Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins,osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley-Liss, Inc.     

Partial characterization of prostaglandin synthetase in the reproductive tract of the male house cricket, acheta domesticus

An active prostaglandin (PG) synthetase was found in the 12100 g pellet of reproductive tract homogenates of the male house cricket, $\text{Acheta domesticus}$. Comparatively, the 12100 g supernatant and the microsomal fractions were inactive. The PG synthetase in the pellet fraction was characterized in terms of cofactor, temperature, pH, and incubation time requirements. Indomethacin, a known inhibitor of mammalian PG synthetase, was not inhibitory to the cricket synthetase. The procedure and findings are relevant to PG synthetase studies of any organism or tissue.     

Models for loosely linked gene duplicates suggest lengthy persistence of both copies

Consider the appearance of a duplicate copy of a gene at a locus linked loosely, if at all, to the locus at which the gene is usually found. If all copies of the gene are subject to non-functionalizing mutations, then two fates are possible: loss of functional copies at the duplicate locus (loss of duplicate expression), or loss of functional copies at the original locus (map change). This paper proposes a simple model to address the probability of map change, the time taken for a map change and/or loss of duplicate expression, and considers where in the spectrum between loss of duplicate expression and map change such a duplicate complex is likely to be found. The findings are: the probability of map change is always half the reciprocal of the population size N, the time for a map change to occur is order NlogN generations, and that there is a marked tendency for duplicates to remain near equi-frequency with the gene at the original locus for a large portion of that time. This is in excellent agreement with simulations.     

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.     

RASCAL Is a New Human Cytomegalovirus-Encoded Protein That Localizes to the Nuclear Lamina and in Cytoplasmic Vesicles at Late Times Postinfection

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.Cytomegalovirus (CMV) is a highly prevalent betaherpesvirus that can cause severe multiorgan disease in immunocompromised individuals (45). The ability of this virus to infect an exceptionally wide variety of different cell types substantially contributes to pathogenesis (5, 68). CMV tropism is largely determined by a finely tuned interplay between cellular and viral factors, many of which act at the earliest stages of infection (30, 68). We recently showed that the cellular protein vimentin is required for efficient onset of infection in primary human foreskin fibroblasts (HF). Interestingly, the degree of reliance on the presence and integrity of vimentin intermediate filaments is dependent on the virus strain, with the broadly tropic strain TB40/E being more negatively affected than the HF-adapted, attenuated strain AD169 (44).Serial passage of clinical isolates in HF or in endothelial cells (EC) has produced strains with different tropisms. The attenuated strains AD169 and Towne were developed as vaccine candidates by propagation in HF for more than 50 (AD169) and 125 (Towne) serial passages (19, 53, 61). During this process, both strains, compared to clinical isolates, accumulated multiple mutations and genomic deletions, resulting in the loss of more than 19 open reading frames (ORFs) (8). The number of serial passages in HF of another commonly used strain, Toledo, has been more moderate (19, 54, 58). This, however, did not prevent the emergence of numerous genomic mutations, including the inversion of an ∼15-kb fragment (8, 16, 56). As a consequence of these changes, productive infections by AD169, Towne, and Toledo are largely restricted to HF. In contrast, propagation of clinical isolates in EC has yielded a series of strains with more-intact genomes and broader tropisms, such as TB40/E, VHL/E, and FIX (VR1814) (25, 60, 71). These strains retain the ability to grow in a wider variety of cell types, including EC, epithelial cells, and dendritic cells (DC), in addition to HF (23, 28, 59, 60, 68).The UL128, UL130, and UL131A gene products were recently identified as essential mediators of CMV infection of EC and epithelial cells (26, 72, 73) and of virus transfer from infected EC to monocyte-derived DC (23). Each of these proteins is independently required for the broader tropisms of EC-propagated CMV isolates (63, 64), and the presence of mutations affecting their functionality has been directly linked to the inability of AD169, Towne, and Toledo to initiate productive infections in EC and epithelial cells (26, 72, 73).We have shown that mature Langerhans-type DC differentiated in vitro from CD34⁺ hematopoietic progenitor cells are highly permissive to direct infection with TB40/E or VHL/E, with 48 to 72% of cells in culture expressing the viral immediate-early genes IE1 and IE2 at 48 h postinfection (hpi) (28). In contrast, only 2 to 5% and 0% of mature Langerhans cells were IE1/IE2 positive after exposure to Towne and Toledo, respectively. However, productive infection was detected in 12 to 17% of cells infected with AD169, despite the fact that this strain lacks expression of the UL131A gene as a consequence of a frameshift mutation (26). These results suggested the existence of additional viral genes with products involved in mediating tropisms for specific cell types, such as DC. To identify possible candidates, we compared the amino acid sequence of each ORF found in the genome of TB40-BAC4, a sequenced clone of the TB40/E strain in a bacterial artificial chromosome (BAC) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EF999921","term_id":"157779983","term_text":"EF999921"}}EF999921) (69), to the sequence of each ORF found in AD169 and AD169-BAC (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"X17403","term_id":"59591","term_text":"X17403"}}X17403 and {"type":"entrez-nucleotide","attrs":{"text":"AC146999","term_id":"38093733","term_text":"AC146999"}}AC146999) (10, 49), Towne and Towne-BAC (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"FJ616285","term_id":"222354438","term_text":"FJ616285"}}FJ616285, {"type":"entrez-nucleotide","attrs":{"text":"AC146851","term_id":"37654163","term_text":"AC146851"}}AC146851, and {"type":"entrez-nucleotide","attrs":{"text":"AY315197","term_id":"124303521","term_text":"AY315197"}}AY315197) (17, 18, 49), and Toledo-BAC (accession number {"type":"entrez-nucleotide","attrs":{"text":"AC146905","term_id":"37777314","term_text":"AC146905"}}AC146905) (49). The product of a putative ORF, originally identified by Murphy et al. and named conserved ORF 29 (c-ORF29) (49), was considered of particular interest because the amino acid sequence of the putative protein encoded by Toledo and Towne was extended by 79 residues compared to the putative protein encoded by TB40/E and AD169. This led to our speculation that that the extended version might result in a nonfunctional version of the c-ORF29-encoded protein. We thus focused our studies on the products of this ORF.Here, we show for the first time that CMV c-ORF29 encodes a protein expressed at early to late times postinfection (p.i.) and localizes to the nuclear rim in peculiar invaginations of the nuclear lamina and in cytoplasmic vesicular structures at late times p.i. Based on this localization pattern, we named this gene product nuclear rim-associated cytomegaloviral protein, or RASCAL. Surprisingly, no difference was observed in the distributions of RASCAL during infection of HF with TB40/E or Towne, suggesting that the intracellular trafficking of this protein is not affected by the presence of the additional residues at the C terminus of RASCAL from strain Towne (RASCAL_Towne). Ectopic expression of RASCAL in human embryo kidney 293T (HEK293T) cells further revealed that this protein requires the presence of the nuclear egress complex (NEC) member UL50 to reach the nuclear rim, while coimmunoprecipitation (co-IP) assays provided evidence for the existence of an interaction between RASCAL and UL50. These findings suggest that RASCAL may be a new component of the NEC with possible roles in remodeling the nuclear lamina during nucleocapsid egress from the nucleus.     

