Chemical synthesis of a gene for human stefin A and its expression in E. coli

A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 306-bp synthetic gene carries signals for the initiation and termination of its translation. The gene was expressed in E. coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E. coli alkaline phosphatase signal sequence, respectively. The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.     

Differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta

The molecular mechanisms and pathologic significance of placental viral infections are poorly understood. We investigated factors that regulate placental infection by adenovirus, which is the most common viral pathogen identified in fetal samples from abnormal pregnancies (i.e., fetal growth restriction, oligohydramnios, and nonimmune fetal hydrops). We also determined the pathologic significance of placental adenovirus infection. Northern hybridization, flow cytometry, and immunostaining revealed that placental expression of the coxsackievirus and adenovirus receptor (CAR) varied with gestational age and trophoblast phenotype. The CAR was continuously expressed in invasive or extravillous trophoblast cells but not in villous trophoblast cells. We postulate that the villous syncytiotrophoblast, which does not express CAR and is resistant to adenovirus infection, limits the transplacental transmission of viral pathogens, including adenovirus. Conversely, extravillous trophoblast cells underwent apoptosis when infected by adenovirus in the presence of decidual lymphocytes (which simulated the maternal immune response to viral infection). Thus, adenovirus infection and/or the maternal immune response to adenovirus infection induced the death of placental cell types that expressed CAR. Consequently, we speculate that adenovirus infection of extra-villous trophoblast cells may negatively impact the process of placental invasion and predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.     

Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity

In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.     

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes

Background

Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.

Methodology/Principal Findings

In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.

Conclusion

The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.     

Complete congruence between morphological and rbcL-based molecular phylogenies in birches and related species (Betulaceae).

Estimations of phylogenies from morphological and molecular data often show contrasting results. We compared morphological and molecular phylogenies in an ancient family of woody dicots, the Betulaceae (birch family). The phylogeny of the family was estimated from parsimony analysis of morphological characters in the genera Alnus, Betula, Carpinus, Corylus, Ostrya, and Ostryopsis and from parsimony and distance-matrix analyses of DNA sequences of the chloroplast gene encoding the large subunit of ribulose-1,5-biphosphate carboxylase (rbcL) in the genera Alnus, Betula, Carpinus, Corylus, and Ostrya and in two outgroups, Quercus and Liquidambar. The topologies obtained by the different methods were completely congruent, and bootstrapping strongly supported the division of the family Betulaceae into two major clades, Betuleae (Alnus and Betula) and Coryleae (other members). Only slightly more homoplasy was present in the rbcL sequence data set than in the morphological set. Relative-rate tests indicated that the Coryleae clade had a faster rate of rbcL evolution than did the Betuleae clade. Heterogeneity of rates of morphological evolution also paralleled those for rbcL.     

Electrical Impedance of Isolated Amnion

The electrical impedance of the guinea pig amniotic membrane was measured, under standardized conditions, over the frequency range of 20 to 7000 cycles/second. This impedance can be represented analytically by a simple frequency-dependent function which is precisely of the form of the Debye relaxation equation. The observed data exhibit a broad dispersion centered at a frequency of 1050 cycles/second and a narrow distribution of time constants centered about 152 microseconds, both effects being due to the polydisperse nature of amniotic tissue. If the narrow time-constant distribution is approximated by a single time constant, amnion impedance can be simulated by a simple electrical circuit of frequency-independent elements. The Maxwell-Wagner interfacial treatment, although successfully adapted for cell suspensions, is shown to lose its quantitative significance in the case of the tightly structured amnion. In addition, determinations were made on the chemical composition of amniotic fluid, fetal blood and urine, and maternal blood and urine; the DC potential across the amniotic membrane was also measured.     

The role of low soil temperature in the inhibition of growth and PSII function during dark chilling in soybean genotypes of contrasting tolerance

Dark chilling affects growth and yield of warm-climate crops such as soybean [ Glycine max (L.) Merr.]. Several studies have investigated chilling-stress effects on photosynthesis and other aspects of metabolism, but none have compared effects of whole-plant chilling (WPC; shoots and roots) with that of aboveground chilling in legumes. This is important because low root temperatures might induce additional constraints, such as inhibition of N₂ fixation, thereby aggravating chilling-stress symptoms. Effects of dark chilling on PSII, shoot growth, leaf ureide content and photosynthetic capacity were studied in two soybean genotypes, Highveld Top (chilling tolerant) and PAN809 (chilling sensitive), in experiments comparing effects of WPC with that of shoot chilling (SC). Both treatments inhibited shoot growth in PAN809 but not Highveld Top. Also, WPC in PAN809 caused a decrease in leaf ureide content followed by severe chlorosis and alterations in O-J-I-P fluorescence-rise kinetics, distinct from SC. A noteworthy difference was the appearance of a ΔK peak in the O-J-I-P fluorescence rise in response to WPC. These genotypic and treatment differences also reflected in the degree of inhibition of CO₂ assimilation rates. The appearance of a ΔK peak, coupled with growth inhibition, reduced ureide content, chlorosis and lower CO₂ assimilation rates, provides mechanistic information about how WPC might have aggravated chilling-stress symptoms in PAN809. We introduce a model explaining how chilling soil temperatures might trigger N-limitation in sensitive genotypes and how characteristic changes in O-J-I-P fluorescence-rise kinetics are linked to changes in carbon and nitrogen metabolism.     

Biological lability of terrestrial DOM increases CO2 outgassing across Arctic shelves

Biogeochemistry - Arctic shelf seas receive greater quantities of river runoff than any other ocean region and are experiencing increased freshwater loads and associated terrestrial matter inputs...     

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.