Proteomic analysis of blastema formation in regenerating axolotl limbs

Background

For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.

Results

Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.

Conclusion

This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.     

Carotenoid-based plumage colouration is associated with blood parasite richness and stress protein levels in blue tits (Cyanistes caeruleus)

Carotenoids are molecules that birds are not able to synthesize and therefore, must be acquired through their diet. These pigments, besides their function of giving birds red and yellow colouration when deposited in feathers, seem to act as immune-stimulators and antioxidants in the organism. Hence, only the healthiest individuals would be able to express carotenoid-based ornaments to a larger extent without compromising the physiological functions of carotenoids. Various studies have reported that birds infected by parasites are paler than those uninfected, but, to our knowledge, none of them has assessed the possible effect of multiple infections by blood parasites on plumage colour. By comparing the yellow colour in the breast plumage of blue tits, Cyanistes caeruleus, between birds infected by different numbers of blood parasite genera, we found that those birds infected by more than one genus were paler than those parasitized just by one. In addition, we examined the potential role of carotenoid-based plumage colour of blue tits as a long-term indicator of other parameters of health status, such as body condition and immunoglobulin and heat shock protein (HSP) levels. Our results indicate that more brightly coloured birds had lower HSP70 levels than paler birds, but we did not find any significant association between colour and body condition or immunoglobulin levels. In addition, we found a positive significant association between Haemoproteus density of infection and HSP60 levels. Overall, these results support the role of carotenoid-based colours as indicators of health status in blue tits and show detrimental effects of parasitism on this character.     

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.     

The Afro-American Biomphalariae

The reports on the occurrence of African planorbids in South America and of South American species in Africa and Asia are reviewed.     

Kinetic Analysis of the ex vivo Expansion of Human Hematopoietic Stem/Progenitor Cells

The total cell expansion of human umbilical cord blood (CB) and adult bone marrow (BM) CD34+-enriched cells cultured in supplemented serum-free media, either over irradiated human feeder layers or in stroma-free systems, were characterized by a simple kinetic model using only two parameters: the specific cell expansion rate, mu, and the death rate constant, k(k). Both CB and BM cells can expand at approximately the same rate (0.21 day(-1)) in this culture system however, cell death depends on the presence of stroma and the environment in which the cells are cultured.     

Heterochrony in Silurian phacopid trilobites as suggested by the ontogeny of Acernaspis

New material of early growth stages of the Silurian (Llandovery) trilobite Acernaspis is described. Pre-adult ontogenetic stages of this genus closely resemble adults of the post-Llandovery genus ***Ananaspis. A heterochronic descent of Ananaspis from Acernaspis is proposed. ***Ananaspis is interpreted as pae-domorphic, having arisen largely through neoteny. Neotenic changes already appear in the lineage in the last Acernaspis species, and Ananaspis then underwent continuous ncotcnic change throughout its known Silurian history. ▭ Heterochrony, neoteny, Silurian, Trilobita, Phacopidae, Acernaspis. Ananaspis.     

Metformin reduces the stimulatory effect of obesity on in vivo Walker-256 tumor development and increases the area of tumor necrosis

AimsThe objective of this study was to analyze the influence of obesity and insulin resistance on tumor development and, in turn, the effect of insulin sensitizing agents.Main methodsMale offspring of Wistar rats received monosodium glutamate (400 mg/kg) (obese) or saline (control) from the second to sixth day after birth. Sixteen-week-old control and obese rats received 5 × 10⁵ Walker-256 tumor cells, subcutaneously injected into the right flank. Some of the obese and control rats received concomitant treatment with metformin (300 mg/kg) by gavage. At the 18th week, obesity was characterized. The percentage of rats that developed tumors, the tumor relative weight and the percentage of cachexia incidence were analyzed. The tumor tissue was evaluated histologically by means of hematoxylin and eosin staining.Key findingsMetformin did not correct the insulin resistance in obese rats. The tumor development was significantly higher in the obese group, whereas metformin treatment reduced it. After pathological analysis, we observed that the tumor tissues were similar in all groups except for adipocytes, which were found in greater quantity in the obese and metformin-treated obese groups. The area of tumor necrosis was higher in the group treated with metformin when compared with the untreated one.SignificanceMetformin reduced Walker-256 tumor development but not cachexia in obese rats. The reduction occurred independently of the correction of insulin resistance. Metformin increased the area of necrosis in tumor tissues, which may have contributed to the reduced tumor development.     

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

Brassinosteroids mitigate cadmium toxicity in cowpea plants

Anthropogenic activities and improper uses of phosphate fertilizers have led to an increase in cadmium concentrations in agricultural soils. Brassinosteroids are steroid hormones that are rapidly assimilated and metabolised with beneficial roles in physiological and biochemical processes in plants. Our aim was to ascertain whether exogenous treatment with 24-epibrassinolide (EBR) can mitigate the Cd toxicity, and whether this substance can reduce the Cd accumulation in plant tissues. Furthermore, the dose response to EBR was determined following exposure to Cd in Vigna unguiculata. The experiment was a completely randomised factorial design with two concentrations of Cd (0 and 500 μM) and three concentrations of EBR (0, 50, and 100 nM). Spraying plants exposed to Cd with EBR significantly reduced the concentrations of Cd and increased nutrient contents in all tissues. The EBR treatment caused significant enhancements in leaf, root, and total dry matter. Foliar application of EBR reduced the negative effects of Cd toxicity on chlorophyll fluorescence and gas exchange parameters. Pretreatment with EBR also increased contents of pigments in plants exposed to Cd, compared with the identical treatments without EBR. Cd elevated contents of oxidant compounds, inducing cell damages, while EBR significantly decreased the concentrations of these compounds. We confirmed that EBR mitigated the negative effects related to Cd toxicity, reduced the absorption and transport of Cd, and increased the contents of essential elements. In plants exposed to Cd, the most apparent dose response was found for 100 nM EBR, with beneficial repercussions on growth, gas exchange, primary photosynthetic processes, and photosynthetic pigments, which were intrinsically connected to lower production of oxidant compounds and cell damage.