Does the Upstream Region Possessing MULE-Like Sequence in Rice Upregulate PsbS1 Gene Expression?

The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.     

Brain-derived neurotrophic factor receptor TrkB exists as a preformed dimer in living cells

Background

Neurotrophins (NTs) and their receptors play crucial roles in the development, functions and maintenance of nervous systems. It is widely believed that NT-induced dimerization of the receptors initiates the transmembrane signaling. However, it is still controversial whether the receptor molecule has a monomeric or dimeric structure on the cell surface before its ligand binding.

Findings

Using chemical cross-linking, bimolecular fluorescence complementation (BiFC) and luciferase fragment complementation (LFC) assays, in this study, we show the brain-derived neurotrophic factor (BDNF) receptor TrkB exists as a homodimer before ligand binding. We have also found by using BiFC and LFC that the dimer forms in the endoplasmic reticulum (ER), and that the receptor lacking its intracellular domain cannot form the dimeric structure.

Conclusions

Most, if not all, of the TrkB receptor has a preformed, yet inactive, homodimeric structure before BDNF binding. The intracellular domain of TrkB plays a crucial role in the spontaneous dimerization of the newly synthesized receptors, which occurs in ER. These findings provide new insight into an understanding of a molecular mechanism underlying transmembrane signaling mediated by NT receptors.     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

ABSTRACT: BACKGROUND: The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. RESULTS: We identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks. CONCLUSIONS: This list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.     

Further purification of a fibroblast growth factor-like factor from chick embryo extract by heparin-affinity chromatography

Summary A mitogenic factor which promotes quail myoblast proliferation has been purified some 10⁵-fold from chick embryo extract by a combination of cation-exchange chromatography and heparin-affinity chromatography. The factor is eluted from heparin-Sepharose with 2M NaCl and is a single-chain polypeptide with an apparent molecular weight of 15000 to 17000. It is active at subnanogram level in triggering the proliferation and thereby delaying temporarily fusion of myoblasts. It also stimulates the proliferation of quail fibroblasts in a similar effective concentration range. For both myoblasts and fibroblasts the dose-response to the factor is quantitatively and qualitatively comparable with that of bovine pituitary fibroblast growth factor. These observations strongly suggest that the factor very probably corresponds to chicken fibroblast growth factor or to a closely related molecule(s) and that it is possibly involved in the regulation of myogenesis. This work was partly supported by a grant from the National Center of Neurology and Psychiatry (NCNP grant 86-01) of the Ministry of Health and Welfare, Japan.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5°C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P_β') and liquid-crystalline (L_α) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of ~100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P_β'(mst), differing from the initial P_β' was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P_β'(mst) into P_β' occurs for over 24 h but only by way of the phase sequence P_β'(mst) → L_β' → P_β'. According to differential scanning calorimetry (DSC), the enthalpy of the P_β'(mst)-L_α transition is by ~5% lower than that of the P_β'-L_α transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3°C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L_c) to its gel (L_β') phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a “sequential” relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one.     

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.     

Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)