Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

Summary To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N⁶-benzyladenine (BA) per L, and 0.8% Noble agar). The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3–4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.     

An endoplasmic reticulum (ER) stress-suppressive compound and its analogues from the mushroom Hericium erinaceum

Three new compounds, 3-hydroxyhericenone F (1), hericenone I (2), and hericenone J (3), were isolated from the mushroom Hericium erinaceum. The structures of 1-3 were determined by the interpretation of spectral data. Compound 1 showed the protective activity against endoplasmic reticulum (ER) stress-dependent Neuro2a cell death, however, compounds 2 and 3 did not.     

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of ��-�� Subunit Interactions in the Catalytic Dwell

The temperature-dependent rotation of F₁-ATPase γ subunit was observed in V_max conditions at low viscous drag using a 60-nm gold bead (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126–4131). The Arrhenius slopes of the speed of the individual 120° steps and reciprocal of the pause length between rotation steps were very similar, indicating a flat energy pathway followed by the rotationally coupled catalytic cycle. In contrast, the Arrhenius slope of the reciprocal pause length of the γM23K mutant F₁ was significantly increased, whereas that of the rotation rate was similar to wild type. The effects of the rotor γM23K substitution and the counteracting effects of βE381D mutation in the interacting stator subunits demonstrate that the rotor-stator interactions play critical roles in the utilization of stored elastic energy. The γM23K enzyme must overcome an abrupt activation energy barrier, forcing it onto a less favored pathway that results in uncoupling catalysis from rotation.F-ATPase (F_oF₁), consisting of the catalytic sector F₁ (α₃β₃γδϵ) and the transmembrane proton transport sector F_o (ab₂c₁₀), synthesizes or hydrolyzes ATP coupled with proton transport (for reviews, see Ref. 1–6). As Abrahams et al. (7) discovered in the first high resolution x-ray structure, a critical feature of the F₁-ATPase is the inherent asymmetry of the three β subunits in different conformations, β_TP, β_DP, and β_E, referring to the nucleotide bound in each catalytic site, ATP, ADP, or empty, respectively. A rotational mechanism has been firmly established mostly based on direct observation in single molecule experiments of the behavior of the rotor complex ϵγc₁₀, relative to the stator complex α₃β₃δab₂ (reviewed in Ref. 1). ATP hydrolysis-dependent rotation of the γ and ϵ subunits in purified bacterial F₁ (8, 9), the ϵγc₁₀ complex in detergent solubilized F_oF₁ (10–13), and the ϵγc₁₀ complexin F_oF₁ in lipid bilayers (14) were shown experimentally by single molecule observations using fluorescent actin filament as a probe. Relative rotation of the single copy F_o a subunit was also shown in F₀F₁, which was immobilized through the ring of ∼10 c subunits, suggesting that the rotor and stator are interchangeable mechanical units (14). ATP synthesis by F-ATPase is believed to follow the reverse mechanism of ATP hydrolysis because mechanically induced rotation of the γ subunit in immobilized F₁ in the presence of ADP and P_i results in net ATP synthesis (15, 16). There remain many questions about the mechanism of coupling between catalysis and transport via mechanical rotation. In particular, the mechanism of coupling H⁺ transport to rotation of the subunit c₁₀ ring is still not well understood (4).In contrast, there is considerably more information on the mechanism of coupling catalysis to γ and ϵ subunit rotation. Observations of γ subunit rotation in the catalytic F₁ sector are consistent with Boyer''s binding change model (17); thus coupling between the chemistry and rotation can be assessed by studies of the soluble F₁, and these findings relate to the mechanism of the entire ATP synthase complex. The γ subunit rotates relative to the α₃β₃ hexamer in distinct 120° steps. A 120° rotation step consisting of pause and rotation substeps appears to correspond to the hydrolysis of one ATP, assuming that three ATP molecules are hydrolyzed per 360° revolution (18). Additional pauses observed at low ATP concentrations are attributed to the “ATP waiting” dwell (19). Yasuda et al. (19) and Shimabukuro et al. (20) further resolved that each 120° step occurred in two substeps: an 80° substep whose onset was dependent upon the Mg·ATP concentration, and a 40° substep, which was not affected by substrate concentration (19). The pause before the 80° substep, the ATP waiting dwell became shorter with increasing [Mg·ATP]. In contrast, the pause duration before the 40° rotation step was modulated by the slow hydrolysis rate of ATPγS² or by the catalytic site mutant βE190D (in the Bacillus PS3 F₁), which was found to significantly increase the length of the catalytic dwell (20). These data together indicate that the dwell before the 40° step is the “catalytic dwell” (20) and defines the order of the substeps during the 120° rotation step observed in high Mg·ATP concentrations (21).In this paper, we address the question of when the rate-limiting step of steady state catalysis occurs, with respect to the rotational behavior. Pre-steady state analysis of the burst kinetics of ATP hydrolysis at nearly V_max conditions demonstrated that the rate-limiting transition state occurs after the reversible hydrolysis/synthesis step and before release of phosphate (P_i) (22, 23). The rate-limiting step is likely associated with a rotation step because a γ-β cross-linked enzyme is still able to undergo the initial ATP hydrolysis, but the rotation-impeded enzyme is unable to release P_i (23). Significantly, the kinetics of steady state hydrolysis can only be assessed when the Mg·ATP concentration is high enough to fill all three catalytic sites. The only model consistent with these data is one that involves all three catalytic sites. During each 120° catalytic cycle, one site binds ATP, a different site carries out reversible hydrolysis/synthesis, and the third site releases product P_i and ADP (22, 23).Steady state analyses, which take advantage of a particular γ subunit mutation γM23K (24), are consistent with this model. Replacement of the conserved γMet-23 with lysine causes an uncoupling between catalysis and γ subunit rotation caused by altered interactions between γ and β subunits (25). Importantly, Al-Shawi and Nakamoto (26) and Al-Shawi et al. (25, 27) found that the γM23K mutation strongly affected the rate-limiting transition state of steady state ATP hydrolysis and ATP synthesis. The slope of the Arrhenius plots and thus the energy of activation were significantly increased in the mutant enzyme. Several second site suppressor mutations, mostly in the γ subunit (28, 29) but also in the β subunits (30, 31), were genetically identified because they restored coupled ATP synthesis. Significantly, all were in the γ-β interface. Thermodynamic analyses found that the second site suppressors generally compensated for the primary γM23K mutations by reducing the increased activation energy (25, 27, 31). Although most of the second site mutations were found distant from the γM23K site, the x-ray crystal structures (7) suggested that γM23K may directly interact with conserved βGlu-381. As expected, replacement of βGlu-381 with aspartate also suppressed the uncoupling effects of γM23K (31).To identify the rate-limiting transition state step in the rotational behavior, we analyzed the temperature dependence of the γM23K mutant in V_max conditions observed in single molecule experiments. Interestingly, direct observation of this mutant using the micron-length actin filaments did not detect differences in the rotation behavior at room temperature (9). In contrast, we find in the data presented here that there is dramatic effect of the mutation on the temperature dependence of the length of the catalytic dwell or pause between the 120° rotation steps. This is likely because of two factors: first, we used a bead small enough not to invoke a drag on the rotation (32), and second, the temperature dependence of the rate of the rotation steps is critical for the analyses of the mechanism.     

