Inhibitory effect of soy saponins on the activity of ß-lactamases, including New Delhi metallo-ß-lactamase 1.

ß-Lactamase-producing bacteria encode enzymes that inactivate ß-lactam antibiotics by catalyzing the hydrolysis of the ß-lactam ring. Crude soy saponins were observed to have synergistic effects on the antimicrobial activity of ß-lactam antibiotics against ß-lactamase-producing Staphylococcus aureus strains. Furthermore, the activities of ß-lactamases derived from Enterobacter cloacae, Escherichia coli, and S. aureus were decreased significantly in the presence of crude soy saponins. This inhibitory effect was also observed against the New Delhi metallo-ß-lactamase 1 (NDM-1), an enzyme whose activity is not inhibited by the current ß-lactamase inhibitors. The synergistic effect on the antimicrobial activity of ß-lactam antibiotics by crude soy saponins was thought to result from the inhibition the ß-lactamase activity. The components of crude soy saponins include several kinds of soyasaponins and soyasapogenols. It was revealed that soyasaponin V has the highest inhibitory activity against NDM-1. The combined use of soy saponins with ß-lactam antibiotics is expected to serve as a new therapeutic modality, potentially enhancing the effectiveness of ß-lactam antibiotics against infectious diseases caused by ß-lactamase-producing bacteria, including those encoding NDM-1.

Nuclear localization of Haa1, which is linked to its phosphorylation status, mediates lactic acid tolerance in Saccharomyces cerevisiae.

Improvement of the lactic acid resistance of the yeast Saccharomyces cerevisiae is important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened for S. cerevisiae genes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genes YGP1, GPG1, and SPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin gene MSN5 led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

2A protease is not a prerequisite for poliovirus replication.

Poliovirus (PV) 2A(pro) has been considered important for PV replication and is known to be toxic to host cells. A 2A(pro)-deficient PV would potentially be less toxic and ideal as a vector. To examine whether 2A(pro) is needed to form progeny virus, a full-length cDNA of dicistronic (dc) PV with (pOME) or without (pOMEDelta2A) 2A(pro) was constructed in the strain PV1(M)OM. RNAs of both pOME and pOMEDelta2A were capable of forming progeny viruses, called OME and OMEDelta2A, respectively. In their ability to induce a cytopathic effect (CPE), the strains ranked as OMEDelta2A < OME falling dots PV1(M)OM. These results suggest that 2A(pro) is not essential for full-length dc PV to form progeny virus and that it contributes to the efficient viral replication and/or induction of a CPE. To clarify whether 2A(pro) is essential for P1-null (lacking the entire coding sequence for capsid proteins) PV, the RNA replication activity of P1-null PV (pOMDeltaP1) or P1-null PV without 2A(pro) (pOMDeltaP1Delta2A) or without both 2A(pro) and 2B (pOMDeltaP1Delta2ADelta2B) was examined. The RNAs of pOMDeltaP1 and pOMDeltaP1Delta2A could replicate and form progeny viruses under a trans supply of P1 protein, whereas the RNA of pOMDeltaP1Delta2ADelta2B could not. These results suggest that 2A(pro) is not needed for the replication of P1-null PV, although it is important for PV RNA replication and inducing a CPE. To know whether a 2A(pro)-deficient PV can be used as a vector, a P1-null PV containing the enhanced green fluorescent protein (EGFP) coding sequence with or without 2A(pro) was examined. It expressed fluorescent protein. This result suggests that 2A(pro)-deficient PV can express foreign genes.

Late-onset hypogonadism (LOH) and androgens: validity of the measurement of free testosterone levels in the diagnostic criteria in Japan.

