Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.     

Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

BackgroundCirculating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.MethodsWe measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.ResultsThe average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005).ConclusionsCirculating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.     

Modular Skeletal Evolution in Sticklebacks Is Controlled by Additive and Clustered Quantitative Trait Loci

Understanding the genetic architecture of evolutionary change remains a long-standing goal in biology. In vertebrates, skeletal evolution has contributed greatly to adaptation in body form and function in response to changing ecological variables like diet and predation. Here we use genome-wide linkage mapping in threespine stickleback fish to investigate the genetic architecture of evolved changes in many armor and trophic traits. We identify >100 quantitative trait loci (QTL) controlling the pattern of serially repeating skeletal elements, including gill rakers, teeth, branchial bones, jaws, median fin spines, and vertebrae. We use this large collection of QTL to address long-standing questions about the anatomical specificity, genetic dominance, and genomic clustering of loci controlling skeletal differences in evolving populations. We find that most QTL (76%) that influence serially repeating skeletal elements have anatomically regional effects. In addition, most QTL (71%) have at least partially additive effects, regardless of whether the QTL controls evolved loss or gain of skeletal elements. Finally, many QTL with high LOD scores cluster on chromosomes 4, 20, and 21. These results identify a modular system that can control highly specific aspects of skeletal form. Because of the general additivity and genomic clustering of major QTL, concerted changes in both protective armor and trophic traits may occur when sticklebacks inherit either marine or freshwater alleles at linked or possible “supergene” regions of the stickleback genome. Further study of these regions will help identify the molecular basis of both modular and coordinated changes in the vertebrate skeleton.     

Differentiation of chronic lymphocytic leukemia B cells into immunoglobulin secreting cells decreases LEF-1 expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.     

Maintaining connexin43 gap junctional communication in v-Src cells does not alter growth properties associated with the transformed phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine "normal" cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Sexual behaviour and paternity in three captive groups of rhesus monkeys (Macaca mulatta)

Each offspring born into three captive groups of rhesus monkeys (Macaca mulatta) during the spring of 1981 was assigned (1) a most probable behavioural father and (2) a subset of all possible behavioural fathers, on the basis of intensive observations of sexual activity during the 1980 fall breeding season. These predictions were then compared with true paternity determined by electrophoretic and serological analyses of polymorphic blood proteins of mothers, infants, and all adult and subadult males. Coincidence between the assignments of paternity based upon behavioural and biochemical data occurred no more frequently than chance would allow. Furthermore, there were no significant relationships between biochemical and behavioural rankings of overall reproductive success. It was concluded that conventional estimates of sexual activity during the breeding season are not a reliable indicator of true paternity and relative reproductive success in rhesus macaques.