Transfection of Reaggregating Embryonic Chicken Retinal Cells with an Antisense 5'-DNA Butyrylcholinesterase Expression Vector Inhibits Proliferation and Alters Morphogenesis

Abstract: The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Protein kinase C is regulated in vivo by three functionally distinct phosphorylations

Background: Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers.Results We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C βII are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site.Conclusion Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.     

Strong teratocarcinoma transplantation loci,Gt-1 and Gt-2, Flank H-2

Evidence is presented for the existence of two strong murine teratocarcinoma transplantation antigens (Gt) on the cell line PCC3. It is shown that the loci governing expression of these antigens are linked to the H-2 complex. These loci have been further mapped with respect to the brachyury marker (T) and H-2: Gt-1 lies 5±2 crossover units proximal to H-2 and 12±2 crossover units distal to T, Gt-2 lies 21±4 crossover units distal to H-2. It is possible that these strong transplantation antigens provide an embryonic analogue to the adult major histocompatibility system.     

Cleavage of Bovine Brain Microtubule-Associated Protein-2 by Human Immunodeficiency Virus Proteinase

The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.     

Species composition,similarity, and structure of mayan homegardens in tixpeual and tixcacaltuyub,yucatan, Mexico

We studied species composition, similarity, and structure of homegardens in two Yucatecan Maya communities, Tixpeual and Tixcacaltuyub, Yucatan, Mexico. The number of gardens sampled per village was 20 and 22; total area sampled was very similar, 45,265 m² and 40,150 m²; the number of trees and shrubs present was 5651 and 5603; and number of species was 135 and 133, respectively. Diversity was low for both sites (H′= 1.6), as were the correlation coefficients (r) for the species-area and individuals-area correlations. The relatively low values obtained for the structural parameters reflect the random pattern of plant incorporation to the gardens, the variability in the proportion of constantly used and not constantly used garden area, and a certain uniformity in the number of species used and number of individuals present, and the relationship between these parameters and garden size. All these reflect the uniqueness of each homegarden, which depends upon the cultural background of the owner. We noticed a trend towards a change in homegarden structure and function in response to the modernization process. Homegardens in villages in the outskirts of cities tend to have more ornamental species and commercial fruit plants than homegardens in isolated villages.     

Correction to: Integrative transcriptomics reveals genotypic impact on sugar beet storability

Plant Molecular Biology - In the above mentioned publication, part of Fig. 6B was distorted (extra diagonal lines appeared). The original article has been corrected and the proper version...     

Cyclin D1 and cyclin D3 show divergent responses to distinct mitogenic stimulation

D‐type cyclins predominantly regulate progression through the cell cycle by their interactions with cyclin‐dependent kinases (cdks). Here, we show that stimulating mitogenesis of Swiss 3T3 cells with phorbol esters or forskolin can induce divergent responses in the expression levels, localization and activation state of cyclin D1 and cyclin D3. Phorbol ester‐mediated protein kinase C stimulation induces S phase entry which is dependent on MAPK activation and increases the levels and activation of cyclin D1, whereas forskolin‐mediated cAMP‐dependent protein kinase A stimulation induces mitogenesis that is independent of MAPK, but dependent upon mTor and specifically increases the level and activation of cyclin D3. These findings uncover additional levels of complexity in the regulation of the cell cycle at the level of the D‐type cyclins and thus may have important therapeutic implications in cancers where specific D‐cyclins are overexpressed. J. Cell. Physiol. 225: 638–645, 2010. © 2010 Wiley‐Liss, Inc.     

Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells

Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.