Schistosoma mansoni: Mechanisms in regulation of glycolysis

Adult pairs of Schistosoma mansoni convert glucose to lactate rapidly and almost quantitatively under aerobic and anaerobic conditions E. Bueding, 1950, Journal of General Physiology33, 475–495). Glycolysis is the principal source of energy of schistosomes and its inhibition by trivalent organic antimonials, at the phosphofructokinase step [EC 2.7.1.11], may be the basis for the chemotherapeutic effects of these agents E. Bueding and J. M. Mansour, 1957, British Journal of Pharmacology and Chemotherapy12, 159–165). We have developed standardized conditions for the comparison of rates of glucose consumption and lactate production by intact schistosomes in vitro and by centrifuged homogenates of worms. The rates of glycolysis of homogenates prepared from freshly isolated worms, and from worms that have been lyophilized immediately after harvesting and stored for prolonged periods at ?80 C were identical, when measured in media containing appropriate concentrations of glucose, NAD, ATP, MgCl₂, KCl, and phosphate. The specific activities of the 11 glycolytic enzymes and of 3 related enzymes (fructose-biphosphatase [EC 3.1.3.11], glycerol-3-phosphate dehydrogenase [EC 1.1.1.8], and malate dehydrogenase [EC 1.1.1.37]) were measured in homogenates under optimal conditions. The profile of the relative activities of glycolytic enzymes of S. mansoni resembles closely that of Ehrlich ascites tumor cells, and differs markedly from that observed in erythrocytes or skeletal muscle. As is the case in many animal tissues, hexokinase [EC 2.7.1.1] was the enzyme of lowest specific activity, and the rate of glycolysis of homogenates was almost the same as the hexokinase activity. Several other lines of evidence support the view that the hexokinase reaction is the rate-limiting step in the glycolysis of worm homogenates. Hexokinase activity was not particulate in schistosome homogenates, and there was no detectable high K_m glucokinase-like activity. The rate of glycolysis by homogenates exceeded that of intact worms by a factor of nearly 5. The contributions of glucose transport, availability of ADP and inorganic phosphate, regulatory enzymes, and a substrate cycle catalyzed by fructose-bisphosphatase are considered as possible mechanisms for the restraint of glycolysis in intact worms. The mechanisms contributing to the rapid rates of glycolysis of adult S. mansoni have not been identified, although several can be excluded (unusually high capacity of the glycolytic enzymes, the presence of mitochondrial hexokinase, the occurrence of glycosomes, and the operation of defective mitochondrial shuttles). In view of the regulatory role of hexokinase in the glycolysis of S. mansoni, inhibition of this enzyme is a potentially important target for the development of new antischistosomal drugs.     

High prevalence of asthma symptoms in Warao Amerindian children in Venezuela is significantly associated with open-fire cooking: a cross-sectional observational study

Background

The International Study on Asthma and Allergies in Childhood (ISAAC) reported a prevalence of asthma symptoms in 17 centers in nine Latin American countries that was similar to prevalence rates reported in non-tropical countries. It has been proposed that the continuous exposure to infectious diseases in rural populations residing in tropical areas leads to a relatively low prevalence of asthma symptoms. As almost a quarter of Latin American people live in rural tropical areas, the encountered high prevalence of asthma symptoms is remarkable. Wood smoke exposure and environmental tobacco smoke have been identified as possible risk factors for having asthma symptoms.

Methods

We performed a cross-sectional observational study from June 1, 2012 to September 30, 2012 in which we interviewed parents and guardians of Warao Amerindian children from Venezuela. Asthma symptoms were defined according to the ISAAC definition as self-reported wheezing in the last 12 months. The associations between wood smoke exposure and environmental tobacco smoke and the prevalence of asthma symptoms were calculated by means of univariate and multivariable logistic regression analyses.

