The immune system is divided into innate and adaptive immunity. Either immunity consists of humoral and cellular responses, and immunity is maximized when both responses coordinately function. Adaptive immunity has been intensively studied, while it was only recently that we gained some understanding of innate immunity. In particular, cellular responses in innate immunity have been poorly understood compared with humoral responses. In addition, the mechanisms and roles of innate immune responses could be distinct between the organisms that possess both innate and adaptive immunity and those possessing only innate immunity. On the other hand, invading pathogenic microbes employ various strategies to inhibit the host immune system for their survival. I here summarize what needs to be known to gain a deeper understanding of the innate immune response. The readers are suggested to refer to the accompanying articles for more detailed description.
One of the fundamental questions in innate immunity is how a large battery of invading pathogens is recognized by a limited number of germ line-encoding receptors. In Drosophila, peptidoglycan recognition protein (PGRP) family members have a crucial role in recognizing invading bacterial pathogens and in inducing immune reactions. PGRP-SA, -SD, and -SC1a are involved in recognizing gram-positive bacteria and in activating the Toll pathway to produce antimicrobial peptides. PGRP-LC and -LE recognize diaminopimelic acid (DAP)-containing peptidoglycans, which are cell wall components of many gram-negative bacteria and some gram-positive bacteria, and activate the imd pathway to produce antibacterial peptides. In addition to the extracellular function of PGRP-LE to activate immune reactions in the hemolymph, PGRP-LE acts as an intracellular receptor for monomeric DAP-type peptidoglycans. Moreover, a version of PGRP-LE containing only the PGRP domain functions extracellularly as a CD14-like accessory factor, capable of enhancing PGRP-LC-mediated peptidoglycan recognition. Subsequent intracellular signaling is transduced through the RHIM-like motif found in PGRP-LC and -LE.
Use of invertebrate models of infection has given exciting insights into host-pathogen interaction for a number of bacteria. In particular, this has revealed important factors of the host response with remarkable parallels in higher organisms. Recently, emerging of multi-drug resistant bacteria raises a requirement of developing new therapies such as controlling host defense system. Finding host factors that can purge bacteria from human body could give us a new concept of pharmaceutical targets. For this purpose, fruit flies, Drosophila melanogaster, has been used as a model animal for human infectious diseases and became a tractable tool for identifying novel gene products that can activate host defense mechanisms. In this review we will discuss about recent progress of Drosophila model of pathogen infection, which could imply a useful genetically tractable model for human infectious diseases.
Pathogenic gram-negative bacteria, including Salmonella typhimurium, remodel their outer membrane to survive within host tissues and phagosomes. The remodeling includes modifications of lipid A, a membrane anchor portion of lipopolysaccharide. Lipid A modifications, such as palmitoylation, deacylation, addition of aminoarabinose, and addition of phosphoethanolamine, are beneficial for salmonellae to resist host innate immunity. Aminoarabinose attachment, phosphoethanolamine attachment, and palmitoylation of lipid A increase salmonellae resistance to cationic antimicrobial peptides. Lipid A deacylation and palmitoylation reduce its ability to activate the Toll-like receptor 4-MD-2 complex, suggesting that these modifications are beneficial for salmonellae to evade host innate immune recognition. These modifications are regulated transcriptionally by the two-component regulatory system PhoP-PhoQ, which is essential for S. typhimurium virulence. Lipid A modifications are also regulated posttranslationally. Aminoarabinose modification of lipid A represses deacylation of lipid A by PagL. The posttranslational regulation may be involved in S. typhimurium pathogenesis.
Phagocytosis with macrophages provides a specialized mechanism for regulated ingestion and intracellular destruction of bacteria. Bacteria are first engulfed by endocytosis into a phagosome. The fusion of phagosomes and lysosomes releases toxic products that kill most bacteria and degrade them into fragments. Debris from dead bacteria is then released by exocytosis. However, some bacteria that survive within host phagocytes have evolved strategies to escape the bactericidal mechanisms associated with phagocytosis: i) antiphagocytosis (Yersinia), ii) escaping from the phagosome into cytoplasm (Listeria), and iii) remodeling their phagosome by inhibiting the maturation of phagosomes (Salmonella, Mycobacterium, Legionella). In this review, I first summarize various strategies by bacteria to avoid phagocytosis by emphasizing the steps that have been subverted by bacteria. Then, I highlight the mechanisms for surviving phagocytosis by Salmonella, with a focus on the induction of macrophage-apoptosis and modulation of membrane traffic in host cells.
