Novel -75°C SEM cooling stage: application for martensitic transformation in steel.

Microstructural changes during the martensitic transformation from face-centred cubic (FCC) to body-centred cubic (BCC) in an Fe-31Ni alloy were observed by scanning electron microscopy (SEM) with a newly developed Peltier stage available at temperatures to -75°C. Electron channelling contrast imaging (ECCI) was utilized for the in situ observation during cooling. Electron backscatter diffraction analysis at ambient temperature (20°C) after the transformation was performed for the crystallographic characterization. A uniform dislocation slip in the FCC matrix associated with the transformation was detected at -57°C. Gradual growth of a BCC martensite was recognized upon cooling from -57°C to -63°C.

X-ray photoelectron spectroscopy and transmission electron microscopy analysis of silver-coated gold nanorods designed for bionanotechnology applications.

Multicomponent nano-agents were designed and built via a core-shell approach to enhance their surface enhanced Raman scattering (SERS) signals. These nano-agents had 36 nm × 12 nm gold nanorod cores coated by 4 nm thick silver shell films and a subsequent thin bifunctional thiolated polyethylene glycol (HS-PEG-COOH) layer. Ambient time-lapsed SERS signal measurements of these functionalized nanorods taken over a two-week period indicated no signal degradation, suggesting that large portions of the silver shells remained in pure metallic form. The morphology of the nanorods was characterized by transmission electron microscopy (TEM) and ultra-high resolution scanning TEM. X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) were utilized to assess the oxidation states of the silver shells covered by HS-PEG-COOH. The binding energies of Ag 3d XPS spectra yielded very small chemical shifts with oxidation; however, the AES peak shapes gave meaningful information about the extent of oxidation undergone by the nano-agent. While the silver shells without HS-PEG-COOH coatings oxidized significantly, the silver shells with HS-PEG-COOH remained predominantly metallic. In fact, six month-old samples still retained mostly metallic silver shells. These findings further demonstrate the stability and longevity of the nanostructures, indicating their significant potential as plasmonically active agents for highly sensitive detection in various biological systems, including cancer cells, tissues, or even organisms.

Imaging of surface spin textures on bulk crystals by scanning electron microscopy.

Direct observation of magnetic microstructures is vital for advancing spintronics and other technologies. Here we report a method for imaging surface domain structures on bulk samples by scanning electron microscopy (SEM). Complex magnetic domains, referred to as the maze state in CoPt/FePt alloys, were observed at a spatial resolution of less than 100 nm by using an in-lens annular detector. The method allows for imaging almost all the domain walls in the mazy structure, whereas the visualisation of the domain walls with the classical SEM method was limited. Our method provides a simple way to analyse surface domain structures in the bulk state that can be used in combination with SEM functions such as orientation or composition analysis. Thus, the method extends applications of SEM-based magnetic imaging, and is promising for resolving various problems at the forefront of fields including physics, magnetics, materials science, engineering, and chemistry.

Morphological and chemical analysis of bainite in Cu-17Al-11Mn (at.%) alloys by using orthogonal FIB-SEM and double-EDS STEM.

In this study, new microscopy techniques were developed for understanding the mechanism for the bainitic transformation in a Cu-17Al-11Mn (at%) alloy. An orthogonally arranged focused ion beam and a scanning electron microscope were employed to observe three-dimensional (3D) morphology of the bainite phase, in addition to compositional analysis by using a scanning transmission electron microscope equipped with a double-detector energy-dispersive X-ray spectrometer system. The 3D morphology of these samples was observed at different aging times and aging temperatures; the results obtained indicated that with increasing aging time and/or aging temperature, the bainite phase at the initial stage of formation exhibits a plate-like shape, which changes to a lenticular form. A habit plane was uniquely determined as ∼{9 3 2} by the combination of 3D image reconstruction and an electron back-scattered diffraction technique. The compositional analysis revealed the spatial distribution of the compositional variation between the bainite and matrix phases in the initial stages of the transformation. In the bainite phase, the Cu concentration was higher, while the concentrations of Al and Mn were lower than those in the surrounding matrix, indicative of the diffusion of the constituent elements with the growth of the bainite phase.

