Extracellular Ca2+ transients affect poly-(R)-3-hydroxybutyrate regulation by the AtoS-AtoC system in Escherichia coli

Escherichia coli is exposed to wide extracellular concentrations of Ca2+, whereas the cytosolic levels of the ion are subject to stringent control and are implicated in many physiological functions. The present study shows that extracellular Ca2+ controls cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis through the AtoS-AtoC two-component system. Maximal cPHB accumulation was observed at higher [Ca2+]e (extracellular Ca2+ concentration) in AtoS-AtoC-expressing E. coli compared with their DeltaatoSC counterparts, in both cytosolic and membrane fractions. The reversal of EGTA-mediated down-regulation of cPHB biosynthesis by the addition of Ca2+ and Mg2+ was under the control of the AtoS-AtoC system. Moreover, the Ca2+-channel blocker verapamil reduced total and membrane-bound cPHB levels, the inhibitory effect being circumvented by Ca2+ addition only in atoSC+ bacteria. Histamine and compound 48/80 affected cPHB accumulation in a [Ca2+]e-dependent manner directed by the AtoS-AtoC system. In conclusion, these data provide evidence for the involvement of external Ca2+ on cPHB synthesis regulated by the AtoS-AtoC two-component system, thus linking Ca2+ with a signal transduction system, most probably through a transporter.     

Sequential Activation of Metabolic Pathways: a Dynamic Optimization Approach

The regulation of cellular metabolism facilitates robust cellular operation in the face of changing external conditions. The cellular response to this varying environment may include the activation or inactivation of appropriate metabolic pathways. Experimental and numerical observations of sequential timing in pathway activation have been reported in the literature. It has been argued that such patterns can be rationalized by means of an underlying optimal metabolic design. In this paper we pose a dynamic optimization problem that accounts for time-resource minimization in pathway activation under constrained total enzyme abundance. The optimized variables are time-dependent enzyme concentrations that drive the pathway to a steady state characterized by a prescribed metabolic flux. The problem formulation addresses unbranched pathways with irreversible kinetics. Neither specific reaction kinetics nor fixed pathway length are assumed. In the optimal solution, each enzyme follows a switching profile between zero and maximum concentration, following a temporal sequence that matches the pathway topology. This result provides an analytic justification of the sequential activation previously described in the literature. In contrast with the existent numerical approaches, the activation sequence is proven to be optimal for a generic class of monomolecular kinetics. This class includes, but is not limited to, Mass Action, Michaelis–Menten, Hill, and some Power-law models. This suggests that sequential enzyme expression may be a common feature of metabolic regulation, as it is a robust property of optimal pathway activation.     

Do Botanical Pesticides Alter the Structure of the Soil Microbial Community?

The effects of synthetic pesticides on the soil microbial community have been thoroughly investigated in the past mostly by culture-dependent methods and only few recent studies have used culture-independent approaches for this purpose. However, it should be noted that most of these studies have been conducted in microcosms where the soil microbial community is exposed to unrealistic concentrations of the pesticides, providing an unrealistic exposure scheme for soil microorganism. On the other hand, little is known regarding the potential impact of botanical pesticides on the soil microbial community. Therefore, a laboratory study and a field study were conducted to investigate the effects of synthetic (metham sodium [MS], sodium tetrathiocarbonate [SoTe], and fosthiazate) and botanical pesticides (azadirachtin, quillaja, and pulverized Melia azedarach fruits [PMF]) on the soil microbial community using phospholipid fatty acids (PLFA) analysis. Principal component analysis (PCA) on the results of the laboratory study indicated that the application of PMF resulted in significant changes in the soil microbial community. This was obvious by the proportional increase in the abundance of fatty acids 18:1ω9cis, 18:1ω9trans, which are common in gram-negative bacteria and saprotrophic fungi, and 18:2ω6,9, which is a fungal indicator. This response was attributed to the release of copious amounts of organic carbon and nutrients in the soil by the PMF. On the other hand, MS inhibited fungi and gram-negative bacteria, while fosthiazate and the botanical pesticides quillaja and azadirachtin did not impose significant changes in the soil microbial community. Similar results were obtained by the field study where application of the fumigants MS and SoTe significantly altered the structure of the soil microbial community with the former having a more prominent effect. Fosthiazate imposed mild changes in the soil microbial community, whereas quillaja and azadirachtin again did not show a significant effect. Overall, botanical pesticides, at their recommended dose, did not alter the structure of the soil microbial community compared to synthetic nonfumigant and fumigant pesticides which induced significant changes.     

