A caged progesterone analog alters intracellular Ca2+ and flagellar bending in human sperm.

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.

Photoregulation of cytochrome P450 activity by using caged compound.

Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP(+) resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 µm; height, 50 µm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.

Design of stable alpha-helices using global sequence optimization.

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix-coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of alpha-helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix-coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable alpha-helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 degrees C is expected to be approximately 70-75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them.

Development of a novel antimicrobial peptide, AG-30, with angiogenic properties.

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an alpha-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo, we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.

Effect of phospholipids on conformational structure of bovine pancreatic trypsin inhibitor (BPTI) and its thermolabile mutants.

The effects of negatively charged phosphatidylserine-prepared membranes (PS) and neutral phosphatidylcholine-prepared membranes (PC) on the structure of wild-type and mutant bovine pancreatic trypsin inhibitor (BPTI) at neutral pH were investigated. The presence of PC did not have any effect on the protein structure while PS induced a non-native structure in three mutant BPTI proteins. However, the negatively charged membrane did not have any effect on wild-type BPTI. The findings revealed that (i) elimination of some disulphide bonds results in dramatic change in protein structure, and, (ii) that this biochemical interaction is surface-driven and electrostatic interactions may play a very strong role in influencing the fore-stated changes in protein structure. Of further interest were the results obtained from investigating the possible role of PS fluidity and concentration in altering mutant. When the value of Gibbs free-energy change of unfolding (DeltaG(U)) was positive, various non-native structures were formed in a concentration-dependent manner. However, when the value of DeltaG(U) was negative, only two types of non-native structures were formed: one with high beta structure content at low PS fluidity state, and the other with a high alpha-helical content at high PS fluidity state. Our study reveals how particular combinations of phospholipid:protein interactions can induce a protein conformation transition from a native to a non-native one at neutral pH, especially when the native structure is predestabilized by amino acid substitutions. This revelation may open up opportunities to explore alternative ways in which phospholipids may play a role in protein mis-folding and the related pathologies.

A highly bone marrow metastatic murine breast cancer model established through in vivo selection exhibits enhanced anchorage-independent growth and cell migration mediated by ICAM-1.

To understand the mechanisms underlying bone marrow metastasis precisely, we established the highly metastatic 4T1E/M3 murine breast cancer cell line. 4T1 murine breast cancer cells were transfected with the neomycin resistance gene, selected in G418, intravenously injected into mice, and harvested from bone marrow. By repeating this protocol three times, we established the 4T1E/M3 cells. The clonality of 4T1E/M3 cells was markedly high confirmed by genomic southern analysis using neo-gene probe. When tissues harvested from mice after intravenous injection of 4T1E/M3 cells were examined histologically, markedly enhanced bone marrow metastasis was observed; 77% of spines from 4T1E/M3-injected mouse showed metastasis as compared to 14% metastasis seen with the parent cells. In vitro, 4T1E/M3 cells attached more strongly to the plastic plate and to bone marrow-derived endothelial cells. DNA micro arrays, real time RT-PCR and FACS analyses revealed that the expression of ICAM-1 and beta2 integrin was upregulated in 4T1E/M3 cells at both the mRNA and cell surface protein levels. 4T1E/M3 cells also showed greater anchorage-independent proliferation in soft agar, and migrated markedly faster than the parent cells in wound healing assays. Anti-ICAM-1 antibodies strongly inhibited both the colony formation and the migration activity of 4T1E/M3 suggesting the importance of the role of ICAM-1. Our newly established highly metastatic 4T1E/M3 cells may provide a potentially powerful tool to study the molecular mechanisms of bone marrow metastasis and to identify new molecular targets for therapeutic interventions.

Loading and unloading of molecular cargo by DNA-conjugated microtubule.

We developed a novel method to load and unload molecular cargos to and from microtubules (MTs) that move on kinesin-coated surfaces. Quantum dots (Qds) (molecular cargo) connected to 21-mer DNA can be selectively loaded on DNA-conjugated MTs through DNA hybridization. The average velocity of the Qd-loaded MTs (0.43 +/- 0.06 microm s(-1) at 25 degrees C) was comparable to that of control MTs. In addition, MTs conjugated with two types DNA sequences can achieve multiloading of Qds. To unload Qd molecular cargos from MTs, the DNA double helix connecting Qds to MTs were cleaved by an appropriate restriction enzyme. This biomolecular motors-based transport system should enable us to construct nanometer-scale devices such as nanobiosensor, nanofluidic system, or nanomachine.

Altering the speract-induced ion permeability changes that generate flagellar Ca2+ spikes regulates their kinetics and sea urchin sperm motility.