Results

We included 630 children between two and ten years of age. Asthma symptoms were recorded in 164 of these children (26%). The prevalence of asthma symptoms was associated with the cooking method. Children exposed to the smoke produced by cooking on open wood fires were at higher risk of having asthma symptoms compared to children exposed to cooking with gas (AOR 2.12, 95% CI 1.18 - 3.84). Four percent of the children lived in a household where more than ten cigarettes were smoked per day and they had a higher risk of having asthma symptoms compared to children who were not exposed to cigarette smoke (AOR 2.69, 95% CI 1.11 - 6.48).

Conclusion

Our findings suggest that children living in rural settings in a household where wood is used for cooking or where more than ten cigarettes are smoked daily have a higher risk of having asthma symptoms.     

The long periodicity phase (LPP) controversy part I: The influence of a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP] based stratum corneum modelling system: A neutron diffraction study

This study used neutron diffraction to investigate a ceramide-[NP] C24/[AP] C24 /[EOS]-br C30/cholesterol/lignoceric acid (0.6: 0.3: 0.1: 0.7: 1) based stratum corneum modelling system. By adding specifically deuterated ceramides-[NP]-D₃, [AP]-D₃, and [EOS]-br-D₃, detailed information on the lamellar and the nanostructure of the system was obtained. For the short periodicity phase a natural-like lamellar repeat distance of 5.47?±?0.02?nm was observed, similar to the [NP]/[AP] base system without the [EOS]-br. Unlike in this system the ceramides here were slightly tilted, hinting towards a slightly less natural arrangement. Due to the deuteration it was possible to observe that the long ceramide chains were overlapping in the lamellar mid-plane. This is considered to be an important feature for the natural stratum corneum. Despite the presence of a ceramide [EOS] analogue – able to form a long phase arrangement – no distinct long periodicity phase was formed, despite a slightly higher than natural ω-acyl ceramide ratio of 10?mol%. The deuterated variant of this ceramide determined that the very long ceramide was integrated into the short periodicity phase, spanning multiple layers instead. The – compared to the base system – unchanged repeat distance highlights the stability of this structure. Furthermore, the localisation of the very long ceramide in the short periodicity phase indicates the possibility of a crosslinking effect and thus a multilayer stabilizing role for the ceramide [EOS]. It can be concluded, that additionally to the mere presence of ceramide-[EOS] more complex conditions have to be met in order to form this long phase. This has to be further investigated in the future.     

Use of human embryonic stem cell derived-mesenchymal cells for cardiac repair

Human mesenchymal stem cells (hMSC) have proven beneficial in the repair and preservation of infarcted myocardium. Unfortunately, MSCs represent a small portion of the bone marrow and require ex vivo expansion. To further advance the clinical usefulness of cellular cardiomyoplasty, derivation of "MSC-like" cells that can be made available "off-the-shelf" are desirable. Recently, human embryonic stem cell-derived mesenchymal cells (hESC-MC) were described. We investigated the efficacy of hESC-MC for cardiac repair after myocardial infarction (MI) compared to hMSC. Because of increased efficacy of cell delivery, cells were embedded into collagen patches and delivered to infarcted myocardium. Culture of hMSC and hESC-MCs in collagen patches did not induce differentiation or significant loss in viability. Transplantation of hMSC and hES-MC patches onto infarcted myocardium of athymic nude rats prevented adverse changes in infarct wall thickness and fractional area change compared to a non-viable patch control. Hemodynamic assessment showed that hMSCs and hES-MC patch application improved end diastolic pressure equivalently. There were no changes in systolic function. hES-MC and hMSC construct application enhanced neovessel formation compared to a non-viable control, and each cell type had similar efficacy in stimulating endothelial cell growth in vitro. In summary, the use of hES-MC provides similar efficacy for cellular cardiomyoplasty as compared to hMSC and may be considered a suitable alternative for cell therapy.     

Label free imaging of cell‐substrate contacts by holographic total internal reflection microscopy

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label‐free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label‐free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion.