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.     

NADP-Dependent Alcohol Dehydrogenase from Tea Seeds

d-Coronamic acid was deaminated by 1-aminocyclopropane-1-carboxylate (ACPC) deaminase to produce α-keto-n-caproic acid. This deaminase which was purified from Pseudomonas sp. ACP was active to only d-coronamic acid among its stereoisomers. l-Coronamic acid or dl-allocoronamic acid was inactive or negligibly poor as the substrate. In addition, both deamination of ACPC and d-coronamic acid were inhibited by l-alanine, not by d-isomer and the inhibition of ACPC deamination by l-alanine was competitive. On the basis of these results, stereoselectivity of the enzymatic deamination was discussed.     

Hydrodynamic interactions between two swimming bacteria

This article evaluates the hydrodynamic interactions between two swimming bacteria precisely. We assume that each bacterium is force free and torque free, with a Stokes flow field around it. The geometry of each bacterium is modeled as a spherical or spheroidal body with a single helical flagellum. The movements of two interacting bacteria in an infinite fluid otherwise at rest are computed using a boundary element method, and the trajectories of the two interacting bacteria and the stresslet are investigated. The results show that as the two bacteria approach each other, they change their orientations considerably in the near field. The bacteria always avoided each other; no stable pairwise swimming motion was observed in this study. The effects of the hydrodynamic interactions between two bacteria on the rheology and diffusivity of a semidilute bacterial suspension are discussed.     

Reorganization of membrane ergosterol during cell fission events of Candida albicans: a freeze-fracture study of distribution of filipin-ergosterol complexes

The redistribution of ergosterol molecules which occurs during bud and germ tube formation (dimorphism) in Candida albicans was studied using filipin, a sterol-specific antibiotic, and examined by the freeze-fracture technique. When cells were fixed in a glutaraldehyde solution containing 50 micrograms/ml of filipin, filipin-ergosterol complexes, which were recognized as either pits on the exoplasmic fracture face or protuberances on the protoplasmic fracture face, were homogeneously distributed on the yeast plasma membranes. The plasma membrane of young budding yeast cells demonstrated few filipin-ergosterol complexes compared to the parent yeast plasma membrane. In addition, at a certain time during enlargement of budding yeast cells, the complexes became virtually absent from the constricted region between daughter and parent yeast cell. On the other hand, when germ tubes emerged as cylindrical outgrowths from the parent yeast cells, filipin-ergosterol complexes were heterogeneously redistributed on the plasma membrane. These results suggest that ergosterol molecules may be in lower concentration in the plasma membrane at the constricted region of yeast cell than elsewhere on the plasmalemma of the yeast cell.     

An HRP study in the monkey of olivary projections from the mesodiencephalic structures with particular reference to pretecto-olivary neurons

The cells of origin of the olivary projection from mesodiencephalic structures have been demonstrated in the Japanese monkey (Macaca fuscata) with the horseradish peroxidase (HRP) method. Particular attention has been paid to the pretecto-olivary projection which is entirely ipsilateral and originates from the nucleus of the optic tract (NOT), the posterior (or principal) pretectal nucleus (PPN), the sublentiform nucleus (SL), and the (pretectal) ventral lateral zone (VLZ). Pretecto-olivary cells are small-medium sizes of oval or fusiform types. To facilitate comparison among the findings of several experiments, a diagram of the macaque olive, as visualized unfolded, was constructed (Fig. 1). Although a topographic correlation has not emerged clearly from the present experiments, the pattern of the pretecto-olivary projection in the monkey appears to be similar to that found in the cat. Participation of some of the pretectal nuclei in the optokinetic nystagmus and the vestibulo-ocular reflex is discussed in connection with the pretecto-olivary and olivocerebellar projections.