The basis of diagnosis of late-onset hypogonadism (LOH) is the measurement of androgen levels. Traditionally, total testosterone (total T) was also used as the primary indicator of gonadal function in Japan. In 1998, the International Society for the Study of the Aging Male was founded to conduct basic and clinical research on this issue internationally. As a result, it is said that bioavailable testosterone levels should be measured in the diagnosis of LOH. At present, however, there are a number of problems for bioavailable testosterone to become a routine diagnostic tool. Here, we will explain the various measurement indicators of androgens, measurement problems, standard values of total T, and free testosterone (free T) in Japan, and the diagnostic methods for LOH overseas. In Japan, the Japanese Urological Association and the Japanese Association of Men's Health recommend the measurement of free T levels in the diagnosis of LOH, for the following reasons: (i) It has been demonstrated that free T is more strongly correlated with calculated bioavailable testosterone, than total T, showing a statistically significant difference; (ii) The behavior of free T is highly consistent with that of bioavailable testosterone, with free T levels being markedly decreased due to aging; (iii) For the measurement of free T, a method that allows the measurement of free T only, without affecting the balance between free T and protein-bound testosterone in blood, is available; and 4) The mean total T by age range decreases only to 80% of the young adult mean even during presenile and senile periods, when LOH occurs most frequently, but the free T level shows a linear decrease with aging and drops to 50% of the young adult mean. For these reasons, we will describe the validity of the recommendation for the measurement of free T levels.

Antifungal cyclic depsipeptide, eujavanicin A, isolated from Eupenicillium javanicum.

In the course of searching for new antifungal agents, a new cyclic depsipeptide, eujavanicin A (1), was isolated from Eupenicillium javanicum as an antifungal agent against the human pathogenic filamentous fungus Aspergillus fumigatus. The structure of 1 was established by spectroscopic and chemical investigations. The absolute stereochemistry was elucidated by Marfey's method and by chiral HPLC analysis.

Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations.

BACKGROUND: In immunoassay kits for determination of urinary free cortisol (UFC) concentrations, the results vary markedly from kit to kit, so we compared in this study the reaction specificity among 4 commercially available immunoassay kits to determine the applicability of these assays in routine determination of UFC concentrations. METHOD: Using 4 commercially available kits, cross-reaction was investigated. In addition, urine samples were fractionated by HPLC to investigate endogenous immunoreactive cortisol responses. HPLC fractions were subjected to gas chromatography-mass spectrometry (GCMS) to identify substances causing inter-kit assay discrepancies. RESULTS: Among the 4 kits, cortisol Kit "TFB" (Immunotech; IOT-RIA method) showed the lowest cross-reaction (2.5%) for prednisolone. Furthermore, on HPLC, 87.8% of the reaction of the entire fraction was seen in the fractions corresponding to the elution position of standard cortisol with the IOT-RIA method; this was the highest percentage among the 4 kits. GCMS revealed that the substance that showed a cross-reaction with the other 3 kits was 5alpha-tetrahydrocortisol (5alpha-THF) glucuronide. CONCLUSIONS: The IOT-RIA method was found to be the most specific for UFC. The other 3 commercially available kits showed cross-reaction with a conjugate of 5alpha-THF, found to be one of the causes of inter-kit assay discrepancies.

[Reference ranges of total serum and free testosterone in Japanese male adults].

PURPOSE: To establish reference range of serum Total Testosterone (T-T) and Free Testosterone (F-T) in Japanese male adults. SUBJECTS AND METHOD: Among 1,172 male adults, who daily lived their healthy life, 1,143 subjects in the year range from 20 to 77 years old, who had serum LH concentration within its reference range (For 20-70 years old: 1.1-25.9 mIU/mL), were selected. As diurnal rhythm of both T-T and F-T was observed, blood samples were collected in the morning when T-T and F-T concentration were relatively stable at their high concentration levels. The collected samples were stored at -20 degree C until they were used for assays. RESULTS: Reference range for T-T has decided to express by the mean +/- 2SD calculated from the entire test results, because influence of aging on the results was negligible, Reference range of T-T has established as 2.01-7.50 ng/mL. The other hand, reference ranges of F-T classified for every decade have decided to express by the mean +/- 2SD of each decade subgroup, because great influence of aging on F-T was observed. For each decade from 20 years on and > 70 years, reference ranges of F-T have established as: 8.5-27.9 pg/mL, 7.6-23.1 pg/mL, 7.7-21.6 pg/mL, 6.9-18.4 pg/mL, 5.4-16.7 pg/mL, and 4.5-13.8 pg/ mL, respectively. CONCLUSION: Reference ranges of serum T-T and F-T in Japanese male adults have established. And the value of Young Adult Mean (YAM) of F-T calculated for a group of the ages between 20 and 39 years have been proposed as a guideline of requirement for the Androgen Replacement Therapy (HRT). The value of 80% and 70% of YAM were 12.4 pg/mL and 10.9 pg/mL, respectively.

