Combination of Sonodynamic and Photodynamic Therapy against Cancer Would Be Effective through Using a Regulated Size of Nanoparticles.

Nanoparticles have been used for many functional materials in nano-sciences and photo-catalyzing surface chemistry. The titanium oxide nanoparticles will be useful for the treatment of tumor by laser and/or ultrasound as the sensitizers in nano-medicine. We have studied the combination therapy of photo- and sono-dynamic therapies in an animal tumor model. Oral-administration of two sensitizers titanium oxide, 0.2%-TiO2 nanoparticles for sono-dynamic and 1 mM 5-aminolevulinic acid for photodynamic therapies have resulted in the best combination therapeutic effects for the cancer treatment. Our light microscopic and Raman spectroscopic studies revealed that the titanium nanoparticles were distributed inside the blood vessel of the cancer tissue (1-3 µm sizes). Among these nanoparticles with a broad size distribution, only particular-sized particles could penetrate through the blood vessel of the cancer tissue, while other particles may only exhibit the side effects in the model mouse. Therefore, it may be necessary to separate the optimum size particles. For this purpose we have separated TiO2 nanoparticles by countercurrent chromatography with a flat coiled column (1.6 mm ID) immersed in an ultrasonic bath (42 KHz). Separation was performed with a two-phase solvent system composed of 1-butanol-acetic acid-water at a volume ratio of 4:1:5 at a flow rate of 0.1 ml/min. Countercurrent chromatographic separation yielded fractions containing particle aggregates at 31 and 4400 nm in diameter.

An NMR method for the determination of protein-binding interfaces using dioxygen-induced spin-lattice relaxation enhancement.

Using oxygen as a paramagnetic probe, researchers can routinely study topologies and protein-binding interfaces by NMR. The paramagnetic contribution to the amide (1)H spin-lattice relaxation rates (R(1)(P)) have been studied for uniformly (2)H,(15)N-labeled FB protein, a 60-residue three-helix bundle, constituting the B domain of protein A. Through TROSY versions of inversion-recovery experiments, R(1)(P) could be determined. R(1)(P) was then measured in the presence of a stoichiometric equivalent of an unlabeled Fc fragment of immunoglobulin (Ig) G, and the ratio of R(1)(P) of the FB-Fc complex to that of free FB [i.e., R(1)(P)(complex)/R(1)(P)(free)] was determined for each observable residue. Regions of helix I and helix II, which were previously known to interact with Fc, were readily identified as belonging to the binding interface by their characteristically reduced values of R(1)(P)(complex)/R(1)(P)(free). The method of comparing oxygen-induced spin-lattice relaxation rates of free protein and protein-protein complexes, to detect binding interfaces, offers greater sensitivity than chemical shift perturbation, while it is not necessary to heavily deuterate the labeled protein, as is the case in cross saturation experiments.

Allele frequency data for 16 STR loci in the Vietnamese population.

The short tandem repeat systems ACTBP2, D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX and FGA were studied in a population sample from Vietnam (178 individuals, mainly from the Hanoi area). The 16 loci met Hardy-Weinberg expectations and possess a combined power of discrimination greater than 0.9999999999999999998 and a combined power of exclusion greater than 0.99999994 in this Vietnamese population.

Kurdish population data for 11 STR loci (ACTBP2, CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D13S317 and D21S11).

In a Kurdish population sample composed of 950 unrelated individuals from Northern Iraq, 11 tetrameric short tandem repeat (STR) loci from 10 different chromosomes (i.e., ACTBP2, CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D13S317 and D21S11) were typed to establish a database for immigration cases. The combined power of discrimination (PD) and the combined power of exclusion (PE) of all 11 loci were 0.99999999999994 and 0.99996, respectively.

Identification and characterization of an antibacterial peptide of the 26-kDa protease of Sarcophaga peregrina with antibacterial activity.

Previously, we purified a serine protease with a molecular mass of 26 kDa that exhibits potent antibacterial activity from a pupal extract of Sarcophaga peregrina (flesh fly). We divided this protease into 12 peptides and examined their antibacterial activity. A peptide corresponding to residues 155 to 174 (peptide 9) was found to exhibit antibacterial activity comparable to that of the 26-kDa protease. When Escherichia coli was treated with peptide 9, the permeability of both the outer and inner membranes increased, and substrates for beta-lactamase and beta-galactosidase entered the cells, but beta-galactosidase did not leak out of the cells under these conditions. It was suggested that residues 6 to 18 of peptide 9 form an amphiphilic alpha-helix under hydrophobic conditions with an N-terminal basic loop and then interact with acidic phospholipids in the bacterial membranes.

