Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.     

Induction of cell cycle arrest in lymphocytes by Actinobacillus actinomycetemcomitans cytolethal distending toxin requires three subunits for maximum activity

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene. In this study, we used rCdt peptides to study the contribution of each subunit to toxin activity. As previously reported, CdtB is the only Cdt subunit that is capable of inducing cell cycle arrest by itself. Although CdtA and CdtC do not exhibit activity alone, each subunit is able to significantly enhance the ability of CdtB to induce G2 arrest in Jurkat cells; these effects were dependent upon protein concentration. Moreover, the combined addition of both CdtA and CdtC increased the ED50 for CdtB >7000-fold. In another series of experiments, we demonstrate that the three Cdt peptides are able to form a functional toxin unit on the cell surface. However, these interactions first require that a complex forms between the CdtA and CdtC subunits, indicating that these peptides are required for interaction between the cell and the holotoxin. This conclusion is further supported by experiments in which both Jurkat cells and normal human lymphocytes were protected from Cdt holotoxin-induced G2 arrest by pre-exposure to CdtA and CdtC. Finally, we have used optical biosensor technology to show that CdtA and CdtC have a strong affinity for one another (10(-7) M). Furthermore, although CdtB is unable to bind to either CdtA or CdtC alone, it is capable of forming a stable complex with CdtA/CdtC. The implications of our results with respect to the function and structure of the Cdt holotoxin are discussed.     

An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Microbial population dynamics on Golden Delicious apples from bud to harvest and effect of fungicide applications

The microbial population dynamics on apples cv. Golden Delicious were analysed every 15 days between bud and harvest in a fully replicated experiment in northern Spain in 1994 and 1995. The total microbial populations varied with developmental stage, and with prevailing climatic conditions. The predominant mycroflora were the filamentous fungi Cladosporium and Alternaria spp. and white and pink yeasts. Other genera isolated included mainly species of Epicoccum, Fusarium and Acremonium. However, the most important post-harvest pathogens Penicillium expansum and Botrytis cinerea were seldom isolated from ripening apples. Maximum total filamentous fungal populations occurred after fruit set and during early ripening [2 × 10⁴cfu (colony-forming units) g^-1 approximately] while those of bacteria were maximum at bud stage (3.5 × 10⁵and 3.0 × 10⁴ cfu g^-1 in 1994 and 1995 respectively). White yeasts were more numerous than pink yeasts. Endophytic infection of apple buds by Alternaria spp., responsible for core rot, was found in almost all bud tissue. By contrast, Cladosporium spp. were initially isolated later from 12.5–50% of tissue samples during blooming and fruit set. The impact of a four-spray fungicide regime during apple development significantly decreased the total filamentous fungal populations in both years, and that of Cladosporium spp. in 1994. However, bacterial populations were often higher on apples from fungicide-treated plots. Fungicide sprays decreased populations of Cladosporium, Alternaria and white yeasts for a maximum of up to 15–30 days after application. Fungicide application had little effect on endophytic infection of apples by Alternaria spp. between bud and harvest.     

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca²⁺ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca²⁺ in the medium. The observed Ca²⁺-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca²⁺ in the medium. Thus, the ability of Ca²⁺ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca²⁺ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca²⁺ channel blockers tested, such as verapamil and LaCl₃, inhibited the observed Ca²⁺-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m -chlorophenyl hydrazone, inhibited this Ca²⁺-mediated response, whereas monensin, an inhibitor of the Na⁺/H⁺ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca²⁺ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.     

Conversion of dihydroorotate to orotate in parasitic protozoa.

The conversion of dihydroorotate to orotate, one of the key reactions in the de novo pyrimidine biosynthetic pathway, has been studied in a number of parasitic protozoa. Enzyme activities capable of carrying out this reaction were detected in six members of the Kinetoplastida (Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Trypanosoma lewisi, Trypanosoma cruzi, Leishmania enriettii) and three members of the genus Plasmodium (P. knowlesi, P. berghei, P. gallinaceum). The mechanism of the reaction in the two groups of protozoa were quite distinct. In the Kinetoplastida, the enzyme is an hydroxylase which occurs in the soluble fraction of the cell and probably requires tetrahydrobiopterin for activity. In contrast, in Plasmodium, the enzyme is a dehydrogenase which is particulate, probably mitochondrial, and intimately connected to the electron transport chain to which it passes electrons directly, probably at the ubiquinone level. Neither activity is regulated by fully formed pyrimidines. The enzyme in Plasmodium is similar in mechanism to the isofunctional mammalian enzyme. However, since malarial ubiquinones are apparently different from those in the mammal and since menoctone, which is active in vivo in experimental malaria, is a good inhibitor of the malarial enzyme, it could represent a useful target for chemotherapeutic attack. The enzyme in the Kinetoplastida is quite distinct from that in the mammal so that it too apparently falls into this category, though none of the currently used antitrypanosomal drugs appears to block it activity at physiological concentrations.     

A brief overview of amyloids and Alzheimer’s disease

Amyloid fibrils are self-assembled fibrous protein aggregates that are associated with a number of presently incurable diseases such as Alzheimer’s and Parkinson’s disease. Millions of people worldwide suffer from amyloid diseases. This review summarizes the unique cross-β structure of amyloid fibrils, morphological variations, the kinetics of amyloid fibril formation, and the cytotoxic effects of these fibrils and oligomers. Alzheimer’s disease is also explored as an example of an amyloid disease to show the various approaches to treat these amyloid diseases. Finally, this review investigates the nanotechnological and biological applications of amyloid fibrils; as well as a summary of the typical biological pathways involved in the disposal of amyloid fibrils and their precursors.