Ceramide limits phosphatidylinositol-3-kinase C2ß-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Transferrin facilitates the formation of DNA double-strand breaks via transferrin receptor 1: the possible involvement of transferrin in carcinogenesis of high-grade serous ovarian cancer.

Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation.

BACKGROUND: In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. METHODS: Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. RESULTS: Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. CONCLUSIONS: In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Molecular cloning of a class IV chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen and competitive inhibition of its immunoglobulin E-binding capacity by latex C-serum.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified. OBJECTIVE: The aim of this study was to characterize a novel allergen with a high frequency of IgE binding. METHODS: The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA of C. japonica pollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition. RESULTS: The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients with C. japonica pollinosis and/or latex allergy to purified CJP-4. CONCLUSION: We identified CJP-4 as a novel and fourth C. japonica chitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile between C. japonica chitinase and latex C-serum indicated that C. japonica chitinase should be an important pan-allergen in C. japonica pollen.

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea.

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.

Differential response to low temperature of two Delta6 fatty acid desaturases from Mucor circinelloides.

A recently identified Delta6 fatty acid desaturase in Mucor rouxii shows a low sequence homology (approximately 24% at the amino acid level) to that isolated from Mortierella alpina, but is phylogenetically closer to a plant enzyme, suggesting the occurrence of Delta6 desaturase isozymes in Mucorales molds. In the present study, two types of Delta6 desaturases, mcD6-1 ( Mo. alpina type) and mcD6-2 ( M. rouxii type), were cloned from Mucor circinelloides. When the cloned genes were expressed in the yeast Saccharomyces cerevisiae in the presence of a linoleic acid substrate (C18:2Delta9, 12), a newly generated gamma-linolenic acid (C18:3Delta6, 9, 12) was detected in the cells, which confirmed the suspected enzymatic function of the recombinant protein. This is the first report of Delta6 desaturase isozymes present in one organism. Northern analysis demonstrated that the amount of mcD6-2 mRNA was less than half of that of mcD6-1 mRNA in cells grown at 28 degrees C. However, upon cultivation of the cells at 15 degrees C for 0.5-1 h, mcD6-2 mRNA rapidly increased by up to 1.5-fold and then gradually decreased. By contrast, mcD6-1 transcripts levels did not fluctuate significantly for 1 h after the temperature shift, but declined by 75% over the next 2 h. The gamma-linolenic acid content in total fatty acid from M. circinelloides decreased at 28 degrees C, but was maintained at approximately 30% at 15 degrees C. These data suggest that Delta6 desaturase isozymes play physiologically distinct roles in the maintenance of cellular lipids and adaptation to low temperature.

Synthesis and antiherpesvirus activities of 5-alkyl-2-thiopyrimidine nucleoside analogues.

Twenty 5-alkyl-2-thiopyrimidine nucleosides were newly synthesized and examined for antiviral activities against herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). In this study, 2'-deoxy-5-alkyl-2-thiocytidine analogues had lower 50% effective concentration (EC50) values against HSV-1, and 2'-deoxy-5-alkyl-2-thiouridine analogues showed lower EC50 against VZV than their congeners of arabinoside form. Among the compounds examined, 2'-deoxy-5-ethyl and 5-propyl-2-thiocytidine (TN-53 and TN-54) were most potent and selective anti-HSV compounds. Their EC50s were 0.04 and 0.15 microM, and selectivity indexes were more than 7,215 and 1,849, respectively. On the other hand, 2'-deoxy-5-propyl-2-thiouridine (TN-51), 5-bromovinyl-2-thiouracil arabinoside (TN-65) and 5-styryl-2-thiouracil arabinoside (TN-67) were most potent and selective anti-VZV compounds. Their EC50s were 3.1, 3.8 and 2.6 pM for CaQu strain of VZV, respectively, and 2.1 to 3.0 times lower than that of acyclovir. All 2-thiopyrimidine nucleoside analogues did not show antiviral activities against thymidine kinase (TK) negative strains of HSV-1 and VZV. Only three 2-thiocytosine arabinoside compounds showed marginal anti-CMV activities (EC50s were 57-159 pM). All of the five alkyl-2-thio-pyrimidine nucleoside analogues examined were not cytotoxic to human lymphoblastoid cells (RPM18226) and human embryonic fibroblast cells (MRC-5) at 240 microM (100 microg/ml) or more. Regarding the structure-activity relationship of 5-alkyl-2-thiopyrimidine nucleoside analogues, the following remarks will be noted. Elongation of 5-alkyl chain (methyl to ethyl) of 2-thiocytosine in both deoxyribosyl and arabinosyl nucleosides increased anti-HSV-1 activity but not anti-VZV activity. Furthermore, elongation of the same chain (ethyl to propyl) of 2-thiodeoxyuridine increased anti-VZV activity whereas it did not in the case of 2-thiouracil arabinosides.

