Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) in photosystem II

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10 days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, flourescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.     

Competitive dominance of the cyanobacterium Microcystis aeruginosa in nutrient-rich culture conditions with special reference to dissolved inorganic carbon uptake

The aim of the present study was to determine what factors contribute to the competitive advantage of cyanobacteria in eutrophic conditions. Mixed species batch culture experiments were conducted at three pHs (8.2, 8.8, 10.2) and irradiances (30, 90, 180 μmol photons m^?2 s^_1) between Microcystis aeruginosa Kützing and Staurastrum dorsidentiferum W. et West or Synedra ulna (Nitzsch) Ehrenberg, which always resulted in the dominance of M. aeruginosa. The final yields of competitors were often significantly lower than when cultured singly. The dominance of the surface‐loving M. aeruginosa appears to be related to its advantageous C0₂ uptake. To clarify the importance of dissolved inorganic carbon (DIC) as a selective factor, the effect of aeration was examined with M. aeruginosa. The growth of M. aeruginosa quickly stopped in nonaerated conditions, while it continued growing throughout the culture when aerated. This result indicates that DIC limitation easily occurs in static conditions and the viewpoint of C0₂ exchange efficiency would be helpful in discussing the competitive advantage of M. aeruginosa. When the three species were cultured at various ratios of surface area to volume (s/v), postulating it as an index of the gas exchange efficiency, the increases in population densities strongly correlated with s/v, and the relationships between the specific growth rate and s/v corresponded well to a Monod type saturation function. We found that M. aeruginosa had the lowest half‐saturation constant among the three, reflecting its high affinity for DIC.     

Chemistry and biology of the compounds that modulate cell migration

Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. Extensive studies have attempted to reveal the molecular mechanisms behind cell migration; however, they remain largely unclear. Bioactive compounds that modulate cell migration show promise as not only extremely powerful tools for studying the mechanisms behind cell migration but also as drug seeds for chemotherapy against tumor metastasis. Therefore, we have screened cell migration inhibitors and analyzed their mechanisms for the inhibition of cell migration. In this mini-review, we introduce our chemical and biological studies of three cell migration inhibitors: moverastin, UTKO1, and BU-4664L.     

Multiplex PCR/liquid chromatography assay for screening of subtelomeric rearrangements

In idiopathic or nonspecific mental retardation, the overall rate of cryptic subtelomeric rearrangements is estimated to be about 5%. Development of cost-effective screening for subtelomeric deletions would help clinical geneticists to make specific diagnoses in children with idiopathic mental retardation. Current screening modalities include fluorescence in situ hybridization (FISH) using subtelomeric probes and PCR-based quantitative analyses. Reductions in the cost and turnaround time will make the complete screening of subtelomeric rearrangements more widely used in clinical settings. Recently, a versatile method, called the multiplex PCR/liquid chromatography assay (MP/LC), was developed to assess copy numbers in this assay. Multiple genomic regions are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography. In the present study, we developed an MP/LC-based subtelomeric screening system that involves 21 multiple reactions and validated the protocol by analyzing 16 publicly available cell lines with known cytogenetic abnormalities involving at least one subtelomere per patient. To confirm the validity of the MP/LC method, we analyzed these cell lines concurrently with array-based comparative genomic hybridization (array-CGH), which gives higher resolution than the conventional G-banding technique. Among those 16 samples, the results from MP/LC and array-CGH agreed with each other perfectly. In 2 of the 16 samples, MP/LC correctly revealed subtelomeric duplications that were detected by array-CGH but were undetected by conventional cytogenetics, demonstrating the sensitivity of the MP/LC assay. This system is expected to be useful for making specific diagnoses and in genetic counseling for children with idiopathic mental retardation, a sizable fraction of whom have subtelomeric rearrangements.     

Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.     

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.     

Annual Change of Primary Resistance to Clarithromycin among Helicobacter pylori Isolates from 1996 through 2008 in Japan

Background: Recent studies have shown that the combination of proton pump inhibitor, amoxicillin and clarithromycin is one of the best choices for Helicobacter pylori eradication therapy. However, increasing number of cases of H. pylori infection showing resistance to clarithromycin therapy has been reported and this is currently the main cause of eradication failure. We investigated the annual changes of the antimicrobial susceptibility to clarithromycin, amoxicillin and minocycline during a period of 12 years in Japan. Methods: This study comprised 3521 patients (mean age (SD), 55.4 (13.7) years‐old, 2467 males and 1054 females) positive for H. pylori as assessed by microaerobic bacterial culture from 1996 through 2008. All patients were previously untreated for H. pylori and were enrolled in the study to assess primary resistance to the three antibiotics. Results: The overall primary resistance to clarithromycin, amoxicillin and minocycline were 16.4%, (577/3521), 0.03% (1/3521) and 0.06% (2/3521), respectively. From1996 through 2004, the resistance rate to clarithromycin increased gradually to approximately 30% and then it remained without marked fluctuation since 2004. Analysis by gender showed a significant increase (p < .0001) in resistance rate to clarithromycin among females (217/1057, 20.6%) compared to males (360/2467, 14.6%). Analysis by age, disclosed significantly (p < .0001) higher resistance rate to clarithromycin in patients of more than 65‐years‐old compared to the younger population. Conclusions: The resistance rate of H. pylori infection to clarithromycin in Japan has increased gradually to approximately 30% from 1996 through 2004, and remained unchanged since 2004. Elderly and females were at high risk of having resistance to clarithromycin. Our results suggested that the level of clarithromycin resistance in Japan has now risen to the point where it should no longer be used as empiric therapy.