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.     

Long-term release of clodronate from biodegradable microspheres

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions. Published: July 11, 2001.     

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.     

Environmental controls over carbon exchange of three forest ecosystems in eastern China

Net ecosystem productivity (NEP) was continuously measured using the eddy covariance (EC) technique from 2003 to 2005 at three forest sites of ChinaFLUX. The forests include Changbaishan temperate mixed forest (CBS), Qianyanzhou subtropical coniferous plantation (QYZ), and Dinghushan subtropical evergreen broad‐leaved forest (DHS). They span wide ranges of temperature and precipitation and are influenced by the eastern Asian monsoon climate to varying extent. In this study, we estimated ecosystem respiration (RE) and gross ecosystem productivity (GEP). Comparison of ecosystem carbon exchange among the three forests shows that RE was mainly determined by temperature, with the forest at CBS exhibiting the highest temperature sensitivity among the three ecosystems. The RE was highly dependent on GEP across the three forests, and the ratio of RE to GEP decreased along the North–South Transect of Eastern China (NSTEC) (i.e. from the CBS to the DHS), with an average of 0.77 ± 0.06. Daily GEP was mainly influenced by temperature at CBS, whereas photosynthetic photon flux density was the dominant factor affecting the daily GEP at both QYZ and DHS. Temperature mainly determined the pattern of the interannual variations of ecosystem carbon exchange at CBS. However, water availability primarily controlled the interannual variations of ecosystem carbon exchange at QYZ. At DHS, NEP attained the highest values at the beginning of the dry seasons (autumn) rather than the rainy seasons (summer), probably because insufficient radiation and frequent fog during the rainy seasons hindered canopy photosynthesis. All the three forest ecosystems acted as a carbon sink from 2003 to 2005. The annual average values of NEP at CBS, QYZ, and DHS were 259 ± 19, 354 ± 34, and 434 ± 66 g C m⁻² yr⁻¹, respectively. The slope of NEP that decreased with increasing latitude along the NSTEC was markedly different from that observed on the forest transect in the European continent. Long‐term flux measurements over more forest ecosystems along the NSTEC will further help verify such a difference between the European forest transect and the NSTEC and provide insights into the responses of ecosystem carbon exchange to climate change in China.     

Geographical variation in size in the European rabbit Oryctolagus cuniculus (Lagomorpha: Leporidae) in western Europe and North Africa

Craniometric data of nine characters from 260 skulls of Oryctolagus cuniculus from western Europe and northern Africa were gathered to examine phenotypic variation in size, using both univariate and multivariate analyses. Although animals from the northern part of Europe were found to be larger than those from southern Europe, variation in size occurred in a continuous cline. Skull size was positively correlated with latitude and negatively correlated with ambient temperature. On the basis of skull morphology, there is no evidence for separation of O. cuniculus into the two or more subspecies commonly described. Further data, particularly from genetic material, are required to clarify the taxonomic status of the rabbit in Europe.     

GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease

Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).     

Reactive oxygen species scavenging by catalase is important for female Lutzomyia longipalpis fecundity and mortality

The phlebotomine sand fly Lutzomyia longipalpis is the most important vector of American visceral leishmaniasis (AVL), the disseminated and most serious form of the disease in Central and South America. In the natural environment, most female L. longipalpis are thought to survive for less than 10 days and will feed on blood only once or twice during their lifetime. Successful transmission of parasites occurs when a Leishmania-infected female sand fly feeds on a new host. Knowledge of factors affecting sand fly longevity that lead to a reduction in lifespan could result in a decrease in parasite transmission. Catalase has been found to play a major role in survival and fecundity in many insect species. It is a strong antioxidant enzyme that breaks down toxic reactive oxygen species (ROS). Ovarian catalase was found to accumulate in the developing sand fly oocyte from 12 to 48 hours after blood feeding. Catalase expression in ovaries as well as oocyte numbers was found to decrease with age. This reduction was not found in flies when fed on the antioxidant ascorbic acid in the sugar meal, a condition that increased mortality and activation of the prophenoloxidase cascade. RNA interference was used to silence catalase gene expression in female Lu. longipalpis. Depletion of catalase led to a significant increase of mortality and a reduction in the number of developing oocytes produced after blood feeding. These results demonstrate the central role that catalase and ROS play in the longevity and fecundity of phlebotomine sand flies.     

Workers dominate male production in the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae)

Background

Cooperation and conflict in social insects are closely linked to the genetic structure of the colony. Kin selection theory predicts conflict over the production of males between the workers and the queen and between the workers themselves, depending on intra-colonial relatedness but also on other factors like colony efficiency, sex ratios, cost of worker reproduction and worker dominance behaviour. In most bumblebee (Bombus) species the queen wins this conflict and often dominates male production. However, most studies in bumblebees have been conducted with only a few selected, mostly single mated species from temperate climate regions. Here we study the genetic colony composition of the facultative polyandrous neotropical bumblebee Bombus wilmattae, to assess the outcome of the queen-worker conflict over male production and to detect potential worker policing.

Results

A total of 120 males from five colonies were genotyped with up to nine microsatellite markers to infer their parentage. Four of the five colonies were queen right at point of time of male sampling, while one had an uncertain queen status. The workers clearly dominated production of males with an average of 84.9% +/- 14.3% of males being worker sons. In the two doubly mated colonies 62.5% and 96.7% of the male offspring originated from workers and both patrilines participated in male production. Inferring the mother genotypes from the male offspring, between four to eight workers participated in the production of males.

Conclusions

In this study we show that the workers clearly win the queen-worker conflict over male production in B. wilmattae, which sets them apart from the temperate bumblebee species studied so far. Workers clearly dominated male production in the singly as well the doubly mated colonies, with up to eight workers producing male offspring in a single colony. Moreover no monopolization of reproduction by single workers occurred.