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.

Pharmacokinetics of a benzodithiin (RD3-0028) following aerosol treatment in rat.

1. RD3-0028, a benzodithiin compound, has potent antiviral activity against respiratory syncytial virus (RSV) in cell culture. The compound also inhibits growth of RSV and improves pathologic changes of interstitial pneumonia in the immunosuppressed mouse when delivered by small-particle aerosol. 2. In the present study, the absorption, distribution and excretion of 14C-RD3-0028 were compared in rat following either a single aerosol treatment or oral administration. 3. The plasma concentration was maintained at the same level from 5 min to 1 h, and decreased with a half-life of 2.2 +/- 0.1 h for 1-8 h. 4. The excretion of radioactivity in the urine and faeces at 24 h after aerosol treatment was 89.3 and 4.5%, respectively, indicating that almost all the radioactivity was rapidly excreted in the urine. The excretion of total radioactivity was 98.9% within 168 h. 5. The concentrations of radioactivity in the lung and trachea following aerosol treatment were higher than those in other tissues, and were detected even at 72 h. 6. These results suggest that the aerosol treatment might be useful for delivering RD3-0028 to the respiratory tract of RSV-infected patients.

Herpesvirus DNA in peripheral blood mononuclear cells of some patients with Meniere's disease.

Herpes simplex virus-1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV-1, 2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV-1 DNA or VZV DNA was detected in PBMCs of 50 age- and sex-matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV- 1 or VZV may be associated with the development of some cases of Meniere's disease.

Antiviral activity of NMSO3 against adenovirus in vitro.

NMSO3, a sulfated sialyl lipid, was evaluated for its efficacy against adenovirus (AdV) in vitro. The median effective concentration (50% effective concentration, EC(50)) of NMSO3 against replication of AdV type 2 (AdV2), type 4 (AdV4), type 8 (AdV8) and type 37 (AdV37) was 0.21-0.71 microg/ml in HEp-2 cells and 1.01-1.41 microg/ml in MKN-28 cells. The EC(50) values of NMSO3 were lower than those of HPMPC and ddC, which were also evaluated. NMSO3 exhibited minimal cytotoxicity against HEp-2 cells and MKN-28 cells, both for which the median cytotoxic concentration (50% cytotoxic concentration, CC(50)) was more than 1000 microg/ml. NMSO3 was the most potent and selective anti-AdV compound of those examined. NMSO3 inhibited AdV infection of HEp-2 cells only when present during the virus adsorption period. A virus binding assay using radiolabeled AdV4 revealed that NMSO3 inhibited viral binding to the HEp-2 cells. NMSO3 itself bound to the virus particles, but not to the HEp-2 cell membrane. Thus, the mechanism of anti-AdV activity by NMSO3 involves inhibition of virus adsorption to cells by NMSO3 binding to viral particles.

Targets of anti-influenza chemotherapy other than neuraminidase and proton pump.

Antiviral chemotherapy for influenza started with treatment with amantadine and then progressed with finding the clinical efficacy of neuraminidase (NA) inhibitors. Beside amantadine and NA inhibitors, there are several compounds that attack novel targets of influenza virus (FluV) replication. Binding and penetration of FluV to cell membrane are important stages in the process of virus replication, and several compounds that inhibit these functions have been reported, although most of them have yet to be examined for clinical use. A polyoxometalate (PM523) was shown to be potent inhibitor of FluV A, respiratory syncytial virus and measles virus, and was shown to inhibit membrane fusion between FluV envelope and the cellular membrane. Strains of virus with acquired resistance to PM523 had mutations in the amino acids substrates in HA1 head, and amino acid changes occurred in the interface peptide of the trimers of HA. Cap formation of FluV-encoded mRNA is unique; it utilizes 5'-mGpppXm of host mRNA. Several substances which inhibit the cap formation of FluV (they are inhibitors of PB2 enzyme activity of FluV) are introduced and reviewed in this article. A metabolic product of ribavirn, 1,2,4 triazole carboxamide (T-CONH2) is inhibitory for FluV A growth in vitro. Peroral administration of TCONH2 also showed therapeutic effect in an experimental mouse infection model of FluV A as well as ribavirin. TCONH2 seems to be less toxic than ribavirin for mice, and may be useful as alternative chemotherapy of ribavirin. Other anti-FluV substances that have been reported to be effective for FluV infection in the mouse are discussed with respect to the possibility of their clinical potential.

Mechanism of selective inhibition of respiratory syncytial virus by a benzodithiin compound (RD3-0028).

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.

[Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (1999). I. Susceptibility distribution].