Speract, an egg-derived sperm-activating peptide, induces changes in intracellular Ca2+, Na+, pH, cAMP, cGMP, and membrane potential in sperm of the sea urchin Strongylocentrotus purpuratus. Ca2+ is a key regulator of motility in all sperm and, in many marine species, is required for generating turns interspersed with straighter swimming paths that are essential for chemotaxis towards the egg. We show that speract triggers a train of increases in flagellar Ca2+, and that each individual Ca2+ fluctuation induces a transient increase in flagellar asymmetry that leads to a turn. We also find that modifying the amplitude, duration and interval between individual Ca2+ fluctuations by treating sperm with niflumic acid, an inhibitor of Ca2+-activated Cl(-) channels, correspondingly alters the properties of the sperm turns. We conclude that Ca2+ entry through a fast flagellar pathway not only induces sperm turns, but the kinetics of Ca2+ entry may shape the nature of these turns, and that these kinetics are tuned by other channels, possibly including Cl(-) channels. In addition, the speract-induced changes in sperm motility closely resemble those seen during chemotaxis in other marine organisms, yet speract is not a chemoattractant. This implies the Ca2+-induced motility changes are necessary but not sufficient for chemotaxis.

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes.

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.

Photo-control of kinesin-microtubule motility using caged peptides derived from the kinesin C-terminus domain.

To design a nanoscale biodevice that can be controlled by an external stimulus, we have introduced photochemical switching peptides derived from the kinesin C-terminus domain into the kinesin-microtubule in vitro motility system.

Selective detection and transport of fully matched DNA by DNA-loaded microtubule and kinesin motor protein.

DNA-loaded microtubules (MTs) moving on a kinesin motor protein-coated substrate can selectively hybridize with a target fully matched DNA over single-base mismatched DNA and transport it. This technique is capable of collecting target biomolecules toward one point site to design new methodology of DNA analysis.

Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability.

We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel.

Mechanism of tail-mediated inhibition of kinesin activities studied using synthetic peptides.

We used a truncated form of human conventional kinesin (K560) and a set of synthetic tail-derived peptides to investigate the mechanism by which the kinesin tail domain inhibits the protein's ATPase and motor activities. A peptide that spans residues 904-933 (C3) exhibited the strongest inhibitory effect on steady-state motility and ATPase activity. This inhibition reflected diminished binding of the ADP-bound kinesin head to the microtubule. Although peptide C3 bound to both K560 and microtubules, gliding assays using subtilisin-treated microtubules suggested that the binding to the microtubule contributes only little to the inhibition if there is sufficient affinity between the peptide and kinesin. We suggest that tail-mediated inhibition of kinesin activity is mainly the product of allosteric inhibition induced by the intramolecular binding of the kinesin tail domain to the motor domain, but simultaneous binding of the tail to the microtubule also may exert a minor effect.

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns.

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.

Effects of conditions for preparing nanoparticles composed of aminoethylcarbamoyl-beta-cyclodextrin and ethylene glycol diglycidyl ether on trap efficiency of a guest molecule.

Nanoparticles comprising copolymers of aminoethylcarbamoyl-beta-cyclodextrin (AEC-beta-CD) and ethylene glycol diglycidyl ether (EGDGE) are prepared by an interfacial polyaddition reaction in a miniemulsion system. Polymers are formed in a W/O emulsion containing 0.25-10.0% (w/w) water and 5.0% (w/w) surfactant (MO-3S, tetraglycerin monoester, HLB 8.8), where simple particles are predominantly obtained when the water content is 1.0% and 5.0%. Notably, nano-size small particles (diameter: 0.3 microm) are formed under the condition of 5.0% water and 5.0% surfactant, which have the highest beta-CD contents (75.5 wt.%) and the most positive zeta-potential (53.6 mV). The zeta-potential measurement indicates that the obtained particles have positive charge due to protonation of their amino groups below around pH 10. Actually, uptake of 8-anilino-1-naphthalenesulfonic acid (ANS) bearing negative charge (SO(3)(-)) and moderate hydrophobicity depends on the magnitude of zeta-potential of the particles; viz., the particles with zeta-potential of 53.6 mV show the highest efficiency of uptake. The diameter and the beta-CD contents are closely related with the water/surfactant ratio, and the zeta-potentials are dependent on both the diameter and the beta-CD contents. Inclusion of ANS into the CD cavity of EGDGE/AEC-beta-CD particles can be controlled by electrostatic interaction between ANS (negatively charged) and the particle (positively charged). Namely, synergistic effect of cavity-inclusion and electrostatic interaction can dominate the uptake of guest molecules by the particles.

Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry.

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.

Generation and application of a monoclonal antibody that detects a wide variety of protein tyrosine kinases.

To investigate expression profiles of the entire family of protein tyrosine kinases (PTKs), we attempted to generate an antibody that detects a variety of PTKs. For production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens, a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody with a broad cross-reactivity to PTKs. We established a hybridoma cell line producing a monoclonal antibody, YK34, which appeared to cross-react with at least 68 PTKs in the human genome, as evidenced by its reactivity with the recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of the other PTKs. When differentiation of HL-60 cells was induced by 12-O-tetradecanoylphorbol-13-acetate, on Western blotting we observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in the morphology of the cells. These results suggest that the YK34 antibody will be a powerful tool for analysis of a variety of cellular PTKs.

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures.

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of beta(2)-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the beta-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular beta-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular beta-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.

Two thimet oligopeptidase-like Pz peptidases produced by a collagen-degrading thermophile, Geobacillus collagenovorans MO-1.

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in G. collagenovorans MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.