Relationship of CSF neurotransmitter metabolite levels to disease severity and disability in multiple sclerosis

Axonal degeneration and brain tissue loss occur during disease progression in multiple sclerosis (MS) and are expected to influence neurotransmitter activities, with consequences on neurologic and psychiatric symptomatology. We searched for relationships of disease duration, disability, and severity of MS patients to CSF levels of the major metabolites of noradrenaline, dopamine, and serotonin, MHPG, methoxyhydroxyphenylglycol (MHPG), homovanillic acid, and 5-hydroxyindoleacetic acid (5-HIAA), respectively, in 39 patients with relapsing–remitting (RR) MS in remission, and 26 patients with progressive (PR) MS. Disability and Disease Severity were assessed by the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score (MSSS). Compared with the levels of 50 control subjects, MHPG levels were not different in either MS group, correlated negatively to duration of illness and number of relapses in the RRMS group, but not to EDSS score or to MSSS. Homovanillic acid levels were significantly lower only in the PRMS group, with a negative correlation to duration of illness, and a strong negative correlation to EDSS score, but not to MSSS. 5-HIAA was significantly lower in both RRMS and PRMS groups. In the RRMS group, 5-HIAA levels were negatively related to EDSS and to MSSS. Multiple regression analyses revealed a significant association of MHPG to duration of illness, and a strong negative association of 5-HIAA to MSSS rather than to EDSS. The strong negative correlation of MSSS to CSF 5-HIAA levels in RRMS group of patients indicates that deficits in central serotonergic activity are related to the rate of disability accumulation in RRMS, and could be linked to the reported reduction of disease activity by serotonergic drugs.     

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.     

Endogenous FGF‐2 is critically important in PTH anabolic effects on bone

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2?/? mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2?/? osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2?/? osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2?/? osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2?/? osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2?/? osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2?/? osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2?/? osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2?/? mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009. © 2008 Wiley‐Liss, Inc.     

PBOND： Web Server for the Prediction of Proline and Non-Proline cis / trans Isomerization

PBOND is a web server that predicts the conformation of the peptide bond between any two amino acids. PBOND classifies the peptide bonds into one out of four classes, namely cis imide (cis-Pro), cis amide (cis-nonPro), trans imide (trans-Pro) and trans amide (trans-nonPro). Moreover, for every prediction a reliability index is computed. The underlying structure of the server consists of three stages: (1) feature extraction, (2) feature selection and (3) peptide bond clas- sification. PBOND can handle both s...     

Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg(2 + ) which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.     

Synthesis and pharmacochemical study of novel polyfunctional molecules combining anti-inflammatory, antioxidant, and hypocholesterolemic properties

We have designed and synthesized a series of novel molecules having a residue of a classical NSAID and an antioxidant moiety, both attached through amide bonds to a known nootropic structure, an L-proline, trans-4-hydroxy-L-proline or DL-pipecolinic acid residue. The compounds were found to retain anti-inflammatory and antioxidant activities, to acquire hypocholesterolemic action, and to possess a greatly reduced gastrointestinal toxicity. The novel molecules could find useful applications, among others, in slowing the progression or delaying the onset of neurodegenerative diseases.     

Rapid expression of human immunodeficiency virus following activation of latently infected cells

The host cell activation state impacts the nature of human immunodeficiency virus infection. Activated cells facilitate productive infections; quiescent cells enable the virus to enter a latent state, the major obstacle to viral clearance. We wanted to understand how these differences affected viral gene expression. In quiescent cells activated prior to infection, viral RNA was seen 12 h postinfection; when cells were stimulated postinfection, viral RNA was not seen until 36 h postinfection. Up-regulation of viral RNA in latently infected cells occurred within 2 h poststimulation. This hierarchy also held true for viral protein production. These results may explain the rapid reemergence of viremia following termination of therapy.