Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

<p>Abstract</p> <p>Background</p> <p>The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α₂-antiplasmin (α₂AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α₂AP residues 13-42 linked to human serum albumin (HSA) weakened <it>in vitro </it>clots but failed to become specifically incorporated into <it>in vivo </it>clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α₂AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.</p> <p>Results</p> <p>Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H₆NQEQVSPLTLLAG₄Y (designated XL1); H₆DQMMLPWAVTLG₄Y (XL2); H₆WQHKIDLPYNGAG₄Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed <it>Pichia pastoris </it>yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α₂AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α₂AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride <it>vena cava </it>thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.</p> <p>Conclusions</p> <p>Fusion protein XL5-HSA (DQMMLPWAVTLG₄Y-HSAH₆) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins <it>in vitro</it>. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into <it>in vivo </it>clots formed in thrombosis models in both mice and rabbits.</p

Symptoms of anxiety and depression are related to cardiovascular responses to active, but not passive, coping tasks

Objective: Anxiety and depression have been linked to blunted blood pressure (BP) and heart rate (HR) reactions to mental stress tests; however, most studies have not included indices of underlying hemodynamics nor multiple stress tasks. This study sought to examine the relationships of anxiety and depression with hemodynamic responses to acute active and passive coping tasks. Methods: A total of 104 participants completed the Hospital Anxiety and Depression Scales and mental arithmetic, speech, and cold pressor tasks while BP, HR, total peripheral resistance, and cardiac output (CO) were assessed. Results: After adjustment for traditional risk factors and baseline cardiovascular activity, depression scores were negatively associated with systolic BP, HR, and CO responses to the mental arithmetic task, while anxiety scores were inversely related to the systolic BP response to mental arithmetic. Conclusion: High anxiety or depression scores appear to be associated with blunted cardiac reactions to mental arithmetic (an active coping task), but not to the cold pressor test or speech tasks. Future research should further examine potential mechanisms and longitudinal pathways relating depression and anxiety to cardiovascular reactivity

Non-Overlapping Functions for Pyk2 and FAK in Osteoblasts during Fluid Shear Stress-Induced Mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2+/+ and Pyk2−/− primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2+/+ and Pyk2−/− osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK−/− osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts

The complete mitochondrial genome of the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) currently is one of the economically most destructive pest species of stone and pome fruits worldwide. Here we sequenced the complete mitochondrial genome of this pest. This genome is 15,776 bp long, with an A + T content of 81.24%, containing 37 typical animal mitochondrial genes and an A + T-rich region. All gene are arranged as hypothesized ancestral gene order of insects except for trnM, which was shuffled from 3′ downstream of trnQ to 5′ upstream of trnI. cox1 gene uses unusual CGA start codon, as that in all other sequenced lepidopteran mitochondrial genome. The secondary structures for the two rRNA genes were predicted. All helices typically present in insect mitochondrial rRNA genes are generated. A microsatellite sequence was inserted into the region of H2347 in rrnL in G. molesta and two other sequenced tortricid mitochondrial genomes, indicating that the insertion event in this helix might occurred anciently in family Tortricidae. All of the 22 typical animal tRNA genes have a typical cloverleaf structure except for trnS2, in which the D-stem pairings in the DHU arm are absent. An intergenic sequence is present between trnQ and nad2 as well as in other sequenced lepidopteran mitochondrial genomes, which was presumed to be a remnant of trnM gene and its boundary sequences after the duplication of trnM to the upstream of trnI in Lepidoptera. The A + T-rich region is 836 bp, containing six repeat sequences of “TTATTATTATTATTAAATA(G)TTT.

Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched

Nutrient Administration and Resistance Training

Skeletal muscle tissue is tightly regulated throughout our bodies by balancing its synthesis and breakdown. Many factors are known to exist that cause profound changes on the overall status of skeletal muscle, some of which include exercise, nutrition, hormonal influences and disease. Muscle hypertrophy results when protein synthesis is greater than protein breakdown. Resistance training is a popular form of exercise that has been shown to increase muscular strength and muscular hypertrophy. In general, resistance training causes a stimulation of protein synthesis as well as an increase in protein breakdown, resulting in a negative balance of protein. Providing nutrients, specifically amino acids, helps to stimulate protein synthesis and improve the overall net balance of protein. Strategies to increase the concentration and availability of amino acids after resistance exercise are of great interest and have been shown to effectively increase overall protein synthesis. [1-3] After exercise, providing carbohydrate has been shown to mildly stimulate protein synthesis while addition of free amino acids prior to and after exercise, specifically essential amino acids, causes a rapid pronounced increase in protein synthesis as well as protein balance.[1,3] Evidence exists for a dose-response relationship of infused amino acids while no specific regimen exists for optimal dosing upon ingestion. Ingestion of whole or intact protein sources (e.g., protein powders, meal-replacements) has been shown to cause similar improvements in protein balance after resistance exercise when compared to free amino acid supplements. Future research should seek to determine optimal dosing of ingested intact amino acids in addition to identifying the cellular mechanistic machinery (e.g. transcriptional and translational mechanisms) for causing the increase in protein synthesis