The bacterial strains isolated from patients diagnosed as having urinary tract infections (UTIs) in 9 institutions in Japan were supplied between the period of August 1999 to July 2000. Then, the susceptibilities of them to many kinds of antimicrobial agents were investigated. The number of them were 499 strains. The breakdown of these strains was Gram-positive bacteria as 31.3% and Gram-negative bacteria as 68.7%. Susceptibilities of these bacteria to antimicrobial agents were as follows; vancomycin (VCM), ampicillin (ABPC) and imipenem (IPM) showed strong activities against Enterococcus faecalis. The increase of low-susceptible strains which was noticed in the former year showed a slight recovery in this year. VCM showed a strong activity against MRSA preventing growth of all strains with 1 microgram/ml. In addition, the activity of arbekacin (ABK) was also strong with the MIC90 of 2 micrograms/ml against MRSA. However, MSSA and MRSA showing low susceptibilities were detected in one strain each (MIC: 16 micrograms/ml and 32 micrograms/ml, respectively). Carbapenems showed high activities against Citrobacter freundii and Escherichia coli. Meropenem (MEPM) prevented growth of all strains within 0.125 microgram/ml. Quinolone resistant E. coli decreased in this year compared with those in the last year, that percentage was less than 5%. Almost all drugs showed strong activities against Klebsiella pneumoniae and Proteus mirabilis. MEPM and carumonam (CRMN) prevented growth of all strains within 0.125 microgram/ml. On the other hand, one strain of K. pneumoniae showing resistance to cefaclor (CCL) and one strain of P. mirabilis showing low susceptibility to most of cephems were detected. Against Pseudomonas aeruginosa, almost drugs were not so active. The MIC90s of carbapenems were 8 micrograms/ml and those of all other drugs were more than 16 micrograms/ml.

[Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (1999). II. Background of patients].

Five-hundred forty four bacterial strains isolated from 412 patients diagnosed as having urinary tract infections (UTIs) in 9 institutions in Japan were supplied between the period of August 1999 to July 2000. Then, the clinical background of patients were investigated such as sex, age and type of infections, infections and kind of bacteria, frequency of isolation of bacteria by age and infections, bacteria and infections by timing of administration of antibiotics, and bacteria and infections by surgical procedures. About the relationship between age and sex of patients and type of infections, the number of male patients aged less than 50 years was few, and complicated UTIs without indwelling catheter was the most frequent. In females, the number of patients aged less than 20 years was few. Complicated UTIs without indwelling catheter was the most frequent among female patients aged between 40 to 59 years, in other age groups, uncomplicated UTIs was most frequent. As for type of infections and kind of bacteria, Escherichia coli decreased when the infections became complicated, and Pseudomonas aeruginosa and Enterococcus faecalis increased when the infection became complicated. Considering this result by age of patients, isolation frequency of E. coli was gradually decreased with aging in patients aged more than 20 years with uncomplicated UTIs or complicated UTIs without indwelling catheter. The isolation frequencies of E. faecalis and Staphylococcus aureus were gradually increased with aging in complicated UTIs without indwelling catheter. In patients with complicated UTIs with indwelling catheter, there was no difference between age group, and P. aeruginosa and E. faecalis were frequently isolated. As for type of causative organisms in UTIs before and after the administration of antibiotics, the isolation of bacteria was remarkably decreased after administration in patients with uncomplicated UTIs and complicated UTIs without indwelling catheter. E. coli decreased after administration of antibiotics, and P. aeruginosa and E. faecalis increased after administration in patients with all infections. As for type of causative organisms in UTIs and surgical procedures, E. coli were more frequently isolated in patients with uncomplicated UTIs when surgical procedures were experienced. Also, Klebsiella spp. and E. faecalis were more frequently isolated in patients with surgical procedures. However, in complicated UTIs, type of causative organisms had no relationship with surgical procedures.

[Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (1999). III. Secular changes in susceptibility].

