CSP41, a sequence-specific chloroplast mRNA binding protein, is an endoribonuclease.

Correct 3' processing of chloroplast precursor mRNAs (pre-mRNAs) requires a stem-loop structure within the 3' untranslated region. In spinach, a stable 3' stem-loop-protein complex has been shown to form in vitro between petD pre-mRNA, encoding subunit IV of the cytochrome b6/f complex, and chloroplast proteins. This complex contains three chloroplast stem-loop binding proteins (CSPs), namely, CSP29, CSP41, and CSP55. Here, we report the purification of CSP41 and cloning of the csp41 gene and show that CSP41 is encoded by a single nuclear gene. Characterization of bacterially expressed CSP41 demonstrates that this protein binds specifically to the 3' stem-loop structure and a downstream AU-rich element of petD pre-mRNA and that its binding affinity is enhanced by associating with CSP55. Our data also show that CSP41 has substantial nonspecific endoribonuclease activity. These data suggest that CSP41 could be involved in 3' processing of petD pre-mRNA and/or in RNA degradation. The fact that different reaction conditions favor RNA binding over ribonuclease activities suggests a possible mode of in vivo regulation.     

Solution Structure and Backbone Dynamics of the Pleckstrin Homology Domain of the Human Protein Kinase B (PKB/Akt). Interaction with Inositol Phosphates

The programmed cell death occurs as part of normal mammalian development. The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 3'-OH kinase (PI3K)/Protein Kinase B (PKB, also named Akt) cascade. Several targets of the PI3K/PKB signaling pathway have been identified that may underlie the ability of this regulatory cascade to promote cell survival. PKB possesses a N-terminal Pleckstrin Homology (PH) domain that binds specifically and with high affinity to PtIns(3,4,5)P(3) and PtIns(3,4)P(2), the PI3K second messengers. PKB is then recruited to the plasma membrane by virtue of its interaction with 3'-OH phosphatidylinositides and activated. Recent evidence indicates that PKB is active in various types of human cancer; constitutive PKB signaling activation is believed to promote proliferation and increased cell survival, thereby contributing to cancer progression. Thus, it has been shown that induction of PKB activity is augmented by the TCL1/MTCP1 oncoproteins through a physical association requiring the PKB PH domain. Here we present the three-dimensional solution structure of the PH domain of the human protein PKB (isoform beta). PKBbeta-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH-domains, consisting of a beta-sandwich of seven strands capped on one top by an alpha-helix. The opposite face presents three variable loops that appear poorly defined in the NMR structure. Measurements of (15)N spin relaxation times and heteronuclear (15)N[(1)H]NOEs showed that this poor definition is due to intrinsic flexibility, involving complex motions on different time scales. Chemical shift mapping studies correctly defined the binding site of Ins(1,3,4,5)P(4) (the head group of PtIns(3,4,5)P(3)), as was previously proposed from a crystallographic study. More interestingly, these studies allowed us to define a putative alternative low-affinity binding site for Ins(1,4,5)P(3). The binding of this sugar to PKBbeta-PH might also involve non-specific association that could explain the stabilization of the protein in solution in the presence of Ins(1,4,5)P(3).     

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

Purpose

Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.

Methods

To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.

Results

Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.

Conclusion

Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.     

The psychophysiology of nausea

Nausea is an unpleasant sensation usually referred to the stomach and sometimes followed by vomiting. Little is known about the subjective aspects of nausea because like pain and fatigue, it is a private sensation. We conceive of nausea as a complex control mechanism that signals us when not to eat. Our research in the areas of motion sickness and chemotherapy has led us to propose that we each have a dynamic threshold for nausea, which depends on the interaction of inherent factors and more changeable psychological factors, and that this threshold effects the individual's cognitive appraisal of both the nauseogenic stimulus and his/her bodily change in response to the nauseogenic stimulus. Inherent factors that are described are age, gender and race; psychological factors that are included are anxiety, expectation, anticipation and adaptation. The physiological responses that have been found to accompany nausea include an increase in sympathetic nervous system activity, a decrease in parasympathetic activity, an increase of abnormal dysrhythmic gastric activity, and an increase in plasma vasopressin. It is concluded that beneficial selective reduction of nausea will depend on a greater knowledge of the interaction of the psychological and physiological variables.     

