Two-dimensional analysis of proteinase activity

A method was developed to separate proteinases in a complex mixture in two dimensions followed by activity detection using class specific substrates. Using this method, serine proteinase activity was evaluated in gut extracts from a stored-product pest, Plodia interpunctella. With the substrate n-alpha-benzoyl-l-arginine rho-nitroanilide, three major groups of at least six trypsin-like activities were identified, consisting of proteinases with estimated molecular masses of 25-27, 40-41, and 289 kDa, and all with an acidic pI of 4.7-5.5. With the substrate, n-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, two groups of at least five chymotrypsin-like activities were detected, with estimated molecular masses of 28 and 192 kDa and pI values ranging from 6.1 to 7.3. Using the 2-DE activity blot method, information was obtained on the relative number and physical properties of serine proteinases in a mixture of insect gut proteinases without prior fractionation.     

Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3

The endothelial cell (EC)-derived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following EC-pericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. EC-pericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in EC-pericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)-, MMP-10-, and ADAM-15 (a disintegrin and metalloproteinase-15)-dependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events.     

BNP-induced activation of cGMP in human cardiac fibroblasts: interactions with fibronectin and natriuretic peptide receptors

Cardiac remodeling involves the accumulation of extracellular matrix (ECM) proteins including fibronectin (FN). FN contains RGD motifs that bind integrins at DDX sequences allowing signaling from the ECM to the nucleus. We noted that the natriuretic peptide receptor A (NPR-A) sequence contains both RGD and DDX sequences. The goal of the current investigation was to determine potential interactions between FN and NPR-A on BNP induction of cGMP in cultured human cardiac fibroblasts (CFs). Further, we sought to determine whether a Mayo designed NPR-A specific RGD peptide could modify this interaction. Here we reconfirm the presence of all three natriuretic peptide receptors (NPR) in CFs. CFs plated on FN demonstrated a pronounced increase in cGMP production to BNP compared to non-coated plates. This production was also enhanced by the NPR-A specific RGD peptide, which further augmented FN associated cGMP production. Addition of HS-142-1, a NPR-A/B antagonist, abrogated the responses of BNP to both FN and the NPR-A specific RGD peptide. Finally, we defined a possible role for the NPR-C through non-cGMP mechanisms in mediating the anti-proliferative actions of BNP in CFs where the NPR-C antagonist cANF 4-28 but not HS-142-1 blocked BNP-mediated inhibition of proliferation of CFs. We conclude that NPR-A interacts with components of the ECM such as FN to enhance BNP activation of cGMP and that a small NPR-A specific RGD peptide augments this action of BNP with possible therapeutic implications. Lastly, the NPR-C may also have a role in mediating anti-proliferative actions of BNP in CFs.     

Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters

To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells.     

Characterization of the yeast amphiphysins Rvs161p and Rvs167p reveals roles for the Rvs heterodimer in vivo

We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response.     

In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide

Oleamide (cis-9-octadecenamide) is a member of an emerging class of lipid-signaling molecules, the primary fatty acid amides. A growing body of evidence indicates that oleamide mediates fundamental neurochemical processes including sleep, thermoregulation, and nociception. Nevertheless, the mechanism for oleamide biosynthesis remains unknown. The leading hypothesis holds that oleamide is synthesized from oleoylglycine via the actions of the peptide amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The present study investigated this hypothesis using pharmacologic treatments, physiologic assessments, and measurements of serum oleamide levels using a newly developed enzyme-linked immunosorbant assay (ELISA). Oleamide and oleoylglycine both induced profound hypothermia and decreased locomotion, over equivalent dose ranges and time courses, whereas, closely related compounds, stearamide and oleic acid, were essentially without effect. While the biologic actions of oleamide and oleoylglycine were equivalent, the two compounds differed dramatically with respect to their effects on serum levels of oleamide. Oleamide administration (80mg/kg) elevated blood-borne oleamide by eight-fold, whereas, the same dose of oleoylglycine had no effect on circulating oleamide levels. In addition, pretreatment with the established PAM inhibitor, disulfiram, produced modest reductions in the hypothermic responses to both oleoylglycine and oleamide, suggesting that the effects of disulfiram were not mediated through inhibition of PAM and a resulting decrease in the formation of oleamide from oleoylglycine. Collectively, these findings raise the possibilities that: (1) oleoylglycine possesses biologic activity that is independent of its conversion to oleamide and (2) the increased availability of oleoylglycine as a potential substrate does not drive the biosynthesis of oleamide.     

Small interfering RNAs containing full 2'-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary single-strand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2'-O-methyl (2'-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2'-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2'-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.     

Associations among education, cortisol rhythm, and BMI in blue-collar women

Objective: This study sought to test whether a biological measure of chronic stress, Δ cortisol, was related to BMI and whether the relationship between Δ cortisol and BMI varied according to education and positive affect. Research Methods and Procedures: One hundred fifty‐four women from a blue‐collar women's health project in 11 industrial sites in rural North Carolina provided saliva for cortisol assays for a substudy on “stress.” Δ Cortisol, the difference between awakening and midday cortisol measures representing diurnal decline, was calculated (lower values = greater stress). BMI was regressed on Δ cortisol, education, and positive affect. Analyses were controlled for age, race, and worksite. Standardized β‐coefficients were calculated. Results: For participants with complete data (n = 129), BMI was greater (β 95% confidence interval) for women with less than high school education (0.56; 0.18, 0.94) and those who completed high school (0.26; ?0.05, 0.57) relative to women with greater than a high school education (p = 0.009). Δ Cortisol was inversely related to BMI (?0.32; ?0.59, ?0.05; p = 0.022). Education positively modified the inverse relationship between Δ cortisol and BMI (p = 0.047). Positive affect was negatively associated with BMI (?0.44; ?0.82, ?0.06; p = 0.026) and positively modified the inverse association between Δ cortisol and BMI (0.33; ?0.03, 0.69; p = 0.074). Discussion: Education and Δ cortisol were inversely related to BMI, and the magnitude of the association between Δ cortisol and BMI was buffered by higher education. Positive affect was inversely related to BMI. Chronic stress is associated with higher BMI, with this relation attenuated by higher education and, possibly, a positive affect.