Lacunarity as a novel measure of cancer cells behavior

An important goal in many branches of science, especially in molecular biology and medicine is the quantitative analysis of the structures and their morphology. The morphology can be analyzed in many ways, in particular by the fractal analysis. Apart from the fractal dimension, an important part of the fractal analysis is the lacunarity measurement which, roughly speaking, characterizes the distribution of gaps in the fractal: a fractal with high lacunarity has large gaps. In this paper, we present an extension of the lacunarity measure to objects with nonregular shapes that enables us to provide a successful discrimination of cancer cell lines. The cell lines differ in the shape of vacuole (the gaps in their body) which is perfectly suited for the lacunarity analysis.     

Identification of a new co-factor, MOG1, required for the full function of cardiac sodium channel Nav 1.5

The cardiac sodium channel Nav 1.5 is essential for the physiological function of the heart and contributes to lethal cardiac arrhythmias and sudden death when mutated. Here, we report that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and modulates the physiological function of Nav 1.5. The yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domains DII and DIII) of Nav 1.5. The interaction was further demonstrated by both in vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation assays in both HEK293 cells with co-expression of MOG1 and Nav1.5 and native cardiac cells. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities. In neonatal myocytes, overexpression of MOG1 increased current densities nearly 2-fold. Western blot analysis revealed that MOG1 increased cell surface expression of Nav1.5, which may be the underlying mechanism by which MOG1 increased sodium current densities. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues with predominant localization at the intercalated discs. In cardiomyocytes, MOG1 is mostly localized in the cell membrane and co-localized with Nav1.5. These results indicate that MOG1 is a critical regulator of sodium channel function in the heart and reveal a new cellular function for MOG1. This study further demonstrates the functional diversity of Nav1.5-binding proteins, which serve important functions for Nav1.5 under different cellular conditions.     

Cryptosporidium parvum and Giardia lamblia recovered from flies on a cattle farm and in a landfill

Filth flies associated with a cattle barn and a municipal landfill were tested positive by combined immunofluorescent antibody and fluorescent in situ hybridization for Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their guts. More pathogens were carried by flies from the cattle barn than from the landfill; 81% of C. parvum and 84% of G. lamblia pathogens were presumptively viable.     

Phosphatidylglycerol is essential for oligomerization of photosystem I reaction center

Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center.     

Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.     

A nonhydrolyzable analogue of phosphotyrosine, and related aryloxymethano- and aryloxyethano-phosphonic acids as motifs for inhibition of phosphatases

Nonhydrolyzable analogues of both stereoisomers of phosphotyrosine, and a series of related aryloxy (or thio) methyl and aryloxy (or thio) ethyl phosphonic acids of the general formula RX-(CH(2))(n)-PO(3)H(2) (where X=O or S and n=1 or 2), have been tested as nonhydrolyzable mimetics of phosphatase substrates. These compounds were tested against a panel of phosphatases (two alkaline phosphatases, a protein-tyrosine phosphatase, and two serine/threonine phosphatases) with different active site motifs. The compounds exhibit competitive inhibition toward all enzymes tested, with the best inhibition expressed toward the Ser/Thr phosphatases. The stereoisomers of the phosphotyrosine analogues exhibited an unexpected difference in their inhibitory properties toward the protein-tyrosine phosphatase from Yersinia. The K(i) for the d isomer is 33-fold lower than that of the l isomer, and is more than an order of magnitude lower than the reported K(m) of the substrate l-phosphotyrosine.     

On time-space of nonlinear phenomena with Gompertzian dynamics

This paper describes a universal relationship between time and space for a nonlinear process with Gompertzian dynamics, such as growth. Gompertzian dynamics implicates a coupling between time and space. Those two categories are related to each other through a linear function of their logarithms. Moreover, we demonstrate that the spatial fractal dimension is a function of both scalar time and the temporal fractal dimension. The Gompertz function reflects the equilibrium of regular states, that is, states with dynamics that are predictable for any time-point (e.g., sinusoidal glycolytic oscillations) and chaotic states, that is, states with dynamics that are unpredictable in time, but are characterized by certain regularities (e.g., the existence of strange attractor for any biochemical reaction). We conclude that both this equilibrium and volume of the available complementary Euclidean space determine temporal and spatial expansion of a process with Gompertzian dynamics.     

Donor substrate regeneration for efficient synthesis of globotetraose and isoglobotetraose

Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4Glc) and isoglobotetraose (GalNAcbeta1-->3Galalpha1-->3Galbeta1-->4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant beta-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.     

Cytokine secretion by decidual lymphocytes in transient hypertension of pregnancy and pre-eclampsia

BACKGROUND: Transient hypertension (TH) and preeclampsia (PE) are believed to have different pathophysiology. However, 15-25% of pregnant women initially diagnosed as having TH develop PE. To clarify the immuno-pathogenetical connections between the two syndromes, we studied the pattern of T helper cell (Th)1/Th2 cytokine balance disturbances existing inside maternal decidua in normal pregnancy (NP) and pregnancies complicated with TH and PE. METHODS: Third-trimester decidual tissue was obtained by curettage of uterine cavity during elective caesarean sections in NP (n = 11), TH (n = 17) and PE (n = 21) patients. Cell suspensions were prepared by an electromechanical dispersal method and centrifugated using a standard gradient sedimentation technique. Isolated lymphocytes were placed in medium (RPMI 1640, 10% fetal calf serum, L-glutamine, penicillin, streptomycin) and cultured for 72 h with or without mitogen phytohaemaglutinine (PHA). The enzyme-linked immunosorbent assay method was used for estimation of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 and interferon-gamma (IFN-gamma) in culture supernatant. STATISTICAL ANALYSIS: The Kruskal-Wallis and the Mann-Whitney U tests were used (p < 0.05). RESULTS: Both spontaneous and PHA-stimulated secretion of Th2-type cytokines IL-6 and IL-10 was decreased in PE patients compared with TH and NP patients. The concentration of Th1-type cytokine IFN-gamma was increased in patients suffering both from TH and PE. CONCLUSION: On the base of decidual cytokine secretion, both PE and TH are syndromes of local Th1/Th2 cytokine balance disturbances as compared with NP, and TH seems to be an intermediate step to PE.     

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.