Evaluation of a two-dose administration of live oral poliovirus vaccine for wild and virulent vaccine-derived poliovirus type 1, 2, 3 strains in Japan.

We evaluated the efficacy of Japan's vaccination policy, a 2-dose administration of live oral poliovirus vaccine (OPV) against wild and virulent vaccine-derived poliovirus (VDPV) type 1, 2, 3 strains, by investigating the neutralizing antibody titers of residents in Toyama Prefecture, Japan. Seropositivities against the virulent type 1 and 2 strains were more than 90%, but the values against the virulent type 3 strains were approximately 60%. Also, while geometric mean antibody titers against virulent type 1 and 2 strains were more than 180, those against the virulent type 3 strains were 58-59, and 9-12, in particular, at 10 to 19 y of age. A booster dose of the vaccine for the type 3 virus is recommended for adolescents. However, high herd immunity against type 1, 2 and 3 viruses has been maintained for these 22 y, although the seropositivity against type 3 virus was always lower than other types. Our results suggest that Japan's vaccination policy might be enough to prevent an epidemic of poliomyelitis caused by wild and virulent VDPV type 1, 2, 3 strains, even though the titers against type 3 viruses were the lowest.

Eujavanicols A-C, decalin derivatives from Eupenicillium javanicum.

Three new decalin derivatives, eujavanicols A-C (1-3), were isolated from an extract of Eupenicillium javanicum IFM 54704. Their structures were determined by chemical and spectroscopic methods.

Role of the alpha/beta interferon response in the acquisition of susceptibility to poliovirus by kidney cells in culture.

Replication of poliovirus (PV) is restricted to a few sites, including the brain and spinal cord. However, this neurotropism is not conserved in cultured cells. Monkey kidney cells become susceptible to PV infection after cultivation in vitro, and cell lines of monolayer cultures from almost any tissue of primates are susceptible to PV infection. These observations suggest that cellular changes during cultivation are required for acquisition of susceptibility. The molecular basis for the cellular changes during this process is not known. We investigated the relationship between PV susceptibility and interferon (IFN) response in primary cultured kidney and liver cells derived from transgenic mice expressing human PV receptor and in several primate cell lines. Both kidneys and liver in vivo showed rapid IFN response within 6 h postinfection. However, monkey and mouse kidney cells in culture and primate cell lines, which were susceptible to PV, did not show such rapid response or showed no response at all. On the other hand, primary cultured liver cells, which were partially resistant to infection, showed rapid IFN induction. The loss of IFN inducibility in kidney cells was associated with a decrease in expression of IFN-stimulated genes involved in IFN response. Mouse kidney cells pretreated with a small dose of IFN, in turn, restored IFN inducibility and resistance to PV. These results strongly suggest that the cells in culture acquire PV susceptibility during the process of cultivation by losing rapid IFN response that has been normally maintained in extraneural tissues in vivo.

Blockade of the poliovirus-induced cytopathic effect in neural cells by monoclonal antibody against poliovirus or the human poliovirus receptor.

