Argatroban more effectively inhibits the thrombin activity in synovial fluid than naturally occurring thrombin inhibitors.

The purpose of this study was to clarify the precise effect of argatroban on the inhibition of cytokine secretion induced by thrombin on synovial cells. The efficiency of thrombin inactivation by thrombin inhibitors was evaluated in human synovial fluids (SFs). In SFs from 13 osteoarthritis (OA) and 11 rheumatoid arthritis (RA) patients, thrombin, Factor Xa (FXa), plasmin activity, IL-6, MMP-3, VEGF, and D-dimer concentrations were measured. Tissue factor (TF) activity or IL-6, MMP-3, and VEGF secretion of human synovial cells with or without thrombin and argatroban were measured. The efficiency of thrombin inactivation in SFs was compared for thrombin inhibitors: argatroban, antithrombin III (ATIII), or heparin cofactor II (HCII). In SFs, thrombin, FXa, plasmin, D-dimer, IL-6, and MMP-3 were significantly higher in RA than in OA. In synovial cell experiments, TNF-alpha and thrombin enhanced TF activity on the cell surface, and IL-6, MMP-3, and VEGF secretion were enhanced by thrombin. Increased TF activity, and IL-6, MMP-3, and VEGF secretion induced by thrombin were inhibited by argatroban. In SFs, argatroban inactivated thrombin more effectively than ATIII or HCII. Since thrombin plays an important role in the disease activity of OA and RA, it is a potential therapeutic molecular target. Argatroban was the most effective anticoagulant to inhibit thrombin activity in SF. Intra-articular injection is ideal administration because it can deliver high dose of argatroban without high risk of systematic complication.

Locomotor improvement of spinal cord-injured rats through treadmill training by forced plantar placement of hind paws.

STUDY DESIGN: Experimental training model of rats with spinal cord injury (SCI). SETTING: Osaka, JapanObjective:To investigate the effect of forced treadmill training by plantar placement (PP), as compared with dorsal placement (DP), of the dorsal paws on the locomotor behaviors of spinal cord-injured rats. METHODS: The spinal cord was contusion-injured at the thoracic level. Rats were divided into three groups: forced training involving stepping by PP and DP and non-forced training/assistance (nT). Training began 1 week after injury and was conducted for 4 weeks. Locomotor behaviors were estimated using Basso-Beattie-Bresnahan (BBB) scores, dorsiflexion of the hind paws and footprints of the hind paws. Histological and immunohistochemical examinations of the spinal cord lesions were conducted after 4 weeks of training. RESULTS: The values, respectively, of PP, DP and nT groups at 4 weeks of training were as follows: BBB scores were 15.6±0.8, 7.7±1.3 and 10.3±0.4. The paw dorsiflexion angles were 34.1±5.2, 16.4±2.4 and 23.6±3.0 degrees, respectively. The stride angles were 5.1±0.9, 13.7±4.9 and 17.8±4.0 degrees for the left paws. Cavity volumes were 10.3±2.1, 31.0±2.0 and 28.2±4.9%. In addition to cavities, there were astrocyte-devoid areas containing some loose tissues, through which many axons extended longitudinally. CONCLUSIONS: The BBB score, dorsiflexion angle and stride angle were consistently improved in the PP group. Cavity formation was more reduced, and many axons extended through coarse tissues formed in astrocyte-devoid areas at the lesion in the PP group. Forced training by PP of the hind paws promoted the behavioral and histological improvement of rats with SCI.

Aggregative movement of C4 mesophyll chloroplasts is promoted by low CO2 under high intensity blue light.

C4 plants supply concentrated CO2 to bundle sheath (BS) cells, improving photosynthetic efficiency by suppressing photorespiration. Mesophyll chloroplasts in C4 plants are redistributed toward the sides of the BS cells (aggregative movement) in response to environmental stresses under light. Although this chloroplast movement is common in C4 plants, the significance and mechanisms underlying the aggregative movement remain unknown. Under environmental stresses, such as drought and salt, CO2 uptake from the atmosphere is suppressed by closing stomata to prevent water loss. We hypothesized that CO2 limitation may induce the chloroplast aggregative movement. In this study, the mesophyll chloroplast arrangement in a leaf of finger millet, an NAD-malic enzyme type C4 plant, was examined under different CO2 concentrations and light conditions. CO2 limitation around the leaves promoted the aggregative movement, but the aggregative movement was not suppressed, even at the higher CO2 concentration than in the atmosphere, under high intensity blue light. In addition, mesophyll chloroplasts did not change their arrangement under darkness or red light. From these results, it can be concluded that CO2 limitation is not a direct inducer of the aggregative movement but would be a promoting factor of the movement under high intensity blue light.

The influence of climatic oscillations during the Quaternary Era on the genetic structure of Asian black bears in Japan.