Infection with a variety of viruses induces apoptosis in host cells. This phenomenon may be considered to be a self-defense mechanism to avoid viral propagation. However, the growth of influenza virus is completed before host cells become dysfunctional due to apoptosis. To clarify the physiologic consequences of influenza virus-induced apoptosis, the fate of influenza virus-infected cells was examined in vitro as well as in vivo. Influenza virus-infected cells were engulfed by macrophages in vitro, and virus propagation was almost completely inhibited. This phagocytosis was dependent on the specific recognition of the membrane phospholipid phosphatidylserine exposed on the surface of virus-infected apoptotic cells by macrophages. In addition, the activity of viral neuraminidase expressed at the surface of virus-infected cells was necessary for the maximal level of phagocytosis. When mice infected with influenza virus were administered phagocytosis inhibitors, the level of lethality and inflammation in the lung were augmented. These results show that apoptosis makes influenza virus-infected cells susceptible to phagocytosis by macrophages, and that this leads to a reduction in the extent of influenza pathology.
Major cases of chemical incidents and information on chemical agents and chemical terrorist attacks are outlined. Since the late 1990s, major incidents occurred consecutively, such as two cases of sarin attack in 1994 and 1995, an oil spill from a Russian oil tanker in the Japan Sea in 1997, arsenic poisoning in Wakayama in 1998, the criticality incident at Tokai-Mura in 1999 in Japan, and terrorist attacks on September 11, 2001, in New York. The importance of crisis management and cooperation among relevant organizations has been emphasized. To provide information for an appropriate and quick response in emergencies, we prepared a Web portal site for information on chemicals including chemical agents, a chemical incident database, and links to relevant Web sites. In intentional cases of poisoning caused by toxic chemicals in Japan, 111 cases were collected mainly from a newspaper database (1984—1999). Many copy-cat poisonings occurred, especially in 1984—1985 and in 1998 just after an arsenic poisoning incedent in Wakayama. Many cases occurred in the laboratories of institutes, universities, and hospitals where various types of chemicals are used.
In emergency and critical care medicine, it is important to guess which poisons that patients have taken or been exposedto. The assumption and identification of save lives. Therefore an accurate screening system is required to treat acute poisoning patients in clinical toxicology. However, the ability of a medical center is not sufficient to analyze poisonous substances using analytical equipment. Moreover, the handling and maintenance of the equipment are tedious and costly. To improve these problems, a simple detection method should be established to identify poisons and to treat acute patients in emergency and critical care medicine. In our laboratory, various supports have been attempted for the training of analysts who cope with poisoning incidents and accidents due to toxic substances. Moreover, a simple detection method for toxic substances utilized in the medical center was developed without using expensive analysis apparatus. However, it is impossible to detect and identify chemical warfare agents in a clinical laboratory, because of possible secondary exposure to such dangerous substances in insufficient analytical laboratory equipment. Therefore it is necessary to contact related organizations possessing the proper facilities.
Chemical warfare agents (CWAs) are fast acting and sometimes lethal, even at low levels, and can be classified into nerve gases, blister agents, choking agents, blood agents, vomit agents, tear gases, and incapacitating agents. As countermeasures against CWA terrorism, detection and identification are important. In crisis management, monitoring of CWAs in public places and security checks at territorial borders, big event venues, and executive facilities are performed for protection against terrorism. In consequence management, on-site detection by first responders and laboratory analysis after on-site sampling and transfer are performed for minimization of terrorism damage, leading to personal protection, initial investigation, and emergency lifesaving. In incident management, laboratory analysis is performed to provide evidence at court trials for the prevention of future crimes. Laboratory analysis consists of pretreatment of on-site and casualty samples and instrumental analysis using GC-MS. However, CWAs are easily degraded, and thus are difficult to detect. Instead, it is useful to detect their metabolites and degradation products using tert-butyldimethylsilyl derivatization GC-MS or direct LC-MS. Commercially available chemical detection equipment such as gas detection tubes and ion mobility spectrometers are used for on-site detection. We have evaluated the detection performance of such equipment and found that no equipment fulfills the required perfect performance of CWA detection sensitivity, accuracy, response time, return time, and operation. To overcome the drawbacks, we have adopted the monitoring tape method and counterflow introduction atmospheric pressure chemical ionization mass spectrometry and recommend the combination of commercial detection equipment and these new technologies for simultaneous, rapid detection of all CWAs.