The bacteria (Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as having urinary tract infections (UTIs) in 9 institutions in Japan were supplied between the period of August 1999 to July 2000. Then, the susceptibilities of these bacteria to various antimicrobial agents were examined and the results were compared with those obtained between 1991 and 1998. Comparison was made by classifying strains isolated from patients into those with uncomplicated UTIs and those with complicated UTIs (including with or without indwelling catheter). About E. faecalis, increase of low sensitive strains noted in the former year showed a decreasing tendency, however, one strain each with MIC of 4 micrograms/ml to vancomycin (VCM) was detected in patients with both uncomplicated and complicated UTIs. As for S. aureus, many sensitive strains to cephems, imipenem (IPM) and VCM were noted, and each MIC50 was better than that in the former years. S. aureus strains showing low susceptibility to arbekacin (ABK) were detected in patients with complicated UTIs in this year as well as in the former year, and one strain each with MIC of 16 micrograms/ml and 32 micrograms/ml was detected. Susceptibilities of E. coli were effective to all drugs except for penicillins and minocycline (MINO). Decrease of low sensitive strains was also noted in all drugs except for quinolones. Each MIC90 of ciprofloxacin (CPFX) and sparfloxacin (SPFX) in patients with complicated UTIs against E. coli was 3 degrees classes lower than that in patients with uncomplicated UTIs. As for Klebsiella pneumoniae, decrease of low sensitive strains to cephems was noted in patients with uncomplicated UTIs in 1998. In 1999, low sensitive strains decreased also in patients with complicated UTIs, and few were detected. Susceptibilities of K. pneumoniae to quinolones were effective as compared with those in the former years with the MIC80s of 0.125 microgram/ml or below without detection of low sensitive strains. One low sensitive strain of K. pneumoniae with MIC of 8 micrograms/ml was detected for gentamicin (GM). Susceptibilities of P. aeruginosa to carbapenems were notable. The MIC90 of meropenem (MEPM) and IPM was 4 micrograms/ml each which was 2 degrees better than that in 1998. Resistant P. aeruginosa strains to other drugs except for monobactams decreased in 1999.

Antisense therapy of influenza.

The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides (S-ODNs) with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with those directed to the PB2 target sites. The liposomally encapsulated antisense phosphorothioate oligonucleotides exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas the free antisense phosphorothioate oligonucleotides were observed to inhibit viral absorption to MDCK cells. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. Balb/c mice exposed to the influenza virus A (A/PR/8/34) strain at dose of 100 LD(50)s were treated i.v. with various doses (5-40 mg/kg) of liposomally (Tfx-10) encapsulated PB2-AUG or PA-AUG before virus infection and 1 and 3 days postinfection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in days (MDS) and increased the survival rates with a dose-dependent manner. We demonstrate the first successful in vivo antiviral activity of antisense administered i.v. in experimental respiratory tract infections induced with influenza virus A.

Lack of effect of the abnormal fatty acid metabolism in NC/Nga mice on their atopic dermatitis.

Although clinical evidence has suggested that dysregulated fatty acid metabolism is associated with atopic disorders, the molecular basis for such a correlation remains to be demonstrated. In the present study, we analyzed the fatty acid composition in peripheral blood cells of NC/Nga mice, a model for atopic dermatitis (AD). We found that arachidonic acid significantly accumulated in mice with the AD manifestation. In addition, the leucotriene B4-releasing ability upon calcium ionophore A23187 stimulation was potentiated in blood cells. An arachidonic acid accumulation was not apparent in the non-atopic BALB/c strain, but was still observed in healthy NC/Nga mice fed under specific pathogen-free conditions. These results indicate that a disturbed fatty acid metabolism in NC/Nga mice was not a trigger factor for their dermatitis development.

4'-Ethynyl nucleoside analogs: potent inhibitors of multidrug-resistant human immunodeficiency virus variants in vitro.

A series of 4'-ethynyl (4'-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates. Among such analogs examined, 4'-E-2'-deoxycytidine (4'-E-dC), 4'-E-2'-deoxyadenosine (4'-E-dA), 4'-E-2'-deoxyribofuranosyl-2,6-diaminopurine, and 4'-E-2'-deoxyguanosine were the most potent and blocked HIV-1 replication with 50% effective concentrations ranging from 0.0003 to 0.01 microM in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458 to 2,600). These 4'-E analogs also suppressed replication of various drug-resistant HIV-1 clones, including HIV-1(M41L/T215Y), HIV-1(K65R), HIV-1(L74V), HIV-1(M41L/T69S-S-G/T215Y), and HIV-1(A62V/V75I/F77L/F116Y/Q151M). Moreover, these analogs inhibited the replication of multidrug-resistant clinical HIV-1 strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10 or 11 different anti-HIV-1 agents had failed. The 4'-E analogs also blocked the replication of a non-nucleoside reverse transcriptase inhibitor-resistant clone, HIV-1(Y181C), and showed an HIV-1 inhibition profile similar to that of zidovudine in time-of-drug-addition assays. The antiviral activity of 4'-E-thymidine and 4'-E-dC was blocked by the addition of thymidine and 2'-deoxycytidine, respectively, while that of 4'-E-dA was not affected by 2'-deoxyadenosine, similar to the antiviral activity reversion feature of 2',3'-dideoxynucleosides, strongly suggesting that 4'-E analogs belong to the family of nucleoside reverse transcriptase inhibitors. Further development of 4'-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1 is warranted.

High-dose intravenous ribavirin therapy for subacute sclerosing panencephalitis.

Two patients with subacute sclerosing panencephalitis (SSPE) were treated safely and effectively with high doses of intravenous ribavirin combined with intraventricular alpha interferon. The ribavirin concentrations maintained in the serum and cerebrospinal fluid were higher than those which inhibit SSPE virus replication in vitro and in vivo.