     

Stereochemistry of reactions catalysed by arginine decarboxylase and ornithine decarboxylase

The decarboxylations of l-arginine, catalysed by arginine decarboxylase (EC 4.1.1.19) and of l-ornithine, catalysed by ornithine decarboxylase     

Apo[a] size and PNR explain African American-Caucasian differences in allele-specific apo[a] levels for small but not large apo[a

Apolipoprotein [a] (apo[a]) gene size is a major predictor of lipoprotein [a] level. To determine genetic predictors of allele-specific apo[a] levels beyond gene size, we evaluated the upstream C/T and pentanucleotide repeat (PNR) polymorphisms. We determined apo[a] sizes, allele-specific apo[a] levels, and C/T and PNR in 215 Caucasians and 139 African Americans. For Caucasians, apo[a] size affected allele-specific levels substantially greater in subjects with apo[a] < 24 K4; for African Americans, the size effect was smaller than in Caucasians, <24 K4, but did not decrease at higher repeats. In both groups, the level decreased with increasing size of the other allele. Controlling for apo[a] sizes, PNR decreased allele-specific apo[a] levels in Caucasians with increasing PNR > 8. In a multiple regression model, apo[a] allele size and size and expression of the other apo[a] allele (and PNR > 8 for Caucasians) significantly predicted allele-specific apo[a] levels. For a common PNR 8 allele, predicted values were similar in the two ethnicities for small size apo[a]. Allele-specific apo[a] levels were influenced by the other allele size and expression. Observed differences between Caucasians and African Americans in allele-specific apo[a] levels were explained for small apo[a] sizes by the other allele size and PNR; the ethnicity differences remain unexplained for larger sizes.     

A structural assessment of the apo[a] protein of human lipoprotein[a].

Apolipoprotein[a], the highly glycosylated, hydrophilic apoprotein of lipoprotein[a] (Lp[a]), is generally considered to be a multimeric homologue of plasminogen, and to exhibit atherogenic/thrombogenic properties. The cDNA-inferred amino acid sequence of apo[a] indicates that apo[a], like plasminogen and some zymogens, is composed of a kringle domain and a serine protease domain. To gain insight into possible positive functions of Lp[a], we have examined the apo[a] primary structure by comparing its sequence with those of other proteins involved in coagulation and fibrinolysis, and its secondary structure by using a combination of structure prediction algorithms. The kringle domain encompasses 11 distinct types of repeating units, 9 of which contain 114 residues. These units, called kringles, are similar but not identical to each other or to PGK4. Each apo[a] kringle type was compared with kringles which have been shown to bind lysine and fibrin, and with bovine prothrombin kringle 1. Apo[a] kringles are linked by serine/threonine- and proline-rich stretches similar to regions in immunoglobulins, adhesion molecules, glycoprotein Ib-alpha subunit, and kininogen. In comparing the protease domains of apo[a] and plasmin, apo[a] contains a region between positions 4470 and 4492 where 8 substitutions, 9 deletions, and 1 insertion are apparent. Our analysis suggests that apo[a] kringle-type 10 has a high probability of binding to lysine in the same way as PGK4. In the only human apo[a] polymorph sequenced to date, position 4308 is occupied by serine, whereas the homologous position in plasmin is occupied by arginine and is an important site for proteolytic cleavage and activation. An alternative site for the proteolytic activation of human apo[a] is proposed.     

A straightforward kinetic evidence for coexistence of “induced fit” and “selected fit” in the reaction mechanism of a mutant tryptophan indole lyase Y72F from Proteus vulgaris

The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-l-alanine (OIA), with the natural substrate, l-tryptophan, and with a substrate S-ethyl-l-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme (“induced fit”) is realized in the case of mutant Y72F enzyme reaction with OIA. For the reaction of mutant enzyme with l-Trp at lower concentrations of the latter a principally different kinetic scheme is observed. This scheme suggests that binding of the substrate and formation of the quinonoid intermediate are at fast equilibrium, while preceding conformational changes of the holoenzyme proceed with measureable rates (“selected fit”). For the reaction with S-ethyl-l-cysteine the observed concentration dependence of k_obs agrees with the realization of both kinetic schemes, the “selected fit” becoming predominant at lower concentrations of substrate, the “induced fit”— at higher ones. In the reaction with S-ethyl-l-cysteine the formation of the quinonoid intermediate proceeds slower than does catalytic α,β-elimination of ethylthiol from S-ethyl-l-cysteine, and consequently does not play a considerable role in the catalysis, which may be effected by a concerted E2 mechanism.     

