Inhibition of Chk1 by activated PKB/Akt

We have shown recently that DNA damage effector kinase Chk1 is phosphorylated in vitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNA damage in vivo is suppressed in presence of activated PKB. In this study we show that Chk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB on serine 280 correlates with impairment of Chk1 activation by DNA damage. Our results indicate a likely mechanism for the negative effects that phosphorylation of serine 280 has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 does not enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylated by PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR. Phosphorylation by ATM/ATR and association with other checkpoint proteins are essential steps in activation of Chk1. Inhibition of these steps provides a plausible explanation for the observed attenuation of Chk1 activation by activated PKB after DNA damage.     

Familial non-syndromic clear cell renal cell carcinoma

The diagnosis of familial non-syndromic clear cell renal cell carcinoma is one of exclusion. In families presenting with clear cell RCC a germline VHL mutation and a constitutional translocation of chromosome 3 must be excluded before familial non-syndromic clear cell RCC can be diagnosed. Large familial non-syndromic clear cell RCC kindreds are uncommon and a predisposing gene has not been identified. However inheritance is autosomal dominant in most cases and age at onset is earlier than in sporadic cases. Recognition and appropriate screening of familial non-syndromic clear cell RCC cases will reduce morbidity and mortality. Large scale collaborative linkage studies may provide a basis for the identification of familial non-syndromic clear cell RCC susceptibility gene(s).     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Bradykinin receptor gene variant and human physical performance.

Accumulating evidence suggests that athletic performance is strongly influenced by genetic variation. One such locus of influence is the gene for angiotensin-I converting enzyme (ACE), which exhibits a common variant [ACE insertion (I)/deletion (D)]. ACE can drive formation of vasoconstrictor ANG II but preferentially degrades vasodilator bradykinin. The ACE I allele is associated with higher kinin activity. A common gene variant in the kinin beta(2) receptor (B(2)R) exists: the -9 as opposed to +9 allele is associated with higher receptor mRNA expression. We tested whether this variant was associated with the efficiency of muscular contraction [delta efficiency (DE)] in 115 healthy men and women, or with running distance among 81 Olympic standard track athletes. We further sought evidence of biological interaction with ACE I/D genotype. DE was highly significantly associated with B(2)R genotype (23.84 +/- 2.41 vs. 24.25 +/- 2.81 vs. 26.05 +/- 2.26% for those of +9/+9 vs. +9/-9 vs. -9/-9 genotype; n = 25, 61, and 29, respectively; P = 0.0008 for ANOVA adjusted for sex). There was evidence for interaction with ACE I/D genotype, with individuals who were ACE II, with B(2)R -9/-9 having the highest DE at baseline. The ACE I/B(2)R -9 "high kinin receptor activity" haplotype was significantly associated with endurance (predominantly aerobic) event among elite athletes (P = 0.003). These data suggest that common genetic variation in the B(2)R is associated with efficiency of skeletal muscle contraction and with distance event of elite track athletes and that at least part of the associations of ACE and fitness phenotypes is through elevation of kinin activity.     

F-actin capping (CapZ) and other contractile saphenous vein smooth muscle proteins are altered by hemodynamic stress: a proteonomic approach

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.     

Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.     

A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.     

The phenotype of inflammatory macrophages is stimulus dependent: implications for the nature of the inflammatory response

Many diseases are characterized by inflammatory reactions involving both the innate and adaptive arms of the immune system. Thioglycolate medium (TM) injection into the peritoneal cavity has long been used as a stimulus for eliciting inflammatory macrophages for study and for determining the importance of a particular mediator in inflammation. However, the response to this irritant may not be relevant to many inflammatory diseases. Therefore, we have developed an Ag-specific peritonitis model using methylated BSA (mBSA) as the stimulus. Priming mice intradermally with mBSA in adjuvant and boosting 14 days later, followed by an i.p. challenge with mBSA after an additional 7 days, led to an inflammatory reaction equivalent in magnitude to that induced with TM as judged by the number of exudate cells. The inflammatory macrophages elicited by the mBSA protocol differed, being smaller and less vacuolated than TM-elicited macrophages. Also, macrophages from 4-day mBSA-induced exudates expressed more MHC class II than TM-induced exudates, were able to stimulate allogeneic T lymphocytes, and upon in vitro stimulation with LPS secreted greater levels of IL-6 and IL-1beta. Macrophages from 4-day TM-induced exudates, on the other hand, expressed Ly6C and ER-MP58, immature myeloid markers. The inflammatory response elicited using the Ag mBSA may be more relevant for studying the inflammatory responses in many diseases, such as those of autoimmune origin and those involving an acquired immune response.     

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.