The poliovirus (PV)-induced cytopathic effect (CPE) was blocked in neural cells but not in HeLa cells by the addition of monoclonal antibody (MAb) against PV or the human PV receptor (CD155) 2 h postinfection (hpi). Since each MAb has the ability to block viral infection, no CPE in PV-infected neural cells appeared to result from the blockade of multiple rounds of viral replication. Pulse-labeling experiments revealed that virus-specific protein synthesis proceeded 5 hpi with or without MAbs. However, in contrast to the results obtained without MAbs, virus-specific protein synthesis with MAbs was not detected 7 hpi. Shutoff of host translation was also not observed in the presence of MAbs. Western blot analysis showed that 2Apro, the viral protein which mediates the cleavage of eukaryotic translation initiation factor eIF4G, was still present 11 hpi. However, intact eIF4G appeared 11 hpi. An immunocytochemical study indicated that 2Apro was detected only in the nucleus 11 hpi. These results suggest that neural cells possess protective response mechanisms against PV infection as follows: (i) upon PV infection, neural cells produce a factor(s) to suppress PV internal ribosome entry site activity by 7 hpi, (ii) a factor which supports cap-dependent translation for eIF4G may exist in infected cells when no intact eIF4G is detected, and (iii) the remaining 2Apro is not effective in cleaving eIF4G because it is imported into the nucleus by 11 hpi.

Prevalence of vaccine-derived polioviruses in the environment.

A survey of poliovirus in river and sewage water was conducted from October 1993 to September 1995 in Toyama Prefecture, Japan. In this study, 25 isolates differentiated as type 2 vaccine-derived polioviruses (VDPVs) were characterized using mutant analysis by PCR and restriction-enzyme cleavage (MAPREC) to estimate the ratio of 481-G revertants correlated to neurovirulence in a virus population. Of these isolates, 23 (92%) comprised between 44 and 96% 481-G revertants by MAPREC. The other two isolates had revertant percentages close to the 0.6% of the attenuated reference strain. It was presumed that these 23 isolates would be variant with potential neurovirulence by MAPREC analysis. Of the 23 isolates, three were isolated from river water. Moreover, our results by MAPREC showed that type 2 poliovirus was phenotypically more variable than type 1 (69%) or type 3 (55%), as determined in previous studies. The prevalence of virulent-type VDPVs in river and sewage water suggested that the oral poliovaccine itself had led to wide environmental pollution in nature. To terminate the cycle of virus transmission in nature, the ecology of VDPVs should be studied further. A hygiene programme, inactivated poliovirus vaccine immunization and well-maintained herd immunity may play key roles in reducing the potential risk of infection by virulent VDPVs.

Isolation of vaccine-derived type 1 polioviruses displaying similar properties to virulent wild strain Mahoney from sewage in Japan.

Type 1, 2, and 3 vaccine-derived polioviruses were isolated from a sewage disposal plant located downstream of the Oyabe River in Toyama Prefecture, Japan, between October 1993 and September 1995. Neurovirulence was analyzed in 13 type 1 vaccine-derived strains, using mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC). Nine strains (69%) were estimated to have marked neurovirulence. Some of the neutralizing antigenic sites, temperature sensitivity, and plaque-forming ability of two virulent vaccine-derived poliovirus strains were similar to Mahoney strain. The neutralizing activity of human sera obtained after oral poliomyelitis vaccine (OPV) administration against one of the virulent vaccine-derived polioviruses was examined. Although all human sera showed sufficient neutralizing activity for the prevention of poliomyelitis by vaccine-derived poliovirus strains, a lower titer than that against Sabin type 1 strain was observed. Vaccination against virulent vaccine-derived poliovirus will be effective. However, the environmental presence of viruses that have properties similar to those Mahoney strain is a threat. The introduction of inactivated poliovirus vaccine (IPV), and well-maintained herd immunity, together with reinforced environmental surveillance is important for the final phase of the polio eradication program by the World Health Organization (WHO).

Neurovirulence of type 1 polioviruses isolated from sewage in Japan.

Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.