Serum Activity of Platelet-Activating Factor Acetylhydrolase Is a Potential Clinical Marker for Leptospirosis Pulmonary Hemorrhage

Pulmonary hemorrhage has been recognized as a major, often lethal, manifestation of severe leptospirosis albeit the pathogenesis remains unclear. The Leptospira interrogans virulent serogroup Icterohaemorrhagiae serovar Lai encodes a protein (LA2144), which exhibited the platelet-activating factor acetylhydrolase (PAF-AH) activity in vitro similar to that of human serum with respect to its substrate affinity and specificity and thus designated L-PAF-AH. On the other hand, the primary amino acid sequence of L-PAF-AH is homologous to the α1-subunit of the bovine brain PAF-AH isoform I. The L-PAF-AH was proven to be an intracellular protein, which was encoded unanimously and expressed similarly in either pathogenic or saprophytic leptospires. Mongolian gerbil is an appropriate experimental model to study the PAF-AH level in serum with its basal activity level comparable to that of human while elevated directly associated with the course of pulmonary hemorrhage during severe leptospirosis. Mortality occurred around the peak of pulmonary hemorrhage, along with the transition of the PAF-AH activity level in serum, from the increasing phase to the final decreasing phase. Limited clinical data indicated that the serum activity of PAF-AH was likely to be elevated in the patients infected by L. interrogans serogroup Icterohaemorrhagiae, but not in those infected by other less severe serogroups. Although L-PAF-AH might be released into the micro-environment via cell lysis, its PAF-AH activity apparently contributed little to this elevation. Therefore, the change of PAF-AH in serum not only may be influential for pulmonary hemorrhage, but also seems suitable for disease monitoring to ensure prompt clinical treatment, which is critical for reducing the mortality of severe leptospirosis

Bacterial 16S rRNA/rDNA Profiling in the Liquid Phase of Human Saliva

Human saliva can be separated by centrifugation into cell pellet and cell-free supernatant, which are called cellular phase and liquid phase in this study. While it is well documented that the cellular phase of saliva contains hundreds of oral bacteria species, little is known whether the liquid phase of saliva contains any information related to oral microbiota. In this study, we analyzed the bacterial nucleic acid contents of the liquid phase of saliva. Using primers universal to most eubacterial 16S rDNA, we detected large amounts of bacterial 16S rRNA and rDNA in the cell-free phase of saliva. Random sequencing analysis of forty PCR amplicons from the cell-free phase of saliva led to 15 operational taxonomic unit (OTU) groups. Furthermore, using denaturing gradient gel electrophoresis (DGGE), we compared 16S rRNA/rDNA profiles derived from liquid phases and cellular phases of saliva samples, and found positive correlations (Pearson Correlation=0.822, P<0.001) between these sample groups. These findings indicate that the liquid phase of saliva contains numerous bacterial 16S rRNA/rDNA molecules that have correlations with bacteria existing in the cellular phase

Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta - regulation by selective estrogen receptor modulators and importance in breast cancer

Estrogens display intriguing tissue-selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer, for menopausal hormone replacement, and for fertility regulation. Certain compounds that act through the estrogen receptor (ER), now referred to as selective estrogen receptor modulators (SERMs), can demonstrate remarkable differences in activity in the various estrogen target tissues, functioning as agonists in some tissues but as antagonists in others. Recent advances elucidating the tripartite nature of the biochemical and molecular actions of estrogens provide a good basis for understanding these tissue-selective actions. As discussed in this thematic review, the development of optimal SERMs should now be viewed in the context of two estrogen receptor subtypes, ERα and ERβ, that have differing affinities and responsiveness to various SERMs, and differing tissue distribution and effectiveness at various gene regulatory sites. Cellular, biochemical, and structural approaches have also shown that the nature of the ligand affects the conformation assumed by the ER-ligand complex, thereby regulating its state of phosphorylation and the recruitment of different coregulator proteins. Growth factors and protein kinases that control the phosphorylation state of the complex also regulate the bioactivity of the ER. These interactions and changes determine the magnitude of the transcriptional response and the potency of different SERMs. As these critical components are becoming increasingly well defined, they provide a sound basis for the development of novel SERMs with optimal profiles of tissue selectivity as medical therapeutic agents