The Asian black bear (Ursus thibetanus) inhabits two of the main islands, Honshu and Shikoku, in Japan. To determine how climatic oscillations during the Quaternary Era affected the genetic structure of the black bear populations in Japan, we examined their phylogeographic relationships and compared their genetic structure. We analysed an approximately 700-bp sequence in the D-loop region of mitochondrial DNA collected from 589 bears in this study with 108 bears from a previous study. We observed a total of 57 haplotypes and categorized them into three clusters (Eastern, Western and Southern) based on the spatial distribution of the haplotypes. All but 2 of the 41 haplotypes in the Eastern cluster were distributed locally. Genetic diversity was generally low in northern Japan and high in central Japan. Demographic tests rejected the expansion model in northern populations. Haplotypes of the Western and Southern clusters were unique to local populations. We conclude that the extant genetic structure of the Asian black bear populations arose as follows: first, populations became small and genetic drift decreased genetic diversity in the northern area during the last glacial period, whereas large continuous populations existed in the southern part of central Japan. These patterns were essentially maintained until the present time. In western and southern Japan, the effects of climatic oscillations were smaller, and thus, local structure was maintained.

Plutonium, 137Cs and uranium isotopes in Mongolian surface soils.

Plutonium (238Pu and 239,240Pu), 137Cs and plutonium activity ratios (238Pu/239,240Pu) as did uranium isotope ratio (235U/238U) were measured in surface soil samples collected in southeast Mongolia. The 239,240Pu and 137Cs concentrations in Mongolian surface soils (<53 µm of particle size) ranged from 0.42 ± 0.03 to 3.53 ± 0.09 mBq g-1 and from 11.6 ± 0.7 to 102 ± 1 mBq g-1, respectively. The 238Pu/239,240Pu activity ratios in the surface soils (0.013-0.06) coincided with that of global fallout. The 235U/238U atom ratios in the surface soil show the natural one. There was a good correlation between the 239,240Pu and 137Cs concentrations in the surface soils. We introduce the migration depth to have better understanding of migration behaviors of anthropogenic radionuclides in surface soil. We found a difference of the migration behavior between 239,240Pu and 137Cs from 137Cs/239,240Pu - 137Cs plots for the Mongolian and Tsukuba surface soils; plutonium in surface soil is migrated easier than 137Cs.

A new nitroxyl-probe with high retention in the brain and its application for brain imaging.

In order to estimate free radical reactions and image them in the brain of living animals, a nitroxyl spin-probe, carboxy-PROXYL acetoxymethyl ester (CxP-AM) was newly synthesized. CxP-AM was designed to be hydrolyzed by esterase, but not by lipase, so that it would pass through the blood-brain barrier and be retained in the cytosolic phase of parenchymal cells in the brain after intravenous injection. The pharmacokinetics of CxP-AM was compared with those of carboxy-PROXYL (CxP) and its methyl ester (CxP-M). Carboxyl esterase almost completely hydrolyzed CxP-AM within 3 min. After intravenous injection, the brain retained 1.8 times more CxP-AM than CxP-M, and retained it for more than 30 min. Electron spin resonance computed tomographic (ESR-CT) imaging of CxP-AM in the heads of mice produced marked contrast in the encephalon region, while CxP was distributed only in the extracranial region and CxP-M was distributed in both regions, confirming the pharmacokinetics of CxP-AM. The decay rate of CxP-AM determined with time-resolved ESR-CT imaging was different in the two brain regions, suggesting regional differences in the total reducing capability. CxP-AM should become a powerful probe for the investigation and diagnosis of free radical reactions and their imaging in the brain.

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.

Role of carbohydrate moiety in granulocyte colony stimulating factor.

Biological activities of two granulocyte-colony stimulating factor (G-CSF) preparations with (Lenograstim) or without (Filgrastim) sugar moiety were compared. Both G-CSF preparations similarly enhanced the N-Formyl-Met-Leu-Phe-induced-migration of human peripheral blood polymorphonuclear cells, but did not significantly affect the proliferation of human oral tumor cell lines (HSC-2, HSG). However, Lenograstim induced cytotoxicity (accompanied by the production of cytoplasmic vacuoles and large DNA fragments) in human promyelocytic leukemic cells HL-60, more potently than Filgrastim. Lenograstim, but not Filgrastim, enhanced the cytotoxic activity of sodium ascorbate. In contrast to Lenograstim, Filgrastim was degraded gradually, but too slowly to explain its lower biological activity. These data suggest that the carbohydrate moiety in G-CSF might confer unique biological activities.

Interaction between sodium 5,6-benzylidene-L-ascorbate and gallic acid.

The interaction between sodium 5,6-benzylidene-L-ascorbate (SBA) and gallic acid was investigated by two different parameters: radical intensity and cytotoxicity induction. These compounds produced ESR signals of radicals under alkaline conditions. The addition of increasing concentrations of SBA completely scavenged the gallate radical and replaced the latter with its ascorbate radical. On the other hand, gallic acid dose-dependently enhanced the radical intensity of SBA. Both of these two compounds dose-dependently reduced the viable cell number of human squamous carcinoma HSC-2 cells without inducing internucleosomal DNA cleavage. Electron micrographs of the dying cells demonstrate the irreversible degenerative changes especially in the cytoplasm of the cells. The cytotoxic activity of gallic acid was almost completely eliminated by catalase, whereas SBA was totally insensitive to catalase. When these two compounds were mixed together before adding to HSC-2 cells, the cytotoxic activity of gallic acid was significantly reduced by SBA, whereas that of SBA was not reduced by gallic acid. SBA dose-dependently reduced the gallate oxidation in the culture medium. The interaction between SBA and gallic acid may modify their individual biological activity.

