In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.     

In vitro propagation and rhizome formation in Curcuma longa Linn

Turmeric (Curcuma longa Linn.) which is cultivated by underground rhizomes is a slow propagating species. Multiplication and callus induction starting from the rhizome buds and shoot tips of C. longa in MS medium was carried out. A combination of naphthalene acetic acid (NAA; 1.0 mg/l) with kinetin (Kn; 1.0 mg/l) or NAA (1.0 mg/l) with 6-benzylaminopurine (BAP; 2.0 mg/l) was optimum for rapid clonal propagation of turmeric. A concentration of 2.5-3.0 mg/l of 2,4-dichlorophenoxy-acetic acid (2,4-D) was found to be optimum for callus induction. Regeneration of plantlets from a callus was successfully conducted in MS medium supplemented with standard growth hormones for multiplication at 25 +/- 2 degrees C under a 16 h photoperiod. These plantlets were successfully transferred to the field. Plantlets (4-month-old) were incubated in a medium containing different concentrations of sucrose supplemented with NAA (0.1 mg/l) and Kn (1.0 mg/l) at 27 +/- 2 degrees C under an 8 h photoperiod for induction of rhizomes. In vitro rhizome formation was observed in media containing 6 and 8% sucrose.     

Effect of magnesium ions on the tertiary structure of the hepatitis C virus IRES and its affinity for the cyclic peptide antibiotic viomycin

A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg(2+) ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with K(d) values of 25-55 microM. Interestingly, viomycin binding to the two fragments is affected differently by Mg(2+); noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg(2+)-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg(2+) ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

The influenced of PAC, zeolite, and Moringa oleifera as biofouling reducer (BFR) on hybrid membrane bioreactor of palm oil mill effluent (POME)

The main objective of this work was to determine the effectiveness of various biofouling reducers (BFRs) to operational condition in hybrid membrane bioreactor (MBR) of palm oil mill effluent (POME). A series of tests involving three bench scale (100 L) hybrid MBR were operated at sludge retention times (SRTs) of 30 days with biofouling reducer (BFR). Three different biofouling reducers (BFRs) were powdered actived carbon (PAC), zeolite (Ze), and Moringa oleifera (Mo) with doses of 4, 8 and 12 g L⁻¹ respectively were used. Short-term filtration trials and critical flux tests were conducted. Results showed that, all BFRs successfully removed soluble microbial products (SMP), for PAC, Ze, and Mo at 58%, 42%, and 48%, respectively. At their optimum dosages, PAC provided above 70% reductions and 85% in fouling rates during the short-term filtration and critical flux tests.     

Why Don't We Ask? A Complementary Method for Assessing the Status of Great Apes

Species conservation is difficult. Threats to species are typically high and immediate. Effective solutions for counteracting these threats, however, require synthesis of high quality evidence, appropriately targeted activities, typically costly implementation, and rapid re-evaluation and adaptation. Conservation management can be ineffective if there is insufficient understanding of the complex ecological, political, socio-cultural, and economic factors that underlie conservation threats. When information about these factors is incomplete, conservation managers may be unaware of the most urgent threats or unable to envision all consequences of potential management strategies. Conservation research aims to address the gap between what is known and what knowledge is needed for effective conservation. Such research, however, generally addresses a subset of the factors that underlie conservation threats, producing a limited, simplistic, and often biased view of complex, real world situations. A combination of approaches is required to provide the complete picture necessary to engage in effective conservation. Orangutan conservation (Pongo spp.) offers an example: standard conservation assessments employ survey methods that focus on ecological variables, but do not usually address the socio-cultural factors that underlie threats. Here, we evaluate a complementary survey method based on interviews of nearly 7,000 people in 687 villages in Kalimantan, Indonesia. We address areas of potential methodological weakness in such surveys, including sampling and questionnaire design, respondent biases, statistical analyses, and sensitivity of resultant inferences. We show that interview-based surveys can provide cost-effective and statistically robust methods to better understand poorly known populations of species that are relatively easily identified by local people. Such surveys provide reasonably reliable estimates of relative presence and relative encounter rates of such species, as well as quantifying the main factors that threaten them. We recommend more extensive use of carefully designed and implemented interview surveys, in conjunction with more traditional field methods.     

Anion binding and controlled aggregation of human interleukin-1 receptor antagonist

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.     

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay.In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.