Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.     

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.     

Cytolytic T lymphocyte-defined retroviral antigens on normal cells: Encoding by the Akv-1 proviral locus

We previously described the generation and specificity of H-2-restricted cytolytic lymphocytes (CTL) directed against tumors induced by AKR/Gross murine leukemia viruses (MuLV). Such anti-AKR/Gross virus CTL demonstrated type specificity; only tumors induced by endogenous MuLV that expressed the Gross cell-surface antigen were lysed. These CTL and their precursor also recognized normal spleen cells from AKR-H-2 ^b , but not AKR-H-2 ^b , Fv-1 ^b mice, however, suggesting that N-ecotropic, retrovirus-associated antigens were primarily involved. Here, expression of these CTL-defined retroviral antigens by H-2^b-positive AKR × C57L recombinant inbred strains was examined by using normal spleen cells as stimulators in the generation of specific anti-AKR/Gross virus CTL. Analysis of the strain distribution pattern of stimulation indicated that a single proviral locus, Akv-1, was primarily, if not entirely, responsible for CTL-defined retroviral antigen expression. The lack of correlation with two other well-defined proviral loci was interesting. Whereas Akv-3 is known to encode a defective virus, Akv-4 has been shown to code for an infectious virus thought to be very similar or identical to that of Akv-1. Although quantitative differences cannot be formally excluded, dose response experiments argued against this possibility and suggested that Akv-1 and Akv-4 may exhibit qualitative differences germane to antiviral CTL recognition.     

Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha

We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha. When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide. The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct. They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits.     

Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein

Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1. Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of beta gamma subunits. Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that Gs alpha, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.     

Effect of land use on the phytoplankton community of Pampean shallow lakes of the Salado River basin (Buenos Aires Province,Argentina)

Aquatic Ecology - The Salado River basin, Buenos Aires, Argentina, contains numerous permanent shallow lakes under anthropic pressure due to different land uses/land covers (LULC) in their...     

Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost

Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from <40 to >3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R² = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.     

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+-pumping pyrophosphatase in pepper plants

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.     

Phylogenetic analyses of complete mitochondrial genome sequences suggest a basal divergence of the enigmatic rodent <Emphasis Type="Italic">Anomalurus</Emphasis>

Background  

Phylogenetic relationships between Lagomorpha, Rodentia and Primates and their allies (Euarchontoglires) have long been debated. While it is now generally agreed that Rodentia constitutes a monophyletic sister-group of Lagomorpha and that this clade (Glires) is sister to Primates and Dermoptera, higher-level relationships within Rodentia remain contentious.     

Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation

Stromal stem cells from human dental pulp (SBP-DPSCs) were used to study osteogenic differentiation in vitro and in vivo. We previously reported that SBP-DPSCs are multipotent stem cells able to differentiate into osteoblasts, which synthesize three-dimensional woven bone tissue chips in vitro. In this study, we followed the temporal expression pattern of specific markers in SBP-DPSCs and found that, when differentiating into osteoblasts, they express, besides osteocalcin, also flk-1 (VEGF-R2). In addition, 30% of them expressed specific antigens for endothelial cells, including CD54, von-Willebrand (domain 1 and 2), CD31 (PECAM-1) and angiotensin-converting enzyme. Interestingly, we found endotheliocytes forming vessel walls, observing that stem cells synergically differentiate into osteoblasts and endotheliocytes, and that flk-1 exerts a pivotal role in coupling osteoblast and endotheliocyte differentiation. When either SBP-DPSCs or bone chips obtained in vitro were transplanted into immunocompromised rats, they generated a tissue structure with an integral blood supply similar to that of human adult bone; in fact, a large number of HLA-1+ vessels were observed either within the bone or surrounding it in a periosteal layer. This study provides direct evidence to suggest that osteogenesis and angiogenesis mediated by human SBP-DPSCs may be regulated by distinct mechanisms, leading to the organization of adult bone tissue after stem cell transplantation.