per cent at 12 months. Forty-six (59 per cent) limbs avoided bypass surgery or amputation. Laser-assisted angioplasty may offer an alternative to femoropopliteal bypass, although the former procedure is not as durable.

Biologically effective ultraviolet radiation: surface measurements in the United States, 1974 to 1985.

Recent reports of stratospheric ozone depletion have prompted concerns about the levels of solar ultraviolet radiation that reach the earth's surface. Since 1974 a network of ground-level monitoring stations in the United States has tracked measurements of biologically effective ultraviolet radiation (UVB, 290 to 330 nanometers). The fact that no increases of UVB have been detected at ground levels from 1974 to 1985 suggests that meteorological, climatic, and environmental factors in the troposphere may play a greater role in attenuating UVB radiation than was previously suspected.

Preservation of human tissue immersed for five years in fresh water of known temperature.

Two human bodies were recovered from the waters of the Duluth, Minnesota harbor. Extensive adipocere formation resulted in remarkable preservation of gross anatomic features of internal organs. Total time of immersion could be precisely estimated at five years. Water temperature during those five years could also be accurately estimated by direct measurements taken during the year following recovery of the bodies and from information supplied by a local electric power generating company. Immersion occurred at the time of the year when water temperature was highest (70 degrees F [21 degrees C]) facilitating the rapid formation of adipocere. A proposed mechanism for formation of adipocere is described.

The effect of proteolytic degradation of plasma fibronectin on the responses of functional and immunometric assays for intact fibronectin.

Numerous studies have been made into the nature and importance of altered levels of plasma fibronectin seen in a range of clinical disorders. However, fibronectin is highly sensitive to the proteolytic degradation which may accompany some of these conditions. The influence of such degradation on the assays used is frequently unknown. We have investigated the effects of controlled protease degradation on the responses of an electroimmuno and a functional assay, using purified fibronectin. Tryptic digestion influenced the assays more than thrombin, in relation to the degree of breakdown. Both enzymes reduced the functional (gelatin-binding) activity whilst tryptic cleavage increased the apparent concentration by electroimmunoassay. The findings demonstrate that certain assays may be inappropriate for those clinical conditions where levels of intact fibronectin are of most interest. It is also necessary, then, to determine the degree of fibronectin breakdown when measuring pathological levels.