Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand,hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.     

Overexpression of myoglobin and assignment of its amide,Cα and Cβ resonances

Summary Sperm whale apomyoglobin was expressed to high levels on minimal media and isotopically labeled with ¹³C and ¹⁵N nuclei. The isotopically labeled apoprotein was purified to homogeneity in a single step by reversed-phase chromatography and reconstituted with hemin and carbon monoxide gas for NMR analysis. Sequence-specific backbone ¹H^N, ¹⁵N and ¹³C as well as side-chain ¹³C resonance assignments have been made for over 90% of the amino acids in the carbon monoxide complex of the protein. Resonance assignments were made by analysis of a series of 3D triple resonance spectra measured on the uniformly labeled sample. These assignments will provide the basis for analyzing the effects of point site mutations on the structure, stability and dynamics of the protein in solution.     

β-Glucosidase production in agitated solid fermentation,study of its properties

Summary -Glucosidase production by Aspergillus phoenicis was studied in dynamic solid fermentation, on sugar beet pulps. The assays were conducted in agitated tank reactor which allowed for the homogenization of the medium and the regulation of essential parameters: temperature, aeration, pH and humidity.In order to compare some properties of the -glucosidase produced in both liquid and solid cultures, A. phoenicis was also grown on starch in submerged culture. The enzymes produced in solid and liquid cultures have the same optimum pH of about 4.5. -Glucosidase synthetized in solid culture is significantly more thermostable than that from liquid culture and is maximally active at 65°C compared to 60°C for enzyme from liquid culture.     

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.     

Strontium stratigraphy of the Oligocene–Early Miocene shellbeds of the Kutch Basin,western India,and its implications

The Kutch Basin is unique among the western Indian sedimentary basins because of its near-complete sequence of post-Palaeozoic rocks. Due to extensive marine influence, the Oligocene–Early Miocene formations of the basin, namely Maniyara Fort, Khari Nadi and Chhasra, contain numerous shellbeds. Although age assignments of these formations exist based on foraminiferal biostratigraphy, detailed numerical age of the lithounits are yet to be established. We have identified a total of eleven distinct shellbeds (oldest SB 01 to youngest SB11) from this interval primarily containing bivalve fossils. Using ⁸⁷Sr/⁸⁶Sr of selected oyster and pectinid shells with pristine shell characteristics, we report the age of four shellbeds. The ages of SB 01, SB 04, SB 06 and SB 10 are 24.37, 17.31, 16.85 and 15.38 Ma, respectively. Our dates suggest a Chattian (24.37 Ma) age for SB 01 from the Bermoti Member, validating the previous biostratigraphical estimates from the Maniyara Fort Formation. The Chhasra Formation, however, shows a younger range of ages (17.31–15.38 Ma) characterized by a transition from the Burdigalian (SB 04–SB 06) to the Langhian (SB 10) stages. These dates have important implications in the study of sequence stratigraphy, Palaeobiogeography and tectonic history of the Kutch Basin. A surface with subaerial exposure is found in SB 08 (between 16.85 and 15.38 Ma) that corresponds to a global eustatic sea-level decrease (Mi2). Our new dates will also help evaluate the response of marine fauna to the closure of the Tethyan seaway around 19 Ma due to the formation of ‘Gomphotherium Landbridge’. The dated shellbeds enable us to identify pre- and post-closure fauna and assess the effect of biogeographical separation on these fauna. These dates have important implications in evaluating the regional geological record of western India in the context of various global events.     

Studies of fluorescent components of the “heparin” drug and its complexing with phenylalanine, tyrosine, and tryptophan

Impurities of free aromatic amino acids (Phe and Tyr) and the elastin protein were found in the heparin commercial drug (Hep) by spectral luminescent and spectrophotometric methods. The fluorescence quenching of the Trp, Tyr, and Phe amino acids by the Hep drug was studied, and the Stern-Folmer constants (K) that reflected stability of the Hep complexes with amino acids were determined. The stability of AA-Hep complexes increased in the following sequence: Trp < Tyr < Phe (K = 19 ± 2 < 39 ± 3 < 710 ± 70 M^?1, respectively). These values probably determined the dominant contribution of the phenylalanine impurity in the heparin drug. The contamination of animal elastin whose structure differed from that of the human elastin is thought to be a reason for allergic reactions and even anaphylactic shock during medical treatment with this drug.     