The acetylcholinesterase genes of C. elegans: identification of a third gene (ace-3) and mosaic mapping of a synthetic lethal phenotype

In C. elegans, the newly identified ace-3 is the third gene affecting acetylcholinesterase (AChE) activity. ace-3 II specifically affects class C AChE and is unlinked to ace-1 X or ace-2 I, which affect the other two AChE classes (A and B, respectively). Strains homozygous for an ace-3 mutation have no apparent behavioral or developmental defect; ace-1 ace-3 and ace-2 ace-3 double mutants are also nearly wild type. In contrast, ace-1 ace-2 ace-3 triple mutant animals are paralyzed and developmentally arrested; their embryonic development is relatively unimpaired, but they are unable to grow beyond the hatching stage. Based on the analysis of genetic mosaics, we conclude that in the absence of ace-2 and ace-3 function, the expression of ace-1(+) in muscle cells, but not in neurons, is essential for postembryonic viability.     

Role of the intercistronic region in post-transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences

The high-affinity histidine permease of Salmonella typhimurium is encoded by a four-gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30-fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra-operonic control of gene expression, we have isolated MudII-mediated lacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra-operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palindromic) sequences, which are located in the hisJ-hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3' to 5' nuclease degradation (in agreement with data presented by Newbury et al., 1987).     

Role of sodium in determining alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes

In contrast to the absolute Na(+) requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na(+). However, Na(+) (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na(+)-free medium possessed all the enzymes of the citric acid cycle including alpha-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na(+), which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na(+)-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Na(+) to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of alpha-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of alpha-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle.     

A Ddc2-Rad53 fusion protein can bypass the requirements for RAD9 and MRC1 in Rad53 activation

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a “mediator” protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Δrad9 or Δrad9Δmrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Δrad9Δmrc1 cells. In addition, expression of Ddc2-Rad53p in Δrad24Δrad17Δmec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.     

Effects of ginseng components on c-DNA-expressed cytochrome P450 enzyme catalytic activity

Because little is known about the interactions between herbal products and standard medications, the effects of seven ginsenosides and two eleutherosides (active components of the ginseng root) on the catalytic activity of c-DNA expressed cytochrome P450 isoforms were studied in in vitro experiments. Increasing concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 and eleutherosides B and E were incubated with a panel of recombinant human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit the metabolism of the surrogate substrates by 50%) was estimated and this value compared with that obtained for positive control inhibitory drugs furafylline, sulfaphenazole, tryanylcypromine, quinidine, and ketoconizole. Of the components tested, three ginsenosides (Rd, Rc, and Rf) modified the activity of the recombinant enzymes. Ginsenoside Rd produced weak inhibitory activity against the surrogate substrates for CYP3A4 and CYP2D6 and even weaker inhibitory activity against the surrogate substrates for CYP2C19 and CYP2C9. The IC50 values of 58 and 74 uM for the two substrates for CYP3A4 are orders of magnitude higher than that for the potent inhibitor ketoconazole used as a positive control. Ginsenoside Rc produced an increase in the activity of CYP2C9 (70% at 200 uM) and ginsenoside Rf produced an increase in the activity of CYP3A4 (54% at 200 uM). The biological significance of this is unclear at this time. Enzyme "activation", the process by which direct addition of one compound to an enzyme enhances the rate of reaction of the substrate, has been observed in a number of cases with P450 enzymes; however, a matrix effect caused by the test compound fluorescing at the same wavelength as the metabolite of the marker substrate cannot be ruled out. In summary, these studies suggest that the ginsenosides and eleutherosides tested are not likely to inhibit the metabolism of coadministered medications in which the primary route of elimination is via cytochrome P450; the potential of ginsenosides to enhance the catalysis of certain substrates requires further investigation.