Adverse Drug Reactions for Medicines Newly Approved in Japan from 1999 to 2013: Hypertension and Hypotension.

In this survey, the correlation between adverse drug reactions (ADRs) in human and animal toxicities was investigated for 393 medicines which were approved in Japan from September 1999 to March 2013. ADRs were collected from each Japanese package insert. Comparable animal toxicities with ADRs were collected by thorough investigation of common technical documents. The results of this survey show that hypertension and/or hypotension were mainly observed in medicines affecting the central nervous system. Hypertension was also observed in antipyretics, analgesics, anti-inflammatory agents, vasoconstrictors and agents using antibody. Concordance between human ADRs and animal toxicities was analysed. True-positive rate for hypertension and hypotension is 0.29 and 0.52, respectively. Positive likelihood ratio and inverse negative likelihood ratio are 1.98 and 1.21, respectively, in hypertension and 1.67 and 1.44, respectively, in hypotension. Concordance between human ADRs and animal toxicities is not so high in hypertension and hypotension. Identified mechanisms as on-target for hypertension and hypotension are 29.8% and 30.5%, respectively. More than half of the causative factors of hypertension and hypotension were unable to be elucidated. Our results show that the intake of medicines is often linked to blood pressure variations that are not predicted in animal toxicity studies. Improvement of drug development processes may be necessary to provide safer medicines because current animal toxicity studies are insufficient to predict all ADRs in human beings.

In situ heating SEM observation of the bainitic transformation process in Cu-17Al-11Mn (at.%) alloys.

To understand the bainitic transformation behavior in Cu-17Al-11Mn (at.%) alloys, dynamicin situobservation during heating was carried out in a scanning electron microscope (SEM). In this study, after optimizing the sample preparation method and observation conditions, we successfully observed the transformation process with sufficient resolution and contrast. From the observation results, bainite is first formed preferentially at the grain boundaries of the parent phase. Bainite is also formed inside the grains to relax the elastic strain generated by the initial bainite. Regarding the growth mode, in the early stage of the transformation, bainite grows along the longitudinal direction, and in the late stage, it grows along the lateral direction. The growth rate of the bainite was also evaluated by continuous observation of the same plate. Dynamicin situobservation of a martensitic transformation in the same alloy was also performed to compare the growth mode with that of bainite, and it was found that the behavior is considerably different between bainitic and the martensitic transformations.

Potentials and limitations of nonclinical safety assessment for predicting clinical adverse drug reactions: correlation analysis of 142 approved drugs in Japan.

The objective of this study was to elucidate the range of abilities of nonclinical safety assessment for predicting adverse drug reactions (ADRs) in humans. The dataset included 1256 ADRs with an incidence rate of 5% or more collected from 142 drugs approved in Japan from 2001 to 2010 (excluding anticancer agents and vaccines). Gastrointestinal, neurological and hepatobiliary ADRs were relatively common, followed by hematological, cutaneous, systemic and cardiovascular ADRs in the dataset. The analysis revealed that 48% of ADRs were predictable based on a comprehensive nonclinical safety assessment considering animal toxicity. Hematological and ocular ADRs, infection, and application site reactions showed a correlation of more than 70%, while musculoskeletal, respiratory and neurological ADRs showed a correlation of less than 30%. In addition to subjective patient perceptions, several laboratory parameters routinely monitored both in animals and humans showed a lower correlation, e.g., abnormalities in hepatobiliary and metabolic parameters, and blood pressure increase. Large molecule drugs showed lower correlation than small molecule drugs; ADRs were observed in various organs and consideration of pharmacological action did not significantly contribute to the prediction. It was also confirmed that the current standard of toxicology testing regarding dosing duration and dose level is adequate to detect concordant animal toxicity. This study collectively demonstrated a significant value of nonclinical safety assessment in predicting ADRs in humans. It also identified the subset of ADRs with poor predictability, highlighting the need for advanced testing that enables successful translation of animal toxicity to clinical settings with better accuracy and sensitivity.

Interaction of a magainin-PGLa hybrid peptide with membranes: insight into the mechanism of synergism.