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia: II. Biochemistry and spectroscopy

A novel chlorophyll a containing pigment–protein complex expressed by cells of Chromera velia adapted to growth under red/far-red illumination [1]. Purification of the complex was achieved by means of anion-exchange chromatography and gel-filtration. The antenna is shown to be an aggregate of ~ 20 kDa proteins of the light–harvesting complex (LHC) family, unstable in the isolated form. The complex possesses an absorption maximum at 705 nm at room temperature in addition to the main chlorophyll a maximum at 677 nm producing the major emission band at 714 nm at room temperature. The far-red absorption is shown to be the property of the isolated aggregate in the intact form and lost upon dissociation. The purified complex was further characterized by circular dichroism spectroscopy and fluorescence spectroscopy. This work thus identified the third different class of antenna complex in C. velia after the recently described FCP-like and LHCr-like antennas. Possible candidates for red antennas are identified in other taxonomic groups, such as eustigmatophytes and the relevance of the present results to other known examples of red-shifted antenna from other organisms is discussed. This work appears to be the first successful isolation of a chlorophyll a-based far-red antenna complex absorbing above 700 nm unrelated to LHCI.     

Development of hematological respiratory variables in late chicken embryos: the relative importance of incubation time and embryo mass

Oxygen demand increases during embryonic development, requiring an increase in red blood cells (RBCs) containing hemoglobin (Hb) to transport O(2) between the respiratory organ and systemic tissues. A thorough ontogenetic understanding of the onset and maturation of the complex regulatory processes for RBC concentration ([RBC]), Hb concentration ([Hb]), hematocrit (Hct), mean corpuscular indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration ([MCHb])) is currently lacking. We hypothesize that during the last half of incubation when the respiratory organ (the chorioallantoic membrane) envelops most of the egg contents, mean corpuscular indices will stabilize. Accordingly, Hct, [RBC] and [Hb] must also all change proportionally across development. Further, we hypothesize that the hematological respiratory variables develop and mature as a function of incubation duration, independently of embryonic growth. As predicted, a similar increase in Hct (from 18.7±0.6% on day 10 (d10) to 34.1±0.5% on d19 of incubation), [RBC] (1.13±0.03×10(6)/μL to 2.50±0.03×10(6)/μL) and [Hb] (6.1±0.2 g% to 11.2±0.1 g%) occurred during d10-19. Both [RBC] and [Hb] demonstrated high linear correlation with Hct, resulting in constant [MCHb] (~33 g% from d10 to d19). The decrease in MCV (from ~165 μ(3) on d10 to ~140 μ(3) on d13) and MCH (~55 pg to ~45 pg) during d10-13, may be attributed to a changeover from larger primary to smaller secondary and adult-type erythrocytes with MCV and MCH remaining constant (~140 μ(3) and ~45 pg respectively) for the rest of the incubation period (d13-19). Hematological respiratory values on a given incubation day were identical between embryos of different masses using either natural mass variation or experimental growth acceleration, indicating that the hematological variables develop as a function of incubation time, irrespective of embryo growth.     

产异丁醇关键基因在大肠杆菌中表达的研究

[目的]改造大肠杆菌缬氨酸合成途径,使其能够代谢合成异丁醇.[方法]将乳酸乳球菌(Lactococcus lactis) 1.2829的2-酮异戊酸脱羧酶基因(kivD)和醇脱氢酶基因(adhA)串联克隆到大肠杆菌DH5α宿主中表达.[结果]经过改造的宿主菌发酵24 h后异丁醇产量为0.12 g/L.酶活测定实验发现,kivD和adhA基因在宿主菌中均得到表达,但由于KivD的低表达量导致宿主菌最终的异丁醇合成能力偏低.通过研究温度和pH对KivD和AdhA酶活的影响,最终选定二者的最适温度为30℃,最适pH为6.5. [结论]通过向宿主菌导入外源异丁醇合成基因能够改造其自身代谢途径,从而合成异丁醇.     

