Functional Structure of the Oxygen-Evolving Unit of Photosystem II as Determined by Radiation Inactivation

The size of the complex that is essential for the electron-transferactivity from the oxygen-evolving center to the secondary electronacceptor, Q_B, is about 250 kDa, as determined by target-sizeanalysis after the radiation inactivation of functions of photosystemII (PS II). Inter-Chl tranfer of excitation energy was insensitiveto the radiation inactivation indicating that the masses ofCP47, CP43, and light-harvesting Chi a/b proteins are not includedin the functional size of the oxygen-evolving PS II complex.The transfer of electrons from the secondary electron donor,Z, to Q_B was catalyzed by a unit of only 65 kDa. The sizes ofthe complexes involved in these light-induced functions of PSII were dependent on the intensity of actinic light. Under saturatingintensities of light, the functional size of the complex fortransfer of electrons from Z to Q_B was 38 kDa, with a correspondingdecrease in the size of the oxygen-evolving PS II from 250 kDato 125 kDa [Takahashi, Mano and Asada (1985) Plant Cell Physiol.26: 383]. The protein of about 30 kDa functions in the photoreductionof the pheophytin molecule, as well as in the electron transferfrom Z to Q_A. Under low-intensity light, complexes having thesame sizes as those of the basal functional complexes undersaturating-intensity light are further required, probably tostabilize separated charges in the PS II reaction center andthe oxygen-evolving center. (Received June 20, 1990; Accepted September 18, 1990)     

Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor-Like Protein 2

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.     

Persistent Translocation and Inhibition of Ca2+/Calmodulin-Dependent Protein Kinase II in the Crude Synaptosomal Fraction of the Vulnerable Hippocampus Following Hypoglycemia

Abstract: Alterations in the levels and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) were studied in the rat hippocampus during and after insulin-induced hypoglycemic coma. A permanent loss of CaM-kinase II immunohistostaining in the neuronal layer begins at 10 min of isoelectricity in the tip of the dentate gyrus and at 30-min isoelectricity in the CA1 region. The reduction in immunohistostaining in the neurites is less pronounced. Immunoreactivity of CaM-kinase II on western blots increases in the crude synaptosomal fractions and decreases in cytosolic fraction, indicative of a translocation of CaM-kinase II. The translocation persists for at least 1 day of recovery after 30 min of isoelectricity in the vulnerable hippocampus (dorsomedial hippocampus) but not in the resistant hippocampus (dorsolateral hippocampus). Calmodulin binding to western blots shows changes similar to the immunoblots. Ca²⁺/calmodulin-dependent activity of CaM-kinase II in the crude synaptosomal fraction is elevated immediately before isoelectricity and is then inhibited during and after 30 min of isoelectricity, despite the increase of CaM-kinase II immunoreactivity. This was seen in the vulnerable hippocampus. The data indicate that stimulus of translocation and inhibition of CaM-kinase II persist during the recovery phase, preceding neuronal degeneration in the vulnerable hippocampus. This may be of significance for hypoglycemia-induced neuronal death.     

Cutaneous vasodilatation responses synchronize with sweat expulsions

To examine whether cutaneous active vasodilatation is mediated by sudomotor nerve fibres we recorded cutaneous blood flow and sweat rates continuously with laser-Doppler flowmetry and capacitance hygrometry, respectively, from the dorsal and plantar aspects of the foot in 11 male subjects at varying ambient temperatures (T _a) between 22 and 40°C (relative humidity 40%). In a warmer environment (T _a 29–40°C), predominant responses of the blood flow curve from the sole of the foot were transient depressions (negative blood flow responses, NBR), whereas those from the dorsal foot were transient increases (positive blood flow responses, PBR). The PBR on the dorsal foot occurred spontaneously or in response to mental or sensory stimuli, and when PBR did not fuse with each other the rate of PBR was linearly related to tympanic temperature. When dorsal foot sweating was continuous, PBR on the dorsal foot almost entirely synchronized with sweat expulsion. When dorsal foot sweating was intermittent PBR sometimes occurred on the dorsal foot without corresponding sweat expulsions, but these PBR showed a complete correspondence with subthreshold sweat expulsion seen on a methacholine-treated area. The amplitude and the duration of PBR showed a significant linear relationship with the amplitude and the duration of the corresponding sweat expulsion. In a thermoneutral or cooler environment (T _a 22–29°C), PBR occurred on the sole of the foot when mental or sensory stimuli elicited sweating in that area. Thus, PBR occurred when and where sweating appeared. Atropine failed to abolish PBR on the dorsal foot. Blockade of the peroneal nerve eliminated both PBR and NBR on the dorsal foot. The results indicate that an active vasodilatation mechanism is present on the sole of the foot as well as on the dorsal foot, and thus suggest that active vasodilatation is closely related to sudomotor nerve activation.     

Estimation of leaf area index and gap fraction in two broad-leaved forests by using small-footprint airborne LiDAR

In this study, we evaluated methods for reliably estimating leaf area index (LAI) and gap fraction in two different types of broad-leaved forests by the use of airborne light detection and ranging (LiDAR) data. We evaluated 13 estimation variables related to laser height, laser penetration rate, and laser point attributes that were derived from LiDAR analyses. The relationships between LiDAR-derived estimates and field-based measurements taken from the forests were evaluated with simple linear regressions. The data from the two forests were analyzed separately and as an integrated dataset. Among the laser height variables, the coefficient of variation (CV) of all laser point heights had the highest level of accuracy for estimating both LAI and gap fraction. However, we recommend that more evaluations be conducted prior to the use of CV in forests with complex structures. The simplest laser penetration variable, which represents the ratio of the number of ground points to the total number of all points (P _ALL), also had a high level of accuracy for estimating LAI and gap fraction at the study sites regardless of whether the data were analyzed separately or as an integrated data set. Furthermore, P _ALL values showed near 1:1 relationships with the field-based gap fraction values. Hence, the use of P _ALL may be the most practical for estimating LAI and gap fraction in broad-leaved forests, even when the canopies are heavily closed.     

Growth and development, and auxin polar transport of transgenic Arabidopsis under simulated microgravity conditions on a three-dimensional clinostat.

Growth and development, and auxin polar transport in Arabidopsis thaliana transformed with iaaH gene were studied under simulated microgravity conditions on a three-dimensional (3-D) clinostat. Simulated microgravity conditions on a 3-D clinostat did not affect the number of rosette leaves but promoted the growth and development (fresh weight of plant and the elongation of flower stalk) of transformants. Final growth of transformants under simulated microgravity conditions on a 3-D clinostat was almost equivalent to that grown on 1 g conditions in the presence of 1 micromoles IAM (indole-3-acetamide). The activities of auxin polar transport in the segments of flower stalk (inflorescence axis) of transformants grown on 1 g conditions were significantly promoted by the addition of IAM. Interestingly, simulated microgravity conditions on a 3-D clinostat also promoted the activities of auxin polar transport of transformants grown on the medium with or without IAM. Based on the results in this study, transgenic plants may not have an efficient homeostatic mechanism for the control of growth and development, and auxin polar transport activity in microgravity conditions in space.     

Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate

Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate.     

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2′-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-μmol scale corresponds to 1mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.