The antimicrobial peptides magainin 2 and PGLa isolated from the skin of the African clawed frog Xenopus laevis show marked functional synergism. We have proposed that the two peptides form a heterodimer composed of parallel helices with strong membrane permeabilizing activity [Hara, T., Mitani, Y., Tanaka, K., Uematsu, N., Takakura, A., Tachi, T., Kodama, H., Kondo, M., Mori, H., Otaka, A., Fujii, N., and Matsuzaki, K. (2001) Biochemistry 40, 12395-12399]. In this study, to elucidate the molecular mechanism of the synergy, we synthesized a chemically fixed heterodimer and investigated in detail the interaction of the hybrid peptide with bacteria, erythrocytes, and lipid bilayers. The hybrid peptide showed antimicrobial activity and membrane permeabilizing activity against negatively charged membranes, similar to or even stronger than those of a physical equimolar mixture of magainin and PGLa, indicating that the synergy is due to the formation of a parallel heterodimer. The heterodimer assumed a more oblique orientation than the component peptides. In contrast, the cross-linking of the two peptides significantly strengthened the action against erythrocytes and zwitterionic lipid bilayers by enhancing the affinity for membranes without changing the basic mode of action. Thus, the separate production of mutually recognizing peptides without cross-linking appears to be a good way to increase selective toxicity.

Synthesis method of nanomaterials by pulsed plasma in liquid.

A new synthesis method of nanomaterials using pulsed plasma in liquid by the low voltage spark discharge is presented. The fullerene C60 and TiO nanopowder were for the first time synthesized by electric discharge method in liquid. The purity of C60 was >99%, which is much higher than those by the conventional arc plasma in inert gas methods (less than 80% C60 and 20% C70 and other fullerenes). Copper nanoparticles prepared by this method were smaller than those by arc method by a factor of >5. The pulsed plasma in liquid enables us to quench from plasma state, by which we can synthesize nanomaterials, metastable materials, etc. In addition, the applied power is 100 times smaller than those of arc discharge.

Action mechanism of PEGylated magainin 2 analogue peptide.

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic beta-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the alpha-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.

Action mechanism of tachyplesin I and effects of PEGylation.

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.

Expression and role of E-cadherin and CD103beta7 (alphaEbeta7 integrin) on cultured mucosal-type mast cells.

Mucosal-type mast cells (MMC) in the respiratory and/or gut epithelium play pivotal roles in the development of allergic inflammation and nematode clearance. To determine the role of E-cadherin and alphaEbeta7 integrin in MMC localization to the epithelium, we analyzed the epithelial binding of two types of mouse bone marrow-derived mast cells: S3-BMMC, which developed in medium containing stem cell factor (SCF) plus IL-3, and S39T-BMMC, which developed with SCF, IL-3, IL-9 and TGF-beta1. The latter cells were more similar to mature MMC than the former in terms of mouse mast cell protease (mMCP)-1 expression. FACS analyses revealed that S3-BMMC expressed E-cadherin and beta7 integrin but not alphaE integrin, whereas S39T-BMMC expressed alphaEbeta7 integrin as well as E-cadherin. Mn2+ promoted adhesion of S39T-BMMC to the monolayer of E-cadherin+F9 cells. The adhesion was suppressed significantly by the combined addition of blocking antibodies against integrin alphaE and E-cadherin, whereas either blocking antibody alone failed to do so. S3-BMMC adhesion was suppressed by E-cadherin blocking antibody but not by alphaE blocking antibody. These results suggested that E-cadherin and alphaEbeta7 integrin, which are expressed on MMC-analog S39T-BMMC, play an important role in mast cell-epithelial cell interaction through homophilic as well as heterophilic binding to the epithelial E-cadherin molecule.

Protease-activated receptor-2 (PAR-2)-related peptides induce tear secretion in rats: involvement of PAR-2 and non-PAR-2 mechanisms.

