Mechanically driven ATP synthesis by F1-ATPase

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.     

Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.     

Manipulation of elementary charge in a silicon charge-coupled device

The ultimate limit in the operation of an electronic device is the manipulation of a single charge. Such a limit has been achieved in single-electron tunnelling devices. However, these devices are based on multiple tunnel barriers and conductive islands, which are complex structures to fabricate. Here we demonstrate another type of device that can also manipulate elementary charge, but which is more suitable for large-scale integration. The device consists of two closely packed silicon wire-MOSFETs, which are commonly used building blocks of electronic circuits. We have developed a scheme to generate and store holes in the channels of either of these MOSFETs. Subsequently, holes can be transferred between the two MOSFETs at the level of an elementary charge, and their exact position can be monitored. This single-charge transfer device, which is operated at 25 K, is in effect a charge-coupled device. This is also the first realization of a silicon-based device that manipulates elementary charge.     

The role of presenilin cofactors in the gamma-secretase complex

Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). Presenilin is essential for gamma-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and beta-amyloid precursor protein (betaAPP). Cleavage of betaAPP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid beta42 peptides. gamma-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (refs 15-18) and PEN-2 (refs 16, 19). However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Here we show that Drosophila APH-1 (Aph-1) increases the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex.     

Vegetation Spatial Heterogeneity of Different Soil Regions in Inner Mongolia, China

The vegetation spatial heterogeneity and ecological characteristics in different soil regions were analyzed by surveying the vegetation in 12 different soil regions of Inner Mongolia, China, including conifer- ous-broadleaf deciduous forests, shrub, grassland, and desert regions with 1122 large 2 cm × 2 cm quad- rats (actual size 30 km × 30 km, referred to as L-quadrat hereafter) in about 1.18 million km2. Each L- quadrat was divided into four small 1 cm × 1 cm quadrats (actual size 15 km × 15 km, S-quadrat). The vegetation was analyzed based on the beta-binomial distribution to describe the frequency of occurrence and spatial heterogeneity for each kind of vegetation. The weighted average of the heterogeneity of all vegeta- tion in the same soil region provides a measure of the soil regional landscape level heterogeneity which de- scribes the spatial complexity of the regional landscape composition of the existing vegetation. Comparison of the vegetation characteristics in the 12 soil regions shows that, the calcic gray soil has the highest average vegetation type per quadrat. The largest soil region is calcic chestnut soil and has the most vegetation types. Every soil region has its own dominant vegetation sequence which dominates in occurrence and dominant vegetation types which dominates in spatial heterogeneity. For the Inner Mongolian vegetation, the weighted average of the heterogeneity is 0.60 and the vegetation diversity index is 4.47.     

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of alpha 2 beta gamma delta, and this is thought to be also the case for the mammalian skeletal muscle AChR. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four subunits of the Torpedo californica electroplax and calf muscle AChR and of the alpha- and gamma-subunits of the human muscle AChR; the primary structures of the gamma-subunit of the T. californica AChR and the alpha-subunit of the Torpedo marmorata AChR have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the epsilon-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the gamma-subunit than with the other subunits. cDNA expression studies indicate that the calf epsilon-subunit, as well as the calf gamma-subunit, can replace the Torpedo gamma-subunit to form the functional receptor in combination with the Torpedo alpha-, beta- and delta-subunits.     

Location of functional regions of acetylcholine receptor alpha-subunit by site-directed mutagenesis

The availability of cloned cDNAs encoding the four subunits of the Torpedo acetylcholine receptor, which can be expressed to make functional receptors in Xenopus oocytes, has made possible a detailed investigation of the functions of the different structural components of the receptor. The functional analysis of receptors with alpha-subunits altered at specific sites by site-directed mutagenesis of the cDNA has allowed the location of specific regions of the alpha-subunit molecule involved in acetylcholine binding and forming a transmembrane ionic channel.     

Formation of carthamin by a partially purified enzyme from safflower seedlings

Summary An enzyme responsible for synthesizing carthamin from precarthamin was partially purified and the catalytic properties were investigated.Acknowledgments. The authors wish to thank Prof. A. Komamine, University of Tokyo, for reading this paper. We are indebted to Prof. A. Saito, Tokai University, for performing emission spectrochemical analysis of the enzyme protein used in this study.