Role of hydrogen peroxide in antitumor activity induction by sodium 5,6-benzylidene-L-ascorbate.

Role of hydrogen peroxide (H2O2) in the induction of antitumor activity against chemically-induced rat hepatocellular carcinoma by sodium 5,6-benzylidene-L-ascorbate (SBA) was investigated. ESR spectroscopy demonstrated that rat liver homogenate of cancerous tissue significantly enhanced the radical intensity of SBA more potently than that of precancerous or normal tissue. The peroxyoxalate chemiluminescence method demonstrated that SBA significantly enhanced the production of H2O2-derived chemiluminescence intensity in the liver homogenates, and the effect of SBA was greater in cancerous tissue than in precancerous or normal tissue. Addition of ascorbic acid, a degradation product of SBA, showed similar but slightly weaker stimulation effects. These data suggest that antitumor activity of SBA in vivo might, at least in part, be due to H2O2 production.

Prevention of oral diseases by polyphenols (review).

This review summarizes the current data on the effects of natural products on various oral diseases, together with their basic biological activities. We have focused on polyphenols and their secondary metabolites, such as tannins, lignins and flavonoids, and their modulating factors, including saliva proline-rich proteins. These substances are found in a wide variety of plant sources such as vegetables, herbs, nuts and teas, and effectively reduce the incidence of carcinogenesis in the oral cavity, inhibit plaque growth and adsorption of oral bacteria, and inhibit the replication of various viruses. The mechanism of their action includes: the direct inactivation of the bacteria and viruses, inhibition of their replication enzymes, induction of apoptosis in tumor cells, stimulation of monocytes/macrophages to produce cytokines, and stimulation of myeloperoxidase-dependent iodination of neutrophiles. Polyphenols showed biphasic actions, acting as antioxidants at lower doses, but acting as prooxidants at higher doses. The development and progression of oral diseases might be modified not only by these natural products, but also by interaction with saliva, proline-rich proteins, antioxidants, metals and dental materials.

Biological information processing requires quantum logic.

Chaos dynamics, which characterizes biological information processing, generates information along the course of temporal development of the relevant system. In this system, the macroscopic uncertainty principle holds between observation time delta t and phase space volume delta omega determined by this observation. In other words delta t and delta omega cannot simultaneously be small. This principle corresponds to the microscopic uncertainty principle that holds in quantum physics. Through an analogy to this correspondence, it is shown that quantum logic might also govern such macroscopic phenomena as are governed by chaos dynamics.

Electrochemiluminescence in bioanalysis.

The discovery of electrochemiluminescence (ECL) and its development as a means of detection is truly a success story. Although studies describing ECL were published in the early 1960s, most studies using ECL as a means of detection were not widely published until the mid 1990s. Incorporating ECL into assays provides increased sensitivity, several logs of dynamic range and the ability to electronically control the reaction. These characteristics provide advantages over assays that rely on radioisotopic labels, fluorescence and enzymatic activity. There have been many areas of science that have benefited from the use of ECL, including environmental microbiology, virology, neurobiology, molecular biology and immunology. ECL has improved the understanding and treatment of infectious diseases, cancer, neurodegenerative diseases and even sleep apnea disorders. Drug development has also benefited from ECL via improved assessment of pharmacodynamics, pharmacokinetics and determining immune responses against protein-based therapeutics. This review provides an overview of ECL chemistry and principles with a more detailed emphasis on the applications of ECL-based assays in different areas of science and medicine. The primary purpose of this review is to provide an in-depth discussion of the impact that ECL-based analysis has had on microbiology, immunology, virology, neurodegenerative diseases, molecular biology and drug development. Examples of ECL-based bioanalysis in each of these fields are discussed in conjunction with an overview of ECL principles and instrumentation.

Three-dimensional observation of pulp cavities in the maxillary first premolar tooth using micro-CT.

AIM: To observe three-dimensional morphological changes with age in the pulp cavities of maxillary first premolar teeth. METHODOLOGY: The specimens used in this study were 10 maxillary first premolar teeth (five males and five females) obtained from patients in three age groups, namely in their twenties (20s), forties (40s) and sixties (60s). Each specimen was imaged by a micro-CT to reconstruct the three-dimensional structure. Then, using the reconstructed images, the morphological characteristics of the pulp cavity, the volume ratio at the horn region, the floor region and the overall region of the pulp chamber and the diameters of the buccal and lingual orifices of the root canals were compared between the three age groups. RESULTS: The mesio-distal widths and the heights of the pulp cavity decreased with age. The volume ratio and the diameter of the root canal orifices also decreased. The decrease in volume was not constant but showed a large decrease between the 20s and the 40s, compared to those of 40s to 60s. CONCLUSIONS: Morphological features of the pulp cavity of maxillary first premolar teeth in different age groups were observed three dimensionally using micro-CT. Decreases in pulp cavity size and shape with age were clarified using a three-dimensional technique.