Identification of New Diterpenes as Putative Marker Compounds Distinguishing Agnus Castus Fruit (Chaste Tree) from Shrub Chaste Tree Fruit (Viticis Fructus).

Agnus Castus Fruit is defined in the European Pharmacopoeia as the dried ripe fruit of Vitex agnus-castus. In Europe it is used as a medicine targeting premenstrual syndrome and climacteric disorder. In Japan, Agnus Castus Fruit is becoming popular as a raw material for over-the-counter drugs and health food products, though its congenic species, Vitex rotundifolia and Vitex trifolia, have been used as Shrub Chaste Tree Fruit in traditional medicines. Therefore, it is important to discriminate these Vitex plants from the viewpoint of regulatory science. Here we tried to identify putative marker compounds that distinguish between Agnus Castus Fruit and Shrub Chaste Tree Fruit. We analyzed extracts of each crude drug by liquid chromatography-mass spectrometry, and performed differential analysis by comparison of each chromatogram to find one or more peaks characteristic of Agnus Castus Fruit. A peak was isolated and identified as an equilibrium mixture of new compounds named chastol (1) and epichastol (1a). The planar structures of 1 and 1a were determined spectroscopically. Their relative configurations were revealed by nuclear Overhauser effect spectroscopy and differential nuclear Overhauser effect-NMR data. Since avoiding contamination from closely related species is needed for the quality control of natural pharmaceuticals, this information will be valuable to establish a method for the quality control of both, Agnus Castus Fruit and Shrub Chaste Tree Fruit products.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

An aptamer that binds efficiently to the hemagglutinins of highly pathogenic avian influenza viruses (H5N1 and H7N7) and inhibits hemagglutinin-glycan interactions.

Highly pathogenic avian influenza (HPAI) H5 and H7 viruses have ravaged the poultry industry in numerous countries in Asia, Europe, Africa and the Middle East, and have resulted in the deaths of millions of birds. Although HPAI H5N1 viruses currently remain avian viruses, they are continuously evolving and have the potential to become pandemic-type viruses capable of human-human transmission. To develop specific reagents to allow better preparedness against this threat, we selected an aptamer (8-3) from a completely random RNA pool that binds with high affinity (∼ KD 170pM) to the hemagglutinins (HAs) derived from HPAI H5N1 (A/H5N1/Vietnam/1194/2004 and A/H5N1/Indonesia/05/2005) and H7N7 (A/H7N7/Netherlands/219/2003) influenza A viruses. Aptamer 8-3 was able to efficiently distinguish HAs derived from subtypes of influenza A virus other than H5 and H7. Aptamer 8-3 was analyzed further to assess its ability to interfere with HA-glycan interactions using our previously established SPR-based competitive assay, and we found that aptamer 8-3 efficiently interferes with HA-glycan binding (EC50 ∼ 25 nM). To derive shorter variants for other applications, aptamer 8-3 was shortened to a 44-mer by deletion analyses. The shortened aptamer, 8-3S, retains the full-length aptamer's affinity and specificity for its cognate Has, and also interferes with HA-glycan interactions. These studies suggest that aptamer 8-3S should be studied further to explore its potential applications not only in surveillance and diagnosis, but also in the development of H5N1- and H7N7-specific virucidal products that interfere with virus-host interactions to contain future H5N1 and H7N7 pandemics.

Influenza virus surveillance using surface plasmon resonance.

The hemagglutinin (HA) proteins derived from avian influenza viruses bind specifically to the α2-3 sialoglycan (Sia glycan), whereas human-adapted influenza viruses prefer to bind to the α2-6 Sia glycan. A switch of glycan specificity from α2-3 Sia glycan to α2-6 Sia glycan appears to be critical for a virus to become pandemic, therefore, it is important to monitor the influenza virus adaptation to glycan binding. In this article, we described surface plasmon resonance (SPR) methodology for reliable analyses of HA-glycan interactions. The methodology explores the synthetic tetravalent glycans (α2-3 Sia glycan and α2-6 Sia glycans) which facilitates not only the surface capacity of the sensor chip for better SPR signal but also enhance the affinity to the HA resulting an improved sensitivity. To adopt this method routinely for multiple samples of HA or virus, CAP-chip was adopted so that the regeneration of the sensor chip can be achieved. By combining the above developments with BiacoreT100 device, it is possible to program for analyzing multiple samples in continuous fashion under closed environment. Taken together we believe the above methodology is useful in influenza surveillance to monitor the HA adaptations to glycans among influenza viruses.