Isoosmotic type of regulation in the sturgeon, Acipenser güldenstaedti, in the marine period of its life]

Concentration of osmotically active substances in the blood serum of the sturgeon A. guldenstaedti and sevruga A. stellatus from the central part of the Caspian Sea is practically equal to that in the sea water. In the blood serum of the sturgeon and the sevruga, concentration of Na is higher, whereas concentration of K and Mg is lower than in the sea water. Excretion of the urine with relatively low Mg concentration presumably indicated that being isoosmotic to the medium, the fishes studied in the Caspian Sea do not swallow the sea water. Electronmicroscopic studies indicate that with respect to poor development of membranous structures at the base of cells in the proximal tubuli, the kidney of the sturgeon and sevruga stands closer to freshwater than marine teleosts. These data show specific pattern of the osmotic and ionic regulation in the sturgeon, i.e. its isoosmotic type during migration to the Caspian Sea.     

Photodynamic effect of proflavine on ?X174 bacteriophage, its DNA replicative form and its isolated single-stranded DNA: Inactivation, mutagenesis and repair

Summary In contrast to that what is observed with most inactivating agents, proflavine-mediated photoinactivation is about 10 times more efficient on double-stranded X 174 replicative form DNA (RFI) than on isolated single-stranded X 174 DNA. Both X RFI DNA and encapsidated DNA have similar sensitivities to proflavine and light treatment.With the three substrates studied, reactivation can occur through high multiplicity of infection and depends upon the cellular rec A gene product. No effect of the pol A, uvr A or lex A gene mutations has been found on either phage or DNA inactivation rates.The photodynamically induced lesions can be repaired at least in part, by the SOS repair system induced in the host-cells by a 100 J ·m^-2 UV irradiation. SOS repair does not occur with bacteria (or spheroplasts) irradiated in the presence of chloramphenicol.Reversion frequency of the X 174 amber mutations indicates that 1) photodynamically induced lesions are mutagenic whether the rec A gene product is present or not in the indicator bacteria; 2) induction of the SOS repair system is accompanied by a mutagenic process which almost results in a two fold increase of the reversion frequency; and 3) multiplicity reactivation occurs through a recombinational process and is not mutagenic per se.     

Larrea tridentata—Absolute configuration of its epoxylignans and investigations on its antiprotozoal activity

The dichloromethane extract of Larrea tridentata (Creosote bush, Zygophyllaceae) showed activity against the protozoan pathogens Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum (IC₅₀ 2.8, 14.6, 5.2, 2.9 μg/ml, cytotoxicity against L6 rat skeletal myoblasts: 25.4 μg/ml).In search for potentially active constituents, nine lignans (three dibenzylbutanes, four epoxylignans, two aryltetralins), six flavonoids and one ester of ferulic acid (3′-oxohexylferulate) were isolated and identified by spectroscopic methods.Since some ambiguities with respect to the absolute configuration of several chiral lignans from L. tridentata were found in the literature, CD spectra were recorded and correlated with results from quantum mechanical spectra simulations (TD-DFT at the B3LYP/6-31D(d,p) level). Thereby, the absolute stereochemistry of these lignans can now be assigned with certainty.The activity of the isolated constituents against the protozoan parasites was investigated. The major lignan meso-nordihydroguaiaretic acid (NDGA), mainly responsible for the anti-inflammatory effects of this plant, was found to be the most active compound (IC₅₀ values: 4.5, 33.1, 12.0 and 7.7 μM against the mentioned parasites, respectively, 33.1 μM for cytotoxicity against L6 rat skeletal myoblasts). Although its level of activity is only moderate, NDGA can thus also be considered the main active compound for the antiprotozoal activity of L. tridentata.     

Tectoridin,a poor ligand of estrogen receptor α, exerts its estrogenic effects via an ERK-dependent pathway

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17β-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER α as compared to 17β-estradiol and genistein. Despite poor binding to ER α, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER α at Ser¹¹⁸. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.     

