Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system

To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.     

Decreased thermodynamic stability as a crucial factor for familial amyloidotic polyneuropathy

A single mutation in the wild-type transthyretin (WT TTR) such as V30M causes a familial amyloidotic polyneuropathy disease. Comparison of the three-dimensional crystal structures of WT and V30M does not tell much about the reason. High-pressure NMR revealed that at neutral pH both WT and V30M exist as equilibrium between the native tetramer and the dissociated/unfolded monomer. The native tetramer is highly stable in WT (deltaG(0)=104 kJ/mol at 37 degrees C, pH 7.1), but the stability is significantly reduced in V30M (deltadeltaG(0)=-18 kJ/mol), increasing the fraction of the unfolded monomer by a 1000-fold. Significant reduction of thermodynamic stability of WT TTR by mutation could be a crucial factor for familial amyloidotic polyneuropathy.     

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.     

Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls

Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Measurement of bisphenol A in human urine using liquid chromatography with multi-channel coulometric electrochemical detection

Smith-Lemli-Opitz syndrome (SLOS) patients have increased 7- and 8-dehydrocholesterol (DHC) concentrations. Using gas chromatography-mass spectrometry with selected ion monitoring we investigated whether storage time (24 h, 7 and 30 days, and 22 months at room temperature or at 4 degrees C) affected DHC concentrations in whole blood spots (WBSs) from SLOS patients and normal controls. Our results suggest that WBS sterol analysis can be used for SLOS screening and possibly related inborn errors of sterol metabolism with a 100% sensitivity and specificity on specimens stored for up to 30 days, either at room temperature or 4 degrees C. After 22 months of storage at both temperature SLOS samples can be indistinguishable from control samples. Therefore, great caution should be used to exclude SLOS by sterol analysis of WBSs stored for a long time.     

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis> with proteome analysis

RecombinantEscherichia coli strain harboring the λp _R-p _L promotor and heterologus poly-β-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression ofphb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lactate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.     

Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum

Microtubule dynamics in Paramecium caudatum were investigated with an anti-alpha-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-alpha-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.     

A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax

A multiplex PCR was designed for the differential diagnosis of the two parasite species by targeting the 18S rRNA gene with a set of primer combinations, amplifying DNA fragments of 1451-bp and 833-bp for P. falciparum and P. vivax, respectively. The sensitivity of this PCR test was high, as minimal as 0.1 parasite per one microliter of blood sample and a minimum of four copies of the target gene could be detected. For the diagnosis of mixed infection of two Plasmodium spp., there were no apparent competition or cross-reaction between the majority and minority Plasmodium species. The multiplex PCR was evaluated on 210 clinical samples and 60 normal controls. The PCR test yielded highly concordant results with microscopic examination, with the only one exception of a mixed (P. falciparum plus P. vivax) infection case, which was diagnosed as a single infection of P. falciparum by microscopy. We propose that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting P. falciparum and P. vivax.