Spermine, a molecular switch regulating EGFR, integrin β3, Src, and FAK scaffolding

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.     

Reduction of transpiration and altered nutrient allocation contribute to nutrient decline of crops grown in elevated CO2 concentrations

Plants grown in elevated [CO₂] have lower protein and mineral concentrations compared with plants grown in ambient [CO₂]. Dilution by enhanced production of carbohydrates is a likely cause, but it cannot explain all of the reductions. Two proposed, but untested, hypotheses are that (1) reduced canopy transpiration reduces mass flow of nutrients to the roots thus reducing nutrient uptake and (2) changes in metabolite or enzyme concentrations caused by physiological changes alter requirements for minerals as protein cofactors or in other organic complexes, shifting allocation between tissues and possibly altering uptake. Here, we use the meta‐analysis of previous studies in crops to test these hypotheses. Nutrients acquired mostly by mass flow were decreased significantly more by elevated [CO₂] than nutrients acquired by diffusion to the roots through the soil, supporting the first hypothesis. Similarly, Mg showed large concentration declines in leaves and wheat stems, but smaller decreases in other tissues. Because chlorophyll requires a large fraction of total plant Mg, and chlorophyll concentration is reduced by growth in elevated [CO₂], this supports the second hypothesis. Understanding these mechanisms may guide efforts to improve nutrient content, and allow modeling of nutrient changes and health impacts under future climate change scenarios.     

Syntheses of chlorogenin 6alpha-O-acyl-3-O-beta-chacotriosides and their antitumor activities

Chlorogenin 3-O-beta-chacotrioside (1) and its 6alpha-O-acyl derivatives (2-6) were concisely synthesized. Introduction of a hydroxyl or acyloxy group onto the C-6 of the steroidal aglycone of dioscin decreased significantly the cytotoxicity of the parent saponin (dioscin).     

Differential processing of mammalian l-histidine decarboxylase enzymes

In the mammalian species studied so far, the l-histidine decarboxylase (HDC) enzyme responsible for histamine biosynthesis has been shown to undergo post-translational processing. The processing is best characterized for the mouse enzyme, where di-asparate DD motifs mediate the production of active ∼55 and ∼60 kDa isoforms from the ∼74 kDa precursor in a caspase-9 dependent manner. The identification of conserved di-aspartate motifs at similar locations in the rat and human HDC protein sequences has led to proposals that these may represent important processing sites in these species also. Here we used transfected Cos7 cells to demonstrate that the rat and human HDC proteins undergo differential processing compared to each other, and found no evidence to suggest that conserved di-aspartate motifs are required absolutely for processing in this cell type. Instead we identified SKD and EEAPD motifs that are important for caspase-6 dependent production of ∼54 and ∼59 kDa isoforms in the rat and human proteins, respectively. The addition of staurosporine, which is known to pharmacologically activate caspase enzymes, increased processing of the human HDC protein. We propose that caspase-dependent processing is a conserved feature of mammalian HDC enzymes, but that proteolysis may involve different enzymes and occur at diverse sites and sequences.     

The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes

The effect of carbacyclin, a chemically stable analogue of prostacyclin, on the activity of adenylate cyclase in platelet membranes was measured, and compared with the effect of PGE1. When GTP was added in concentrations up to 10 microM the activation of adenylate cyclase by carbacyclin was increased, whereas higher concentrations of GTP were inhibitory. The addition of a non-hydrolysable analogue of GDP, guanosine 5'-[beta-thio]diphosphate (GDP[beta S] ) resulted in a dose-dependent inhibition of adenylate cyclase activation by carbacyclin; this inhibition was relieved by adding increased amounts of GTP.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.