The present paper reviews the use of electrochemical biosensors for detecting organophosphorus pesticides and nerve agents. Acetylcholine esterase (AChE)-immobilized electrodes have been used for detecting AChE inhibitors including organophosphorus and carbamate pesticides. The sensors are composed of AChE and choline oxidase (ChOx) for converting the AChE-generated choline into betaine and hydrogenperoxide (H2O2), which is electrochemically oxidized at the electrode surface to produce the output signal of the sensor. In the presence of AChE inhibitors, the suppressed output signal of the sensor can be observed. If the sensors are operated in the presence of acetylthiocholine as a substrate of AChE, one can eliminate ChOx from the sensor design because enzymatically generated thiocholine is electrochemically active and thus directly oxidized at the electrode without using ChOx. Electron-transfer mediators such as tetracyanoquinodimethane have often been used for catalytically oxidizing thiocholine at the electrode set at less positive potential, which is effective in circumventing possible interference arising from oxidizing compounds in the sample solution. One of the drawbacks of the AChE-based biosensors in detecting organophosphorus pesticides and nerve agents arises from the fact that the sensors indirectly detect the signal based on the inhibition of the AChE-catalyzed reaction. On the other hand, for directly obtaining the output signal, organophosphorus hydrolase (OPH) is immobilized on the electrode surface to prepare amperometric biosensors. OPH catalyzes the hydrolysis reaction of organophosphorus compounds to produce electrochemically active compounds such as p-nitrophenol and thiols from parathion and VX, respectively. Thus OPH-based sensors can be used for detecting these compounds directly. These biosensors would be useful for in-site measurements of organophosphorus pesticides and nerve agents because portable-type biosensors are easily fabricated at relatively low cost.
This paper introduces one of our projects performed at Hokkaido University. During the course of pharmacokinetic studies of SM-12502, which was under development as an anti-platelet-activating factor agent, we found three individuals who showed a slow metabolic phenotype in its pharmacokinetics. Analyzing the genes for CYP2A6 from the three, we discovered that they had the whole CYP2A6 gene deletion (CYP2A6*4C). Genetically engineered Salmonella YG7108 cells expressing human P450 were established to compare the mutagen-producing capacity of the P450 enzymes for various N-nitrosamines. We found that CYP2A6 was involved in the metabolic activation of N-nitrosamines with relatively bulky alkyl chains such as a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which has been known to cause lung tumors in rodents. Thus, to examine the hypothesis that individuals possessing the CYP2A6*4C have a reduced risk of cancer due to the lack of the metabolic activation of certain carcinogens in tobacco smoke, a case-control study was performed. The results clearly indicated a significant association between the CYP2A6 genotype and lung cancer risk in smokers. In contrast, there was no significant relationship between them in nonsmokers. In addition, our results showed that the reduced risk of cancer was caused by the reduced activity of CYP2A6. Thus it was expected that the inhibition of the enzyme would result in a reduced cancer risk caused by smoking. The results of experiments using mice which were treated with NNK, a carcinogenic nitrosamine contained in tobacco smoke, together with 8-methoxypsolaren, a strong inhibitor of CYP2A6, indicated that the inhibition of CYP2A6 completely abolished the occurrence of adenoma.
For the efficient synthesis of divergent nitrogen-containing compounds of pharmaceutical and agricultural importance, the development of efficient, complementary, and new synthetic methodologies is essential. One of the key subjects is how to introduce nitrogen atoms in to organic molecules. This review summaries our recent efforts on this issue, focusing on the use of carbamates, acylhydrazines, and ammonia as nitrogen sources.
An overview is presented on the reports available so far on sweet potato, Ipomoea batatas, cultivated widely in Polynesia in the pre-Columbian era, with reference to possible ways and presumptive dates of transfer from the Americas to Polynesia, such as (1) Polynesian navigators' travel to Peru, (2) Peruvian fishermen's drift westward, (3) vessel drift, (4) seed drift, (5) root-tuber drift, and (6) transport by birds. The author supports the case (1) as most plausible. Ganshu or Ganchu described in the old Chinese herbal books is identified as Dioscorea esculenta. An introduction of the tuber to China and Japan is briefly mentioned.
Since a nutrition support team (NST) began to work in our hospital in March, 2003, we constructed our original nutrition assessment system that supports the prescription formulation of total parenteral nutrition (TPN). However, in daily NST activities, the re-evaluation of this system became necessary because of a high incidence of enteral nutrition (EN) and marked revisions in the dietary reference intakes in Japanese (7th revision). Therefore, we improved this system and added a prescription formulation support function that is also applicable to EN, and also added a function that automatically calculates the necessary doses of nutrients that tend to become deficient in patients with decubituses. This new system allowed the selection/evaluation of EN solutions in a short time with consideration of the 7th revision, and readily identified deficient nutrients and their levels in decubitus patients. We used this system in patients with high-level malnutrition complicated by decubituses and observed certain treatment effects.
