Identification of the molecular mechanisms contributing to polarized trafficking in osteoblasts

The directionality of matrix deposition in vivo is governed by the ability of a cell to direct vesicularflow to a specific target site. Osteoblastic cells direct newly synthesized bone matrix proteins toward the bone surface. In this study, we dissect the molecular mechanisms underlying the polarized trafficking of matrix protein in osteoblasts. We demonstrate using TEM, immunocytochemistry, and cDNA analysis, the ability of osteoblastic cells in culture to form tight junction-like structures and report the expression of the tight junction associated proteins occludin and claudins 1-3 in these cells. We identify intercellular contact sites and the leading edge of migratory osteoblasts as major target sites of vesicular trafficking in osteoblasts. Proteins required for this process, rsec6, NSF, VAMP1, and syntaxin 4, as well as the bone matrix protein, osteopontin, localize to these sites. We demonstrate that osteoblasts in vivo possess VAMP1 and, furthermore, report the expression of two VAMP1 splice variants in these cells. In addition, osteoblasts express the NSF attachment protein alpha-SNAP and the t-SNARE SNAP23. Thus, cell-to-cell contact sites and the leading edge of migratory osteoblasts contain a unique complement of proteins required for SNARE mediated membrane fusion.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Autosomal rearrangements in Graomys griseoflavus (Rodentia): a model of non-random Robertsonian divergence

The south American rodent Graomys griseoflavus exhibits a remarkable chromosome polymorphism as a consequence of four Robertsonian fusions. Focusing on the genetic analysis of the taxon, genome organization of all karyomorphs was studied at chromosome and molecular organization level. Cytogenetic (G, NOR and Re banding) and molecular (satellite and mitochondrial DNAs) events accompanying chromosome divergence allowed tracing a phylogenetic relationship among all karyomorphs. Available data led to propose that chromosome evolution of G. griseoflavus occurred in a non-random sequence of centric fusions, supporting the hypothesis of single origin for Robertsonian karyomorphs.     

The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic,manganese-containing enzyme

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.     

Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, gamma-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1(tr/tr)), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1(tr/tr) animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1(tr/tr) mice are sensitive to ionizing radiation (gamma-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals extensive sequence variation and differential immune recognition

Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 90% protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs from Haemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues of Caenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies.     

Simultaneous binding of two proteins to opposite sides of a single transfer RNA

Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111. The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules. The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA. Here we present RNA footprints that are consistent with this model. This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA. These opposite locations of binding for these two proteins suggest the possibility of a ternary complex. The formation of a tRNA synthetase--tRNA--Trbp111 ternary complex was detected by two independent methods. The results indicate that the tRNA is sandwiched between the two protein molecules. A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex. These results may have implications for how the translation apparatus is linked to other cellular machinery.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.