Protease-activated receptor-2 (PAR-2) plays an extensive role in the regulation of digestive exocrine secretion. The present study examined whether PAR-2-related peptides could modulate tear secretion in rats and analyzed the underlying mechanisms. SLIGRL-NH(2), a PAR-2-activating peptide (PAR-2-AP) derived from mouse/rat PAR-2, when administered i.v. in combination with amastatin, an aminopeptidase inhibitor, evoked tear secretion, whereas LRGILS-NH(2), a PAR-2-inactive reversed peptide, had no such effect. In contrast, LSIGRL-NH(2), a partially reversed peptide known to be inactive with PAR-2, caused tear secretion equivalent to the effect of SLIGRL-NH(2). SLIGKV-NH(2), a human-derived PAR-2-AP, also induced significant tear secretion though to a lesser extent, whereas neither VKGILS-NH(2), a reversed peptide, nor LSIGKV-NH(2), a partially reversed peptide, produced any secretion. In desensitization experiments, after the first dose of SLIGRL-NH(2), the second dose of SLIGRL-NH(2) produced no tear secretion, whereas the response to LSIGRL-NH(2) was only partially inhibited by preadministration of SLIGRL-NH(2). Preadministration of LSIGRL-NH(2) abolished the response to subsequently administered LSIGRL-NH(2) but not SLIGRL-NH(2). The tear secretion induced by LSIGRL-NH(2) but not by PAR-2-APs was blocked by atropine or hexamethonium. Mast cell depletion due to repeated doses of compound 48/80 did not alter the effect of SLIGRL-NH(2) or LSIGRL-NH(2). Finally, IGRL-NH(2), a possible core structure of LSIGRL-NH(2), triggered tear secretion in an atropine-reversible manner. Our findings suggest that the PAR-2-APs SLIGRL-NH(2) and SLIGKV-NH(2) cause tear secretion, most likely via PAR-2 and that LSIGRL-NH(2), a PAR-2-inactive peptide, and IGRL-NH(2), its key structure, trigger tear secretion by stimulating parasympathetic nerves via an unidentified target molecule.

Antimicrobial susceptibility testing of vancomycin-resistant Enterococcus by the VITEK 2 system, and comparison with two NCCLS reference methods.

We evaluated the automated VITEK 2 system (bioMerieux) for antimicrobial susceptibility testing of vancomycin-resistant Enterococcus (VRE). The results obtained with the VITEK 2 system were compared to those obtained using two NCCLS reference methods. The VITEK 2 system produced MICs for penicillin G, erythromycin and vancomycin that were very similar to those of the reference agar-dilution test with all results being within a twofold dilution. When MICs of teicoplanin for these isolates were measured by the agar-dilution method and VITEK 2 system, there was one 'very major' error and seven 'minor' errors. There were no 'major' errors for any of the antibiotics tested. When the results obtained by the micro broth-dilution method were compared with those obtained by the VITEK 2 system, there was one 'very major' error for teicoplanin by the VITEK 2 system, as was the case with the agar-dilution method. There were two 'minor' errors for erythromycin and seven 'minor' errors for teicoplanin. There were no 'major' errors for any of the antibiotics tested. The 35 VRE strains identified phenotypically by the VITEK 2 Advanced Expert System included nine of Enterococcus faecalis and 23 of Enterococcus faecium. Neither Enterococcus avium nor Enterococcus hirae were identified. A total of 32 phenotypes were classified into 22 VanA and 10 VanB strains. PCR genotyping demonstrated 23 vanA+ and nine vanB+ strains. There were differences between the VITEK 2 system results and those of PCR. Overall, 54.3 % of the test results were obtained within 7 h. All MIC values for the 35 VRE isolates were determined within 13 h of completing incubation. The VITEK 2 system is a simple method for accurately detecting vancomycin-resistant strains of Enterococcus and can be used to rapidly determine MICs.

Membrane translocation mechanism of the antimicrobial peptide buforin 2.

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.

Micro-broth dilution method with air-dried microplate for determining MICs of clarithromycin and amoxycillin for Helicobacter pylori isolates.