Effects of plant harvesting and nutrient enrichment on phytoplankton community structure in a shallow urban lake

The utility of shallow water bodies in urban environments is frequently compromised either by dense beds of submerged plants or cyanobacterial blooms associated with nutrient enrichment. Although submerged plants are often harvested to facilitate recreational uses, this activity may alter the phytoplankton community, which in turn, also may restrict the use of the lake. We tested whether (i) plant harvesting reduced the abundance of flagellate algae and increased the abundance of cyanobacteria, and (ii) whether increasing levels of nutrient enrichment caused shifts in the dominance of heterocytous cyanobacteria, non-heterocytous cyanobacteria and Chlorophyta, in a shallow urban lake in Southern Australia as has been observed for shallow Danish lakes in previous studies. These predictions were tested with large (3000 l), replicated mesocosms in a warm, highly productive, shallow lake densely colonised by the submerged angiosperm, Vallisnaria americana Michaux. The heterokont algae, Chlorophyta, Cyanobacteria and Cryptophyta were the most numerous algal divisions in the lake. The Euglenophyta, although uncommon in early summer, became more abundant towards the end of summer. The Dinophyta and Charophyta were rare. The abundance of the heterokont algae and Euglenophyta was significantly reduced by plant harvesting even after plants had partially re-established 18 weeks after initial harvesting. The decline in the Euglenophyta in response to plant harvesting is consistent with earlier findings, that the relative abundance of flagellate algae tends to be greater in the presence of submerged plants. Contrary to our prediction, we found that the Cyanobacteria did not increase in response to plant harvesting, however the response may be altered under higher nutrient levels. Algal responses to nutrient enrichment in the presence of dense V. americana plants generally followed the patterns observed in shallow Danish lakes despite the large differences in climatic conditions. Both studies found that the abundance of heterocytous cyanobacteria declined at higher levels of nutrient enrichment, whereas non-heterocytous cyanobacteria and chlorophytes increased.     

Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.     

Recent advances in shoulder research

Shoulder pathology is a growing concern for the aging population, athletes, and laborers. Shoulder osteoarthritis and rotator cuff disease represent the two most common disorders of the shoulder leading to pain, disability, and degeneration. While research in cartilage regeneration has not yet been translated clinically, the field of shoulder arthroplasty has advanced to the point that joint replacement is an excellent and viable option for a number of pathologic conditions in the shoulder. Rotator cuff disease has been a significant focus of research activity in recent years, as clinicians face the challenge of poor tendon healing and irreversible changes associated with rotator cuff arthropathy. Future treatment modalities involving biologics and tissue engineering hold further promise to improve outcomes for patients suffering from shoulder pathologies.