The primary electron acceptor of green sulfur bacteria,bacteriochlorophyll 663, is chlorophyll a esterified with Δ 2,6-phytadienol

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), ¹H- and ¹³C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7¹ signal was observed at the high-field side of the singlet P3¹ signal in BChl 663, while a doublet peak of P7¹ overlapped that of P11¹ in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13²-epimer of BChl a, BChl a′, were found to be present per reaction center, which may constitute the primary electron donor. This revised version was published online in June 2006 with corrections to the Cover Date.     

RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes

Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that the Rho family of small G proteins regulates L-type Ca2+ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice that overexpress the specific GDP dissociation inhibitor Rho GDI-alpha exhibited significantly decreased basal L-type Ca2+ current density (approximately 40%) compared with myocytes from nontransgenic (NTG) mice. The Ca2+ channel agonist BAY K 8644 and the beta-adrenergic agonist isoproterenol increased Ca2+ currents in both NTG and TG myocytes to a similar maximal level, and no changes in mRNA or protein levels were observed in the Ca2+ channel alpha1-subunits. These results suggest that the channel activity but not the expression level was altered in TG myocytes. In addition, the densities of inward rectifier and transient outward K+ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitches and Ca2+ transients were also similar between the two groups. When the protein was delivered directly into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI-alpha significantly decreased Ca2+ current density, which supports the idea that the defective Ca2+ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant-negative RhoA but not a dominant-negative Rac-1 or Cdc42 also significantly decreased Ca2+ current density, which indicates that inhibition of Ca2+ channel activity by overexpression of Rho GDI-alpha is mediated by inhibition of RhoA. This study points to the L-type Ca2+ channel activity as a novel downstream target of the RhoA signaling pathway.     

Chronic endotoxin exposure does not cause sustained structural abnormalities in the fetal sheep lungs

Chronic early gestational chorioamnionitis is associated with development of bronchopulmonary dysplasia in preterm infants. A single intra-amniotic exposure to endotoxin decreased alveolarization and reduced expression of endothelial proteins in 125-day gestational age preterm lambs. We hypothesized that prolonged exposure to intra-amniotic endotoxin would cause progressive lung inflammation and inhibit alveolar and pulmonary vascular development. Endotoxin (1 mg/day) or saline was administered via an intra-amniotic osmotic pump from 80 to 108 days of gestational age (continuous pump) or by four weekly 10-mg intra-amniotic endotoxin injections starting at 100 days of gestational age (multiple dose). Lung morphometry, lung inflammation, vascular effects, and lung maturation were measured at delivery. The continuous pump lambs delivered at 100 days (approximately 70% of total endotoxin exposure) had lung inflammation, fewer saccules, and decreased endothelial proteins endothelial nitric oxide synthase and VEGF receptor 2 expression compared with controls. The continuous pump (delivered at 138 days) and multiple dose lambs (delivered at 130 and 145 days) had mild persistent lung inflammation and no significant differences in lung morphometry or expression of endothelial proteins compared with controls. Surfactant saturated phosphatidylcholine pool sizes were increased in all endotoxin-exposed groups, but lung function was not changed relative to controls. Contrary to our hypothesis, a prolonged fetal exposure to intra-amniotic endotoxin caused mild persistent inflammation but did not lead to progressive structural abnormalities in lungs of near-term gestation lambs.     

Reversibility of lung inflammation caused by SP-B deficiency

Whereas decreased concentrations of surfactant protein (SP)-B are associated with lung injury and respiratory distress, potential causal relationships between SP-B deficiency and lung inflammation remain unclear. A transgenic mouse in which human SP-B expression was placed under conditional control of doxycycline via the CCSP promoter was utilized to determine the role of SP-B in the initiation of pulmonary inflammation. Adult mice, made SP-B deficient by removal of doxycycline, developed severe respiratory failure within 4 days. Deficiency of SP-B was associated with increased minimal surface tension of the surfactant and perturbed lung mechanics. Four days of SP-B deficiency did not alter SP-C content or surfactant phospholipid content or composition. SP-B deficiency was associated with lung inflammation and increased soluble L-selectin, STAT-3, and phosphorylated STAT-3 in alveolar macrophages and alveolar epithelial cells. Alveolar IL-6, IL-1beta, and macrophage inflammatory protein-2 concentrations were increased after removal of doxycycline, indicating pulmonary inflammation. Restoration of SP-B expression following administration of doxycycline rapidly reversed SP-B-dependent abnormalities in lung mechanics and inflammation. SP-B deficiency is sufficient to cause lung dysfunction and inflammation in adult mice. SP-B reversed inflammation and maintained lung function in vivo, indicating its potential utility for the prevention and treatment of pulmonary injury and surfactant deficiency.     

Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication

Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.     

Allometry and life history of a forest understory palm <Emphasis Type="Italic">Pinanga coronata</Emphasis> (Arecaceae) on Mount Halimun,West Java

We studied the allometry and life history of an understory palm Pinanga coronata (Arecaceae) in a tropical montane rainforest on Mount Halimun, West Java, Indonesia. When the palm was small, the diameter of the stem increased with the minimum amount of vertical growth needed for new leaves to emerge (juvenile stage). After the base diameter of the stem and the length of the leaves reached a sufficient size (3.5cm and 207.7cm, respectively), the palm started to elongate the stem vertically (adult stage). The total mass and area of leaves in the crown increased with increasing stem mass, and approached constant values asymptotically. At the adult stage, the palm increased specific gravity of the lower part of the stem as it grew taller to support the increasing mass of crown and stem. To avoid self-shading, crown architecture changed as the plant grew larger; leaf blades became longer and narrower and petiole length increased to display leaves efficiently away from the stem. The palm also produced clonal sprouting shoots and formed small clumps. Each clump contained an average of 7.5 shoots. The spatial distribution of the palm was strongly affected by topography, but only minimally affected by light conditions. The architectural adaptation to shaded conditions, effective dispersal of fruits and clonal growth are likely to be the factors that allow P.coronata to be one of the most dominant understory plant species in the study area.     

Profile of the oppositely acting enantiomers of the dihydropyridine 202-791 in cardiac preparations: receptor binding, electrophysiological, and pharmacological studies

Receptor binding, electrophysiological, and inotropic effects of the pure dihydropyridine enantiomers (+)S202-791 and (-)R202-791 were studied in cardiac preparations. The KI for (+)S202-791 binding correlated with the ED50's for an increase in contractile force and an increase in calcium current, the latter effect occurring at depolarized as well as resting holding potentials. The KI for (-)R202-791 binding was much lower than the IC50's for inhibition of calcium current measured at holding potentials of -80 or -90 mV and a negative inotropic effect, but correlated closely with the IC50 for inhibition of calcium current measured at -30 mV. Thus, (+)S202-791, is a voltage independent calcium channel activator and (-)R202-791 is a voltage dependent calcium channel inhibitor.     

The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. Evidence for direct regulation independent of phosphorylation by cAMP-dependent protein kinase or stimulation by a dihydropyridine agonist

We demonstrated recently that purified preparations of Gs, the stimulatory G protein of adenylyl cyclase, can stabilize Ca2+ channels in inside-out cardiac ventricle membrane patches stimulated prior to excision by the beta-adrenergic agonist isoprenaline or by the dihydropyridine agonist Bay K 8644 and that such preparations of Gs can restore activity to spontaneously inactivated cardiac Ca2+ channels incorporated into planar lipid bilayers (Yatani, A., Codina, J., Reeves, J.P., Birnbaumer, L., and Brown, A.M. (1987) Science 238, 1288-1292). To test whether these effects represented true stimulation and to further identify the G protein responsible, we incorporated skeletal muscle T-tubule membranes into lipid bilayers and studied the response of their Ca2+ channels to G proteins, specifically Gs, and manipulations known to be specific for Gs. In contrast to cardiac channels, incorporated T-tubule Ca2+ channels exhibit stable average activities over prolonged periods of time (up to 20 min at room temperature), allowing assessment of possible effects of G proteins under steady-state assay conditions. We report that exogenously added human erythrocyte GTP gamma S (guanosine 5'-O-(3-thiotriphosphate]-activated Gs (Gs) or its resolved GTP gamma S-activated alpha subunit (alpha s) stimulate T-tubule Ca2+ channels by factors of 2-3 in the presence of Bay K 8644, and of 10-20 in the absence of Bay K 8644 and that they do so in a manner that is independent of concurrent or previous phosphorylation by cAMP-dependent protein kinase. Activation of purified Gs by cholera toxin increases both its adenylyl cyclase stimulatory and its Ca2+ channel stimulatory effects. Ca2+ channels previously stimulated by the combined actions of Bay K 8644 and cAMP-dependent protein kinase still respond to Gs. We conclude that the responses seen are due to Gs rather than a contaminant, that the effect on Ca2+ channel activity is that of a true stimulation, akin to that on adenylyl cyclase, and show that a given G protein may regulate more than one effector system.