We developed a rapid, simple, and sensitive high-performance liquid chromatography method with UV detection for the quantitative determination of mycophenolic acid (MPA) in human plasma. MPA and the internal standard (naproxen) were separated using a mobile phase of 0.04 M H3PO4-acetonitrile-methanol (3:3:4 v/v/v) over a CAPCELL PAK C18 MG column. A flow-rate of 0.5 ml/min was used at ambient temperature and sample detection was carried out at 254 nm. The assay required only 100 μl of plasma and involved liquid-liquid extraction, which gave high recovery (>94%). The lower limit of quantification for MPA was 0.05 μg/ml. Inter- and intra-day coefficients of variation were less than 9.6% and accuracies were within 9.3%. Additionally, we validated this method in 24-hour monitoring of plasma MPA concentrations after mycophenolate mofetil (MMF) morning and evening administration in 40 Japanese renal transplant recipients with 1.5 g/day MMF. The time to reach the maximum (11.7 μg/ml) and second peak (4.5 μg/ml) of MPA after morning 0.75 g MMF administration was 2.6 h and 9.0 h, and time to reach maximum (10.5 μg/ml) after evening 0.75 g administration was 4.0 h.
To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.
The effect of formulation factors on permeation of diclofenac from some experimental and marketed aqueous eye drops through excised goat cornea was evaluated. Raising the pH of formulation from 6.0 to 8.0 or diclofenac concentration from 0.05 to 0.15% (w/v) or adjusting tonicity with mannitol or addition of viscolizing agent decreased apparent permeability coefficient (Papp). Formulation (pH 7.4) containing sodium metabisulfite or EDTA or combination of methyl and propyl paraben showed significantly (p<0.05) higher Papp whereas benzalkonium chloride (BAC) had no effect and sorbic acid (SA) had reduced permeation. Surprisingly marketed drops containing BAC or SA, showed significantly (p<0.05) higher Papp and decreased in the order of Difen>Voveran>NSAID>Dicol>Diclolab. Lower pH (7.1-7.3) and surface tension of drops indicating presence of surfactant, could mediate increased permeation and presence of buffer could cause irritation on in vivo instillation. The marketed formulations showed corneal hydration >83% suggesting corneal damaging potential.
To compare the antianginal effects of 1,4-dihydropyridine-type calcium-channel blockers, we evaluated the effects of benidipine, amlodipine, nifedipine, and efonidipine on vasopressin-induced myocardial ischemia in rats, an experimental model of angina. Intravenous administration of benidipine (3 μg/kg), amlodipine (1000 μg/kg), and nifedipine (100 μg/kg) suppressed the vasopressin-induced S-wave depression, an index of myocardial ischemia. Efonidipine (100 μg/kg, i.v.) tended to inhibit the S-wave depression. At the antianginal dose of each drug, amlodipine, nifedipine, and efonidipine decreased blood pressure significantly, whereas benidipine had little effect on blood pressure at a dose of 3 μg/kg. These results indicate that benidipine, unlike the other 1,4-dihydropyridine-type calcium-channel blockers examined in this study, inhibits vasopressin-induced coronary vasospasm with fewer undesirable effects such as hypotension in rats, suggesting that benidipine may be useful in the treatment of angina pectoris.
Shikonin, isolated from the plant Lithospermum erythrorhizon Sieb. Et Zucc, has been reported to induce apoptosis in several tumor cells. However, such effect of shikonin on human breast cancer cells has not been reported. Thus, in the present study, whether shikonin could induce MCF-7 human breast cancer cell apoptosis was investigated. The results showed that shikonin (2.5—80 μM) induced MCF-7 cell death in a time- and dose-dependent manner, as measured by MTT assay. The IC50 of a 24 h, 48 h and 72 h time course for MCF-7 cells was 7.4±0.4, 6.3±0.6 and 3.9±0.5 μM, respectively. Cellular morphology observation showed that MCF-7 cells underwent marked apoptotic morphological changes upon treatment with 10 μM shikonin compared with the untreated control. Flow cytometric analysis of shikonin-treated MCF-7 cells showed that the ratio of the apoptotic DNA fragmentation increased in a dose-dependent manner. The present study demonstrated for the first time that the cytotoxic effect of shikonin on MCF-7 cells underwent apoptosis process.