Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.     

Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the alpha-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.     

Dynamic modification of cortical orientation tuning mediated by recurrent connections

Receptive field properties of visual cortical neurons depend on the spatiotemporal context within which the stimuli are presented. We have examined the temporal context dependence of cortical orientation tuning using dynamic visual stimuli with rapidly changing orientations. We found that tuning to the orientation of the test stimulus depended on a briefly presented preceding stimulus, with the preferred orientation shifting away from the preceding orientation. Analyses of the spatial-phase dependence of the shift showed that the effect cannot be explained by purely feedforward mechanisms, but can be accounted for by activity-dependent changes in the recurrent interactions between different orientation columns. Thus, short-term plasticity of the intracortical circuit can mediate dynamic modification of orientation tuning, which may be important for efficient visual coding.     

CD26: an expanding role in immune regulation and cancer

In this review, we highlight major aspects of the biology of CD26, a dipeptidyl peptidase IV (DPPIV)-containing surface glycoprotein with multiple functions. In particular, we discuss findings demonstrating that CD26/DPPIV has an essential role in immune regulation as a T cell activation molecule and a regulator of chemokine function. We also review recent studies that identify key cellular molecules that physically associate with CD26 and the potential consequences of their interaction, including those with clinically-related implications. Furthermore, we present work suggesting a role for CD26 in the pathogenesis and behavior of selected human cancers, both solid tumors and hematological malignancies. We present recent studies that investigate the potential role of CD26 as a molecular target for novel treatment modalities for T cell lymphoid malignancies and possibly other hematological malignancies, with work involving the use of anti-CD26 monoclonal antibody, CD26-transfected cells as well as soluble CD26 molecules.     

Variation in mitochondrial DNA of Vietnamese pigs: relationships with Asian domestic pigs and Ryukyu wild boars

Mitochondrial DNA (mtDNA) sequences (574 bp) of 30 Vietnamese pigs (large and small) were examined and compared with those of 61 haplotypes from wild boars and domestic pigs from various locations in Asia. The large Vietnamese pigs had genetic links to Ryukyu wild boars in southern Japan. The small Vietnamese pigs were closely related to other East Asian domestic pigs. These results indicate that Vietnamese pigs are genetically diverse and may be descendents of wild and domestic pigs from other regions of Asia.     

Thelephantins D-H: five p-terphenyl derivatives from the inedible mushroom Thelephora aurantiotincta

Five p-terphenyl derivatives named thelephantins D-H (1-5) together with nine known compounds, thelephantins A-C (6-8), ganbajunin E (9), p-hydroxylbenzoic acid (10), ganbajunin C (11), thelephorin A (12), 2-O-methylatromentin (13) and atromentin (14), were isolated from the methanolic extract of fruit bodies of the Thelephoraceous Basidiomycete Thelephora aurantiotincta. Their structures were elucidated by high-resolution MS, 2D NMR, IR and UV spectroscopic analysis.     

A Robertsonian translocation, rob(2;28), found in Vietnamese cattle

A new Robertsonian translocation, rob(2;28), was discovered in a local population of the Vietnamese Cattle. The animal (2n = 59, XY) was found by Q- and R-banding to be a heterozygous carrier of a centric fusion translocation involving chromosomes 2 and 28. FISH analysis using a bovine satellite I DNA probe demonstrated that the centromeric heterochromatin block of the rob(2;28) chromosome become much smaller than its ancestors suggesting a monocentric nature of this centric fusion. This is the first report identifying a Robertsonian translocation in Southeast Asian cattle by karyotyping of banded chromosomes.     

G-protein signaling abnormalities mediated by CD95 in salivary epithelial cells

Salivary epithelial cells from patients with primary Sj?gren's syndrome (SS) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressing oncogenes. Very little is known about the role of these oncogene molecules in salivary epithelial cells. To investigate the possible prevention of salivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectants expressing these molecules were made from HSY cells, a human salivary epithelial cell line. HSY cells were transfected with an expression vector for human Bcl-2 or Bcl-xL. Stable transfectants were selected and apoptosis was induced by anti-Fas antibody. Apoptosis was quantified by propidium iodide staining followed by flow cytometry. Caspase activity was detected by immunohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent substrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca2+ mobilization and influx. Fas-mediated apoptosis was significantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity was not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pathway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carbachol and EGF responsiveness (i.e., Ca2+ mobilization and/or influx) by 50-60%. This Fas-mediated inhibition of cell activation was partially or completely restored by specific peptide interference of caspase enzyme activity. The prevention of Fas-mediated apoptosis by the overexpression of Bcl-2 and Bcl-xL in salivary gland epithelial cells results in injured cells expressing caspase activity and unable to respond normally to receptor agonists. Such damaged cells may exist in SS patients and could explain the severe dryness out of proportion to the actual number of apoptotic cells seen on salivary gland biopsy.     

The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.