A study of chiral recognition for NBD-derivatives on a Pirkle-type chiral stationary phase.

A chiral stationary phase (CSP 1) derived from an (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine showed excellent enantiomeric separation for amino acid derivatives with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), in high-performance liquid chromatography (HPLC). We compared elution profiles (separation factor and elution order) of NBD-amino acids and their analogs on HPLC, to determine the diastereomeric complex between the chiral moiety of CSP 1 and NBD-amino acid, which is responsible for the chiral recognition. (1)H-NMR studies of a mixture of model compound of CSP 1 and NBD-Ala suggest that the diastereomeric complex is composed of two hydrogen bonding sites at the amino proton and oxygen atom, and a pi-pi interaction by the benzofurazan structure (2,1,3-benzoxadiazole) of NBD-amino acid. Furthermore CSP 1 was able to separate esters, amides and alpha-methyl amino acids derivatized with NBD-F.

Solution structure of micelle-bound H5 peptide (427-452): a primary structure corresponding to the pore forming region of the voltage dependent potassium channel.

A 26-mer peptide with the sequence of the pore forming region (residues 427-452) of the Shaker K(+) channel (H5 region) was chemically synthesized. Analyses by CD and two-dimensional 1H NMR spectroscopy were used to investigate the structure of the peptide bound to SDS micelles in solution, which are commonly used in biophysical studies. The tertiary structure of the peptide as a monomer was composed of an alpha-helix (431-438), a turn (439-442), and random coils (427-430, 443-452), and was very similar to that of the pore forming region of the native K(+) channel from Streptomyces lividans determined by X-ray analysis. This result suggests that even an isolated peptide forms a native-like conformation for residues from 431 to 442, depending on its intrinsic amino acid sequence and the surrounding environment.

Regional differences in homicide patterns in five areas of Japan.

This article describes regional differences in the homicide patterns which occurred in Sapporo City and the surrounding area, and in Akita, Ibaraki, Chiba and Toyama prefectures in Japan. Information collected from each case of homicide included factors such as age, sex of the victim and assailant, causes of death, disposition of the offender, relationship between assailant and victim, reasons for criminal action, et al. The statistical features of homicidal episodes among the five different regions showed considerable variation, as follows. The mean death rates for homicide (number of victims per 100,000 of population) during the period 1986-1995 were 0.44 (Sapporo), 0.8 (Akita), 0.58 (Toyama), 0.7 (Ibaraki) and 0.75 (Chiba), respectively. Close family relationship between the victim and assailant was observed in the homicidal acts which occurred in Sapporo, Akita and Toyama. Assailant's relationship to victim was commonly extra-familial in Ibaraki and Chiba-neighboring megalopolis Tokyo, where some events of murder by a foreigner occurred. Homicide by female assailant, murder by mentally abnormal killers and homicide-suicide events were closely associated with family members. And these factors contributed to the considerable number of victims in Sapporo, Akita and Toyama. But, this close family relationship of the victim to the assailant did not correspond with the elevation in the number of deaths, and it was rather inversely related to the higher death rates recognized in Ibaraki and Chiba. This comparative study suggested that rapid urbanization considerably affects regional differences in homicide patterns.

Clinical and patho-anatomical factors affecting expansion of thoracic aortic aneurysms.

OBJECTIVE: To examine the expansion of aneurysmal aortic segments (> or = 35 mm) and to assess the impact of clinical and patho-anatomical factors on aneurysm expansion. DESIGN: 87 consecutive patients (mean age 63.6 years, range 22-84 years) were studied using serial (six month intervals) computed tomographic or magnetic resonance imaging to monitor progression of thoracic aortic aneurysms. Aortic diameter was measured at seven predetermined segments and at the site of maximum aortic dilatation (MAX). RESULTS: 780 segment intervals were identified. The median overall aneurysm expansion rate was 1.43 mm/year. This increased exponentially with incremental aortic diameter (p < 0.01) and varied by anatomical segment (p < 0.05). The presence of intraluminal thrombus (p < 0.01) but not dissection or calcification was associated with accelerated growth. Univariate analysis identified thrombus (p < 0.001), previous stroke (p < 0.002), smoking (p < 0. 01), and peripheral vascular disease (p < 0.05) as factors associated with accelerated growth in MAX. Dissection, wall calcification, and history of hypertension did not affect expansion. beta Blocker treatment was not associated with protection. Multivariate analysis confirmed the positive effect of intraluminal thrombus and previous cerebral ischaemia, and the negative effect of previous aortic surgery on aneurysm growth. These findings translated into a mathematical equation describing exponential aneurysm expansion. CONCLUSIONS: Aneurysmal thoracic aortic segments expand exponentially according to their initial size and their anatomical position within the aorta. The presence of intraluminal thrombus, atherosclerosis, and smoking history is associated with accelerated growth and may identify a high risk patient group for close surveillance.