Monitoring influenza hemagglutinin and glycan interactions using surface plasmon resonance.

Hemagglutinin (HA) is a trimeric glycoprotein expressed on the influenza virus membrane. HA of influenza viruses binds to the host's cell surface complex glycans via a terminal sialic acid (Sia), as the first key step in the process of infection, transmission and virulence of influenza viruses. It is important to monitor and evaluate the receptor (glycan) binding preferences of the HAs derived from influenza A viruses, especially those originating from birds and swine, to understand their potential ability for interspecies transmission. From this viewpoint, in the present study, we have developed a protocol for analyzing the glycan-HA interactions efficiently and kinetically, based on surface plasmon resonance (SPR). Our results showed that glycan-HA binding analyses can be performed reliably and efficiently on Biacore-chips in the SPR system, using chemically synthesized biotinylated multivalent-glycans. Using the CAP-chip, we were able to regenerate the surface for multiple analyses, allowing us to derive, for the first time, the precise kinetic parameters for different HA-glycan complexes of newly emerging influenza viruses. These studies suggested that this SPR-based method is suitable for influenza surveillance to define the pandemic scenario as well as to screen of synthetic glycans and other compounds that may interfere with glycan-HA interactions.

Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases.

Chronic inflammation involving constant generation of nitric oxide (*NO) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2'-deoxyinosine (dI) and 2'-deoxyxanthosine are primarily formed by *NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol alpha was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol eta and pol kappaDeltaC (a truncated form of pol kappa) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol alpha, pol eta, and pol kappaDeltaC, promoted preferential incorporation of 2'-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2'-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol alpha, pol eta, and pol kappaDeltaC indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A-->G transition. This ()NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis.

Prestaining method as a useful tool for the agarose gel electrophoretic detection of polymerase chain reaction products with a fluorescent dye SYBR gold nucleic acid gel stain.

Three staining methods using SYBR Gold Nucleic Acid Gel Stain (SYBR Gold) as a fluorescent dye were evaluated for the agarose gel electrophoretic detection of DNA. The methods involve prestain, in-gel stain, and poststain methods. DNA markers and polymerase chain reaction (PCR) products obtained by minisatellite variant repeat-PCR (MVR-PCR) amplification in a D1S8 locus were used as model DNA and practical samples, respectively. Among the three methods tested under the usual electrophoretic conditions, a prestain method using a 10000-fold diluted SYBR Gold solution showed most excellent features regarding cost and rapidity to use with good stainability and resolution over loaded DNA amounts of about 98 ng to 300 ng. The prestain method was found to be applicable to the analysis of DNA in MVR-PCR products from a human hair root.

Evaluation of three methods for effective extraction of DNA from human hair.

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.

Rapid individual identification by minisatellite variant repeat (MVR)-PCR at D1S8 locus using "exponential law".

We describe an efficient and simple minisatellite variant repeat mapping by PCR (MVR-PCR) method based on the assignment of the tandem array of 29 bp repeating units into a-type, t-type and 0-type (a rarely appearing unamplified unit), from the first repeat unit position (code position 0) of 293 bp in D1S8 locus. After microchip electrophoresis of PCR product amplified from the target DNA of a human hair root, each rung of the ladder at the position of 293 + 29 n bp (n: code position) was detected and the type of repeating unit was determined, i.e., aa, a0, tt, t0, at and 00. The peak area of the rungs from PCR product decreased with increase in code position. We found for the first time that the logarithmic plots of the peak area against the code position showed a linear relationship, which implied that peak areas decrease exponentially. The present method was successfully applied to identify 37 individuals using only a hair root as a biological specimen. This "exponential law" is expected to be an effective tool in forensic science.