MICs of clarithromycin and amoxycillin for 253 isolates of Helicobacter pylori were measured by an air-dried microplate method and compared with the results obtained by the agar plate dilution method. The air-dried microplate method is performed by coating each well of a 96-well microplate with the test antibiotic and air-drying it. There were no marked differences between the air-dried microplate method and agar plate dilution methods in the MIC(50) and MIC(90) values or MIC ranges of clarithromycin obtained for the 253 isolates of H. pylori. More specifically, the MICs of clarithromycin for 114 (45.1 %) of the 253 isolates were the same by the air-dried microplate method as the agar plate dilution method, and the differences in the MICs of clarithromycin for a further 114 isolates (45.1 %) varied within one twofold dilution. The MICs of amoxycillin by the former method were in close agreement with the MICs obtained by the latter method: MICs of amoxycillin for 199 (78.7 %) of the 253 isolates were the same by both methods, and the differences in the MICs of amoxycillin for 42 isolates (16.6 %) varied within one twofold dilution. These results indicate that the air-dried microplate method is a useful method for determination of MICs, because the results obtained were in close agreement with those obtained by the standard agar plate dilution method. The air-dried microplate method is, therefore, a convenient and reliable method for determining the MICs of clarithromycin and amoxycillin for H. pylori isolates.

A protective role of protease-activated receptor 1 in rat gastric mucosa.

BACKGROUND AND AIMS: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. METHODS: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. RESULTS: The PAR-1 agonist TFLLR-NH(2), administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH(2) was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH(2) produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH(2) suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. CONCLUSIONS: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

Expression of E-cadherin in human mast cell line HMC-1.

E-cadherin is one of the cell adhesion molecules normally expressed on epithelial cells. We previously reported that murine bone marrow-derived mast cells express E-cadherin that could be involved in homophilic binding with epithelial cell E-cadherin. In the present study we examined whether E-cadherin is also expressed in human mast cell HMC-1. Gene expression of E-cadherin and beta-catenin was observed in HMC-1 by reverse transcription-polymerase chain reaction (RT-PCR), while N-cadherin expression was undetectable. cDNA sequencing of HMC-1 E-cadherin revealed no deletions or mutations. E-cadherin expression in HMC-1 was confirmed by immunoblotting as well as by flow cytometric analyses. In the presence of E-cadherin blocking antibody or a synthetic E-cadherin decapeptide with HAV sequence in culture medium, adhesion of HMC-1 cells to the A431 epithelial cell monolayer was slightly but significantly suppressed. In contrast, N- or P-cadherin decapeptides did not suppress the binding. These results indicated that human mast cell HMC-1 expresses E-cadherin, and is possibly involved in cellular interactions with epithelial cells, while other functions still remain to be elucidated.

Tendency toward increase in the frequency of isolation of beta-lactamase-nonproducing Neisseria gonorrhoeae exhibiting penicillin resistance, and recent emergence of multidrug-resistant isolates in Japan.

Six hundred and ninety-nine strains of Neisseria gonorrhoeae were examined by the agar dilution method according to the M7-A5 guidelines established by the National Committee for Clinical Laboratory Standards (NCCLS) determine to their beta-lactamase production and susceptibility to penicillin G, cefixime, ceftriaxone, tetracycline, spectinomycin, ciprofloxacin, and azithromycin. The frequency of isolation of penicillinase-producing N. gonorrhoeae decreased gradually, from 7.3% of the test isolates (55 isolates) in 1995 to about 1% in 1998 and 1999. In contrast, while beta-lactamase-nonproducing N. gonorrhoeae exhibiting chromosomally mediated penicillin G resistance were not isolated from clinical specimens in 1995, the incidence of isolation of such resistant strains increased markedly, to 8.2% of 159 isolates, in 1997 and 14.9% of 242 isolates in 1999. The incidence of the isolation of tetracycline-resistant strains also increased between 1996 (none detected) and 1998-1999 (each 7%-8%), and a tendency towards an increase in the frequency of isolates of ciprofloxacin-resistant strains was also observed between 1995 (9.8%) and from 1997 to 1999 (more than 20%). There were no isolates resistant to any two antibiotics among penicillin G, tetracycline, and ciprofloxacin until 1997, but, in subsequent surveys in recent years, multidrug-resistant isolates (resistant to penicillin G, tetracycline, and ciprofloxacin) were detected in 1999.