Phylogenetic and molecular clock inferences of cyanobacterial strains within Rivulariaceae from distant environments

Heterocyst-forming cyanobacteria are important players at both evolutionary and ecological scales, but to date it has been difficult to establish their phylogenetic affiliations. We present data from a phylogenetic and molecular clock analysis of heterocystous cyanobacteria within the family Rivulariaceae, including the genera Calothrix, Rivularia, Gloeotrichia and Tolypothrix. The strains were isolated from distant geographic regions including fresh and brackish water bodies, microbial mats from beach rock, microbialites, pebble beaches, plus PCC strains 7103 and 7504. Phylogenetic inferences (distance, likelihood and Bayesian) suggested the monophyly of genera Calothrix and Rivularia. Molecular clock estimates indicate that Calothrix and Rivularia originated ～1500 million years ago (MYA) ago and species date back to 400-300 MYA while Tolypothrix and Gloeotrichia are younger genera (600-400 MYA).     

Alpha-1-antitrypsin deficiency and juvenile liver disease Ultrastructural observations compared with light microscopy and routine liver tests

Sixteen children (aged between 1 month and 20 years) with alpha-1-antitrypsin deficiency (PiZ) were investigated by liver biopsy on one or more occasions. Eight patients had suffered from neonatal cholestasis, and two of them were investigated during the cholestatic period as well. The clinical status and liver function tests were compared with the light and electron microscopical findings. According to the light microscopical analyses at the latest investigation, the cholestatic and noncholestatic patients were classified as healthy, fibrotic or cirrhotic cases. All livers displayed periodic acid-Schiff positive, diastase-resistant globules in some but not all periportally located hepatocytes. By electron microscopy accumulation of retained secretory material was found in all PiZ patients. This accumulation was most conspicuous in the smooth endoplasmic reticulum of hepatocytes. alpha-1-antitrypsin deficiency seems to affect some, but not all hepatocytes. In the affected cells disappearance or hypotrophy of the Golgi complex could be observed. The intracellular transport of very low density lipoproteins (VLDL) was apparently not affected. The migration block in alpha-1-antitrypsin deficiency seems to occur before transportation to the Golgi complex. The extent of the involvement was not strictly age-dependent. There was no ultrastructural evidence of subclinical cholestasis as a possible triggering factor in the development of cirrhosis.     

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity.     

mRNA expression and localization of bNOS,eNOS and iNOS in human cervix at preterm and term labour

Background  

Preterm birth is the primary cause of the neonatal mortality and morbidity. There will be no preterm birth without a cervical softening. Nitric oxide (NO) is shown to be a mediator of term cervical ripening. The aim of this study was to investigate mRNA expression of the three isomers of NO synthases (NOS) and to identify them by immunohistochemistry in the human cervix at preterm birth compared to term.     

Leishmania-induced inhibition of macrophage antigen presentation analyzed at the single-cell level

A number of studies have previously examined the capacity of intracellular Leishmania parasites to modulate the capacity of macrophages to process and present Ags to MHC class II-restricted CD4(+) T cells. However, the bulk culture approaches used for assessing T cell activation make interpretation of some of these studies difficult. To gain a more precise understanding of the interaction between Leishmania-infected macrophages and effector T cells, we have analyzed various parameters of T cell activation in individual macrophage-T cell conjugates. Leishmania-infected macrophages efficiently stimulate Ag-independent as well as Ag-dependent, TCR-mediated capping of cortical F-actin in DO.11 T cells. However, infected macrophages are less efficient at promoting the sustained TCR signaling necessary for reorientation of the T cell microtubule organizing center and for IFN-gamma production. A reduced ability to activate these T cell responses was not due to altered levels of surface-expressed MHC class II-peptide complexes. This study represents the first direct single-cell analysis of the impact of intracellular infection on the interaction of macrophages with T cells and serves to emphasize the subtle influence Leishmania has on APC function.     

Auxin-mediated induction of GAL promoters by conditional degradation of Mig1p improves sesquiterpene production in Saccharomyces cerevisiae with engineered acetyl-CoA synthesis

The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l⁻¹ in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose.     

Ion-binding properties of calbindin D9k: a Monte Carlo simulation study

Monte Carlo simulations are used to calculate the binding constant of two Ca2+ ions to the protein bovine calbindin D9k. The change in binding constant with respect to mutation of charged amino acids, presence of various electrolytes, protein concentration, solution pH, and competitive binding of monovalent ions is investigated. Each of these factors may have a large influence on the binding constant. The simulations are performed in a dielectric continuum model, the so-called primitive model of electrolyte theory, with a fixed protein structure and a uniform dielectric permittivity. The calculated binding constants are in excellent agreement with experimental data and describe changes in the binding constant over six orders of magnitude.