Induction of α1-antitrypsin mRNA and cloning of its cDNA

Local inflammation was inflicted in a baboon by turpentine administration in order to induce the plasma level of α1-antitrypsin, an acute phase protein synthesized in the liver. Comparison of the α1-antitrypsin mRNA activity in the induced and non-induced baboon liver indicated that the “acute phase” response to chemical-inflicted inflammation is mediated through an increase in the steady-state level of cellular mRNA. Alpha-1-antitrypsin was then enriched from the induced baboon liver to a purity of greater than 90% by specific immunoprecipitation of polysomes. Double-stranded DNA was synthesized from the enriched mRNA and inserted into the $\text{Pst}$ I site of pBR322. Recombinant clones containing α1-antitrypsin cDNA sequences were identified by hybridselected translation and confirmed by DNA sequence analysis.     

Can we recover a sequence, just knowing all its subsequences of given length?

The problem tackled here concerns the feasibility of DNA sequencingusing hybridization methods. We establish algorithms for andcomputational limitations to the reconstruction of a sequencefrom all its subsequences having the same length: in other words,the building of a string that contains all the words of a givenset, and only these ones. Generally there are several possiblestrings. We refer to graph theory and propose an algorithm toenumerate all the strings that are solutions. We then carriedout stimulations using real DNA sequences. They provided somenecessary conditions and give some upper bounds to the lengthof the sequence to recover in relation with the length of oligonucleotides.To avoid limiting ourselves to problems that admit a uniquesolution, we introduce another algorithm that produces a signaturefor each solution string. Each signature can be tested to determinewhich one belongs to the correct sequence.     

An α-glucan isolated from root of Isatis Indigotica,its structure and adjuvant activity

The root of Isatis indigotica is a traditional Chinese herbal medicine. An α-glucan (IIP-A-1) was firstly isolated from the roots. In this study we elucidated the chemical structure of IIP-A-1 and determined its adjuvant activity by co-immunizing mice with H1N1 influenza virus split and recombinant hepatitis B surface antigen (HBsAg), respectively. The polysaccharide was pretreated with periodate oxidation, Smith degradation and methylation in order to analyze its structure using GC, HPGPC, FT-IR, NMR and GC-MS. The adjuvant effect was evaluated by determining the antibody titers of serum against H1N1 influenza and HBsAg using ELISA. The proliferation and TNF-α secretion of macrophages administrated with different dose of IIP-A-1 were measured in vitro. The results of this study revealed that IIP-A-1 was an α-glucan with the molecular weight of 3,600 Da. The backbone was α-(1?→?4)-D-glucan with (1?→?6) branch chain. The α-glucan could significantly enhance the immune response of mice immunized with H1N1 influenza or HBsAg in vivo and exert good dose-dependent effects on the proliferation and the TNF-α secretion of macrophages in vitro. These results supported that IIP-A-1 was expected to be an efficacious adjuvant candidate for prophylactic and therapeutic vaccines.     

How much better are females? The occurrence of female advantage,its proximal causes and its variation within and among gynodioecious species

Background

Gynodioecy is a reproductive system of interest for evolutionary biologists, as it poses the question of how females can be maintained while competing with hermaphrodites that possess both male and female functions. One necessary condition for the maintenance of this polymorphism is the occurrence of a female advantage, i.e. a better seed production or quality by females compared with hermaphrodites. Theoretically, its magnitude can be low when sterility mutations are cytoplasmic, while a 2-fold advantage is needed in the case of nuclear sterility. Such a difference is often thought to be due to reduced inbreeding depression in obligatory outcrossed females. Finally, variation in sex ratio and female advantage occur among populations of some gynodioecious species, though the prevalence of such variation is unknown.

Scope

By reviewing and analysing the data published on 48 gynodioecious species, we examined three important issues about female advantage. (1) Are reduced selfing and inbreeding depression likely to be the major cause of female advantage? (2) What is the magnitude of female advantage and does it fit theoretical predictions? (3) Does the occurrence or the magnitude of female advantage vary among populations within species and why?

Conclusions

It was found that a female advantage occurred in 40 species, with a magnitude comprised between 1 and 2 in the majority of cases. In many species, reduced selfing may not be a necessary cause of this advantage. Finally, female advantage varied among populations in some species, but both positive and negative correlations were found with female frequency. The role of reduced selfing in females for the evolution of gynodioecy, as well as the various processes that affect sex ratios and female advantage in populations are discussed.