Protein tyrosine phosphatase CD45 as a molecular biosensor of hydrogen peroxide generation in cell culture media

We have designed a useful method of assessing reactive oxygen species generation in biological fluids. The novel assay utilizes tyrosine phosphatase CD45 as a biosensor of oxidative stress. Applying this new method, we examined oxygen species generation in the following cell culture media: RPMI 1640, DMEM, DMEM enriched with pyruvate and MEM. We discovered that the media (especially RPMI 1640) significantly reduced the activity of protein tyrosine phosphatase. The media-caused inactivation of CD45 was reversible after treatment with dithiothreitol being a powerful reducing agent. Interestingly, the media supplemented with catalase did not exhibit any inhibitory effect on CD45 activity which suggests a hydrogen peroxide-mediated mechanism of the enzyme inactivation. In addition to that, we assessed the impact of oxidative stress level on the activity of CD45 as measured in Jurkat cells cultured in RPMI 1640 either exposed or not exposed to the light of laminar flow cabinet fluorescent lamp. We found that Jurkat cells that were exposed to light displayed ca. 20% lower activity of CD45 than the cells protected against the light. The obtained results indicate that production of hydrogen peroxide in the medium leading to inhibition of CD45 was light-dependent, and that careful protection of cell culture media from the light may help to prevent the artifact in cell studies. Hydrogen peroxide, responsible for CD45 inactivation, can be generated in cell culture media after exposition to light due to photoreactive amino acids present in the media.     

Suppurative thyroiditis caused by Salmonella enteritidis

Bacterial thyroiditis is a rare disease, and one of which the clinical symptoms and signs are frequently misleading. On the other hand, prompt diagnosis is crucial for successful treatment. We report the case of an 82 year-old man with diabetes mellitus type 2 and a history of steroid treatment who presented with severe odynophagia and dysphagia associated with fever, chills, sore throat and right ear pain. Based on the clinical picture, radiological studies, thyroid cytology, blood and thyroid aspirate culture, suppurative thyroiditis caused by Salmonella enteritidis was diagnosed. The patient was successfully treated with antibiotics and surgical drainage.     

The DCX-domain tandems of doublecortin and doublecortin-like kinase

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.     

Structure of the membrane reconstituted transmembrane-juxtamembrane peptide EGFR(622-660) and its interaction with Ca2+/calmodulin

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.     

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.     

Methods for improving the efficiency of estimating total osteon density in the human anterior mid-diaphyseal femur

In order to preserve whole bone integrity and minimize destruction, paleohistologists often rely on histomorphometric data obtained from small areas (1.5–50 mm²) sampled within the anterior mid-diaphyseal femur. Because bone exhibits significant histological variation, the validity of results based on such sampling is questionable. The accuracy of various subareas (columns, rows, squares approximating dimensions and locations assessed by paleohistologists) in predicting total osteon density in the anterior mid-diaphyseal femur is assessed in the present study. Thirty-five specimens (12.7 mm wide, 100 μm thick, average area 56.7 mm²) were chosen at random from a skeletal population of 94 Inuits and Pueblo agriculturists. The specimens were photographed and enlarged; an acetate grid (12 columns, 10 rows, 120 squares, square = 1 mm² of bone surface) was superimposed over the photograph; and secondary osteons and fragments were identified. Alternate columns (50% total area, T.Ar) predicted over 98% of entire section total osteon density. Two column combinations (15% T.Ar), separated by at least one column, predicted 91 to 95% of total osteon density. Individual column (8% T.Ar) predictability ranged from 48 to 86%. Two row combination (32 to 40% T.Ar) predictability values ranged from 86 to 95%. Individual rows (<1 to 20% T.Ar) predicted from 45 to 92% of total variation. Combinations of squares approximating areas and locations assessed by other paleohistologists ranged in predictability values from 80 to 94%. The results demonstrate that subareas of as little as 15% predict 95% of variation in total osteon density in the entire anterior mid-diaphyseal femoral section. A minimization of histological area evaluated without the loss of accuracy allows for a minimization of time invested in data collection and the utilization of partially damaged specimens. Am J Phys Anthropol 107:13–24, 1998. © 1998 Wiley-Liss, Inc.     

Breaking the apple embryo dormancy by nitric oxide involves the stimulation of ethylene production

Mature seeds of apple (Mallus domestica Borb. cv. Antonówka) are dormant and do not germinate unless their dormancy is removed by several weeks of moist-cold treatment. We investigated the effect of short-term (3 h) nitric oxide (NO) pretreatment on breaking of apple embryonic dormancy expressed as inhibition of germination and morphological abnormalities of young seedlings. Imbibition of embryos isolated from dormant apple seeds with sodium nitroprusside (SNP) or S-nitroso,N-acetyl penicillamine (SNAP) as NO donors resulted in enhanced germination. Moreover, NO treatment removed morphological abnormalities of seedlings developing from dormant embryo. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-1-oxyl-3 oxide (cPTIO) removed the above effects. NO-mediated breaking of embryonic dormancy correlated well with enhanced ethylene production. Inhibitor of ethylene synthesis (AOA) reversed the stimulatory effect of NO donors on embryo germination. Additionally SNP reduced embryo sensitivity to exogenously applied ABA ensuing dormancy breakage. We can conclude that NO acts as a regulatory factor included in the control of apple embryonic dormancy breakage by stimulation of ethylene biosynthesis.