Covalent immobilization of cellulose layers onto maleic anhydride copolymer thin films

Thin films of cellulose are advantageous for analytical studies in aqueous environments to investigate various factors determining the performance of cellulose-based products. However, the weak fixation of cellulose layers on common carrier materials often limits this approach. To address this problem, we suggest a novel maleic anhydride copolymer precoating technique which allows for the covalent attachment of cellulose thin films through esterification. Maleic anhydride copolymers were deposited and covalently bound onto planar, aminosilane-modified glass or silicon oxide surfaces. Cellulose was subsequently immobilized on top of the copolymer precoatings by spin coating from N-methylmorpholine-N-oxide/dimethyl sulfoxide solutions. The resulting cellulose films were thoroughly characterized with respect to layer thickness, morphology, chemical constitution, and electrical charging. The stability of the layers against shear stress was demonstrated in aqueous solutions and the covalent attachment of the cellulose to the copolymer films was proven by means of dissolution experiments followed by ellipsometry and high-resolution X-ray photoelectron spectroscopy.     

Myelomonocytic cells are sufficient for therapeutic cell fusion in liver

Liver repopulation with bone marrow-derived hepatocytes (BMHs) can cure the genetic liver disease fumarylacetoacetate hydrolase (Fah) deficiency. BMHs emerge from fusion between donor bone marrow-derived cells and host hepatocytes. To use such in vivo cell fusion efficiently for therapy requires knowing the nature of the hematopoietic cells that fuse with hepatocytes. Here we show that the transplantation into Fah(-/-) mice of hematopoietic stem cells (HSCs) from lymphocyte-deficient Rag1(-/-) mice, lineage-committed granulocyte-macrophage progenitors (GMPs) or bone marrow-derived macrophages (BMMs) results in the robust production of BMHs. These results provide direct evidence that committed myelomonocytic cells such as macrophages can produce functional epithelial cells by in vivo fusion. Because stable bone marrow engraftment or HSCs are not required for this process, macrophages or their highly proliferative progenitors provide potential for targeted and well-tolerated cell therapy aimed at organ regeneration.     

Integrative analysis of the mitochondrial proteome in yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.     

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y₁ receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y₁ receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y₁ receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli.     

Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.     

Biochemical analysis of neutral endopeptidase activity reveals independent catabolism of atrial and brain natriuretic peptide

Recent reports presented contradictory results regarding the catabolism of mature atrial (ANP) and brain (BNP) natriuretic peptides in circulation. Especially the role of neutral endopeptidase (NEP) in BNP degradation was conversely discussed. Our present in vitro-studies characterize the NEP-dependent metabolism of ANP and BNP in different tissues via HPLC-analysis using NEP-deficient mice and specific NEP inhibitors. Our results show a strong tissue-dependent degradation pattern of both peptides, which are not only due to the different NEP activities in these tissues. Whereas NEP rapidly degraded ANP, it had no influence in BNP-metabolism. Additional experiments with purified NEP confirmed this result. Moreover, we describe a degradation of ANP and BNP in NEP-deficient- and NEP-inhibited membranes. Consequently, we postulate the existence of at least one further natriuretic peptide (NP) degrading enzyme, which has not been characterized yet. Thus, the commonly accepted model of the natriuretic peptide system with NEP as the central degrading peptidase has to be partly revised. Moreover, the NEP-independent BNP degradation provides an effective means for achieving a beneficial BNP increase in cardiovascular pathology by inhibiting the assumed novel NP-degrading peptidase(s).     

Contractile function of rat myocardium is less susceptible to hypoxia/reoxygenation after acute infarction

The FHIT (fragile histidine triad) gene located at chromosome 3p14.2 has been proposed as a candidate tumor suppressor gene in human cancers. Fhit protein with the diadenosine 5',5'-P¹,P³-triphosphate (Ap₃A) hydrolase activity is the protein product of FHIT gene. The way in which Fhit exerts its tumor suppressor activity and the relationship of the Ap₃A hydrolase activity to tumor suppression are not known. As a step toward understanding of the Fhit function in the cell we have explored its intracellular localization and distribution in the rat tissues. Data obtained from immunoblot analysis showed that Fhit protein was most abundant in spleen and brain. Moderate amount of Fhit was detected in kidney and liver, whereas the level of Fhit protein in heart, skeletal muscle and kidney glomeruli was undetectable. RT-PCR performed on RNA isolated from these tissues showed no product, whereas the level of Fhit mRNA in spleen, brain, kidney, liver and lung correlated with the Fhit protein level. The immunoblot analysis performed on subcellular fractions of various rat tissues obtained by differential and density-gradient centrifugation showed that Fhit protein was localized exclusively in nucleus and at the plasma membrane. Presented data showing nuclear and plasma membrane localization of Fhit may support the hypothesis concerning Fhit as a signaling molecule.     

Substrate specificity of lipase B from Candida antarctica in the synthesis of arylaliphatic glycolipids

Arylaliphatic glycolipids are known for their pharmaceutical and medicinal properties. We found that a great variety of arylaliphatic esters can be synthesized from non-activated substrates like glucose or the natural occurring drug salicin using lipase B from Candida antarctica (CAL-B). However, esters based on aromatic carboxylic acids or unsaturated arylaliphatic acids, like cinnamic acid and its derivatives, which are known to display anticancer activity, could not be obtained. In this work, we performed computer-aided molecular modeling based on data of our work published recently and syntheses of new glycolipids to understand why some substances are accepted by CAL-B while some are not. For this purpose, we investigated the accessibility of the lipase binding site for the arylaliphatic acyl donors as well as the steric interactions between the aglycons of glucosides and the residues of the alcohol binding pocket in order to elucidate potentials and limitations of CAL-B for the synthesis of aromatic glycolipids.