Cell shape and TGF‐β signaling define the choice of lineage during in vitro differentiation of mouse primary hepatic precursors

Cellular differentiation relies on both physical and chemical environmental cues. The bipotential mouse embryonic liver (BMEL) cells are early progenitors of liver epithelial cells with an apparently infinite proliferative potential. These cells, which remain undifferentiated in a monolayer culture, differentiate upon release from geometrical constraints imposed by growth on a stiff plastic plate. In a complex three dimensional environment of a Matrigel extracellular matrix, BMEL cells form two types of polarized organoids of distinct morphologies: cyst‐like structures suggesting cholangiocyte‐type organization or complex organoids, reminiscent of liver parenchyma and associated with acquisition of hepatocyte‐specific phenotypic markers. The choice of the in vitro differentiation lineage is governed by Transforming Growth Factor‐β (TGF‐β) signaling. Our results suggest that morphological cues initiate the differentiation of early hepatic precursors and confirm the inhibitory role of TGF‐β on hepatocytic lineage differentiation. J. Cell. Physiol. 225: 186–195, 2010. © 2010 Wiley‐Liss, Inc.     

CITRATE OXIDATION IN THE CYTOPLASMIC FRACTION OF RAT BRAIN

Abstract— The cytoplasmic fraction derived from rat brain was shown to possess the ability to oxidize citrate in the presence of NADP, but not in that of NAD. The rate of citrate oxidation is limited by the rate of the aconitase-catalysed isomerization. The dependence of the reaction rate on the protein, citrate and NADP concentrations, and on reaction time, was determined. It was found that 2-oxoglutarate and NADPH, both formed in the citrate oxidation reaction, do not appear in amounts equivalent to the losses of citrate. The possible ways of utilization of the above products in the rat brain cytoplasmic fraction are discussed.
It is suggested that the oxidation of citrate in rat brain cytoplasm may proceed–among other metabolic routes–through its conversion into isocitrate (aconitase) and then 2-oxoglutarate (NADP dehydrogenase) and oxaloacetate (aspartate aminotransferase). In addition it has been shown that hypoxia may markedly effect the activity of the cytoplasmic citrateoxidizing system.     

Array comparative genomic hybridization and genomic sequencing in the diagnostics of the causes of congenital anomalies

The aim of this review is to provide the current state of knowledge about the usefulness of modern genetic technologies in uncovering the causality of isolated and multiple congenital anomalies. Array comparative genomic hybridization and next-generation sequencing have revolutionized the clinical approach to patients with these phenotypes. Both technologies enable early diagnosis, especially in clinically challenging newborn populations, and help to uncover genetic defects associated with various phenotypes. The application of both complementary methods could assist in identifying many variants that may simultaneously be involved in the development of a number of isolated or multiple congenital anomalies. Both technologies carry serious variant misinterpretation risks as well. Therefore, the methods of variant classification and accessible variant databases are mentioned. A useful strategy of clinical genetic testing with the application of both methodologies is presented. Finally, future directions and challenges are briefly commented on in this review.     

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation

A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.     

Lymphokines secreted from sodium periodate-treated lymphocytes

This study was designed to determine if sodium metaperiodate (NaIO₄)-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO₄ induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO₄ induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO₄ was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO₄-treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO₄ stimulation. NaIO₄-stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO₄ as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO₄ is removed, and the lymphokines can be obtained free from the inducer.     

Antimicrobial activity and total content of polyphenols of <Emphasis Type="Italic">Rheum</Emphasis> L. species growing in Poland

In the crude ethanol extracts obtained from the rhizome and roots of Rheum palmatum L., Rheum undulatum L. and Rheum rhaponticum L. growing in Poland concentration of polyphenols ranged from 46.11 to 76.45 mg/g. Concentration of tannins ranged from 7.07% to 8.67%, while anthracene derivatives and anthraquinones varied by species - R. palmatum measured 36.3 and 34 mg/g, while R. undulatum or R. rhaponticum did not exceed 20.4 and 18.1 or 19.8 mg/g and 16.6 mg/g, respectively. Using a broth microdilution method it was found that all of the Rheum spp. extracts were more active against reference strains of Gram-positive bacteria (Staphylococcus spp.) than against those of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis). The strongest inhibitory effect against Staphylococcus spp. was exerted by R. undulatum extract with MIC = 125–250 μg/mL. The moderate in vitro antibacterial activity of R. undulatum suggests that this plant, often used in the European cuisine to improve flavour, may be also important and useful as an alternative or auxiliary medicine remedy in the treatment of uncomplicated superficial infections caused especially by clinically important staphylococci, potentially pathogenic S. aureus or opportunistic S. epidermidis.     

Molecular data reveal high taxonomic diversity of allogromiid Foraminifera in Explorers Cove (McMurdo Sound,Antarctica)

Allogromiids are organic-walled or agglutinated, single-chambered Foraminifera, common in deep-sea and polar benthic communities. The simple forms and paucity of distinctive features make allogromiid identification difficult by traditional means. Molecular phylogenetic methods offer alternative tools for species identification and are used here to investigate allogromiid diversity. We obtained 135 partial small-subunit ribosomal DNA sequences of allogromiids collected in Explorers Cove, McMurdo Sound, Antarctica. In contrast to the 27 morphotypes identified, phylogenetic analysis revealed 49 molecular types (considered separate species) that differ by more than 5% of sequence divergence. The 49 genetic types form 28 molecular supra-groups that differ by more than 20% and probably represent distinct genera or families. Large genetic distances separating the molecular types indicate unexpectedly high taxonomic diversity. Comparison of our data with sequences of non-Antarctic allogromiids suggests that Explorers Cove species might be endemic and only distantly related to comparable northern hemisphere fauna.