Pairing of oligosaccharides in the Fc region of immunoglobulin G.

The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter-chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing.

Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins.

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K(+) channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K(+) channel inhibitors. Unlike most other K(+) channel-blocking toxins, HaTx1 adopts an "inhibitor cystine knot" motif and is composed of two beta-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca(2+) and Na(+) channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels.

A novel NMR method for determining the interfaces of large protein-protein complexes.

Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.

Differential time scale of fluid and solute permeability following hypothermic lung preservation.

BACKGROUND: Assessment of the quality of lung graft preservation by simple functional measures in some laboratory models may fail to detect endothelial injury. The effects of hypothermic preservation in isolation were investigated by measuring the pulmonary capillary filtration coefficient (Kf) and the albumin surface area product (PS) at various cold ischemic intervals. METHODS: Rat lungs were flushed with University of Wisconsin solution at 4 degrees C. Following storage at 4 degrees C, lungs for Kf measurement were subjected to a change in pulmonary arterial pressure. Kf was calculated from the change in rate of weight gain as a function of hydrostatic stress. PS lungs were exposed to Tris buffered Ringer's solution containing 1125 albumin (20 microM) in an isogravimetric state. Following a vascular flush the lungs were homogenized and underwent scintillation counting. Using the Kedem-Katchalsky equation PS was calculated. RESULTS: The Kf for the control, 4-hour, and 7-hour groups were 0.778, 1.816, 4.853 g/ cm H2O/min/100 g wet lung tissue, respectively. There was a significant increase in Kf with each time increment (P,0.01). The Kf for the 24-hour group was 5.587 g/cm H2O/min/100 g wet lung tissue; not an additional significant increase. PS for the control and 4-hour groups (0.0115 and 0.0101 cm3/g wet lung tissue/minute, respectively) were not significantly different. After 7 hours there was a significant increase to 0.171 cm3/g wet lung tissue/min. PS could not be measured after 24 hours. CONCLUSIONS: Significant endothelial injury occurs after 4 hours of cold ischemic preservation. There is progressive injury with time. Increase in water permeability is not secondary to increase in albumin permeability.

Intermittent antegrade warm blood cardioplegia for CABG: extended interval of cardioplegia.

BACKGROUND: Intermittent delivery of warm cardioplegia provides a bloodless surgical field, but it is clinically important to evaluate the periods of normothermic ischemia. The aims of this study are to compare intermittent antegrade warm blood cardioplegia (IAWBC) with intermittent antegrade cold blood cardioplegia (IACBC) groups in terms of myocardial protection, and also to evaluate whether the length of ischemic time in the IAWBC group has an effect on myocardial dysfunction. METHODS: This study is based on a retrospective review of patients who underwent elective coronary artery bypass surgery: 162 consecutive patients with IAWBC and 107 consecutive patients with IACBC. RESULTS: The creatinine kinase peak was smaller in the IAWBC group compared with the IACBC group (p<0.0001). The cardiac index after cardiopulmonary bypass was higher in the IAWBC group (p<0.02), and the amount of inotropic support required to wean from cardiopulmonary bypass was less in the IAWBC group compared with the IACBC group (p<0.0001). CONCLUSIONS: IAWBC with 30 minutes of ischemia provides to be clinically acceptable myocardial protection for coronary bypass surgery.

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer.

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.

Structural basis of the interaction between IgG and Fcgamma receptors.

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor.

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-13C6, 2H7]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with 13C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-13C6, 2H7]glucose.

Progress in the study of ABO blood group system.

Progress in the study of ABO blood group system during the last three decades was reviewed according to following 5 items. 1. Structure of H-, A- and B-active saccharides isolated from the globoside fractions from human erythrocytes. 2. Enzyme characterization of a blood group A-gene specified alpha-N-acetyl-galactosaminyltransferase (A-enzyme), and a blood group B-gene specified alpha-galactosyltransferase (B-enzyme). 3. Immunological properties of the A- and B-enzyme. 4. The cDNA structures of human blood group ABO genes. 5. Transcriptional regulation of the human blood group ABO genes.