Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein

An inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction. Five cDNA clones were isolated from the human placental lambda gt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe. The longest insert consists of 1,566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail. The deduced amino acid sequence was corroborated by chemical analyses of the protein. The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, and endonexin-related proteins. The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.     

Affinity chromatography of urokinase on an agarose derivative coupled with pyroglutamyl-lysyl-leucyl-argininal

Pyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the epsilon-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2 X 10(-4)M.     

Structure and expression of cDNA for calphobindin II, a human placental coagulation inhibitor

Calphobindin II, with Mr 73,000, is one of the human placental anticoagulant proteins. The cDNA encoding calphobindin II was obtained by screening a human placental lambda gt11 cDNA library using a specific antibody as a probe. The longest cDNA insert consisted of 2,361 nucleotides and a 64-nucleotide-long poly(A) tract. An open reading frame encoding 673 amino acids was predicted. The deduced sequence includes an 8-fold repeat of a conserved 70-amino-acid-long segment that has a high degree of sequence identity with the repeated segments in members of the Ca2+-dependent phospholipid binding protein family. The cDNA fragment including the open reading frame was introduced into the expression vector pKK223-3 and subsequently expressed in Escherichia coli JM105 cells. The resulting recombinant protein reacted with the specific monoclonal antibodies to calphobindin II and prolonged the blood coagulation time as did placental calphobindin II.     

Anaerobic coryneforms isolated from human bone marrow and skin. Chemical, biochemical and serological studies and some of their biological activities.

Eighteen isolates if anaerobic coryneforms from human bone marrow and skin and four type strains of Propionibacterium were studied chemically, biochemically and antigenically. All of the isolates were identified as Propionibacterium acnes; of the 18 isolates,16 belonged to sterotype I and two to serotype II. By means of gas liquid chromatography and mass spectral analysis, a large amount of iso-type fatty acids, such as iso-pentadecanoic and iso-heptadecanoic acids were detected in whole cells of isolates and type strains. Antitumor and adjuvant effects of the isolates and type strains were found to differ considerably among the strains. One of the isolates, P. acnes C-7, which showed potent biological activities was fractionated by hot phenol-water extraction. The resulting insoluble middle layer was found the most effective in tumor protection, adjuvant action in immune response and phagocytic activity in mice.     

Sl‐lncRNA15492 interacts with Sl‐miR482a and affects Solanum lycopersicum immunity against Phytophthora infestans

Long non‐coding RNAs (lncRNAs) are involved in the resistance of plants to infection by pathogens via interactions with microRNAs (miRNAs). Long non‐coding RNAs are cleaved by miRNAs to produce phased small interfering RNAs (phasiRNAs), which, as competing endogenous RNAs (ceRNAs), function as decoys for mature miRNAs, thus inhibiting their expression, and contain pre‐miRNA sequences to produce mature miRNAs. However, whether lncRNAs and miRNAs mediate other molecular mechanisms during plant resistance to pathogens is unknown. In this study, as a positive regulator, Sl‐lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants affected tomato resistance to Phytophthora infestans. Gain‐ and loss‐of‐function experiments and RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE) also revealed that Sl‐miR482a was negatively involved in tomato resistance by targeting Sl‐NBS‐LRR genes and that silencing of Sl‐NBS‐LRR1 decreased tomato resistance. Sl‐lncRNA15492 inhibited the expression of mature Sl‐miR482a, whose precursor was located within the antisense sequence of Sl‐lncRNA15492. Further degradome analysis and additional RLM‐5′ RACE experiments verified that mature Sl‐miR482a could also cleave Sl‐lncRNA15492. These results provide a mechanism by which lncRNAs might inhibit precursor miRNA expression through antisense strands of lncRNAs, and demonstrate that Sl‐lncRNA15492 and Sl‐miR482a mutually inhibit the maintenance of Sl‐NBS‐LRR1 homeostasis during tomato resistance to P. infestans.     

Low Child Survival Index in a Multi-Dimensionally Poor Amerindian Population in Venezuela

Background

Warao Amerindians, who inhabit the Orinoco Delta, are the second largest indigenous group in Venezuela.  High Warao general mortality rates were mentioned in a limited study 21 years ago. However, there have been no comprehensive studies addressing child survival across the entire population.

Objectives

To determine the Child Survival-Index (CSI) (ratio: still-living children/total-live births) in the Warao population, the principal causes of childhood death and the socio-demographic factors associated with childhood deaths.

Methods

We conducted a cross-sectional epidemiological survey of 688 women from 97 communities in 7 different subregions of the Orinoco Delta. Data collected included socio-demographic characteristics and the reproductive history of each woman surveyed. The multidimensional poverty index (MPI) was used to classify the households as deprived across the three dimensions of the Human Development Index. Multivariable linear regression and Generalized Linear Model Procedures were used to identify socioeconomic and environmental characteristics statistically associated with the CSI.

Findings

The average CSI was 73.8% ±26. The two most common causes of death were gastroenteritis/diarrhea (63%) and acute respiratory tract Infection/pneumonia (18%).  Deaths in children under five years accounted for 97.3% of childhood deaths, with 54% occurring in the neonatal period or first year of life.  Most of the women (95.5%) were classified as multidimensionally poor.  The general MPI in the sample was 0.56.   CSI was negatively correlated with MPI, maternal age, residence in a traditional dwelling and profession of the head of household other than nurse or teacher.

Conclusions

The Warao have a low CSI which is correlated with MPI and maternal age.  Infectious diseases are responsible for 85% of childhood deaths.  The low socioeconomic development, lack of infrastructure and geographic and cultural isolation suggest that an integrated approach is urgently needed to improve the child survival and overall health of the Warao Amerindians.      

Immune and Stress Responses Covary with Melanin-Based Coloration in the Barn Swallow

Eumelanin and pheomelanin are the main endogenous pigments in animals and melanin-based coloration has multiple functions. Melanization is associated with major life-history traits, including immune and stress response, possibly because of pleiotropic effects of genes that control melanogenesis. The net effects on pheo- versus eumelanization and other life-history traits may depend on the antagonistic effects of the genes that trigger the biosynthesis of either melanin form. Covariation between melanin-based pigmentation and fitness traits enforced by pleiotropic genes has major evolutionary implications particularly for socio-sexual communication. However, evidence from non-model organisms in the wild is limited to very few species. Here, we tested the hypothesis that melanin-based coloration of barn swallow (Hirundo rustica) throat and belly feathers covaries with acquired immunity and activation of the hypothalamic–pituitary–adrenal (HPA) axis, as gauged by corticosterone plasma levels. Individuals of both sexes with darker brownish belly feathers had weaker humoral immune response, while darker males had higher circulating corticosterone levels only when parental workload was experimentally reduced. Because color of belly feathers depends on both eu- and pheomelanin, and its darkness decreases with an increase in the concentration of eu- relative to pheomelanin, these results are consistent with our expectation that relatively more eu- than pheomelanized individuals have better immune response and smaller activation of the HPA-axis. Covariation of immune and stress response arose for belly but not throat feather color, suggesting that any function of color as a signal of individual quality or of alternative life-history strategies depends on plumage region.     

Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation

By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp²⁹–Sec⁵²–Gln⁷²). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser⁵²/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe²⁹/Trp–Val⁷²/Gln mutations enhance the reaction rate via functional cooperation with Sec⁵². Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr¹⁰¹ occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr⁵⁴, Asn⁵⁵, and Gly⁵⁶ to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK _a shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole–dipole interactions among Trp²⁹–Sec⁵²–Gln⁷² lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.