Reactions between dipeptidyl peptidase IV and diacyl hydroxylamines: mechanistic investigations

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of Kcat/Kinact. Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship. Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO2) and the decomposition of the suicide substrate in H2O and D2O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.     

Spectral call features provide information about the aggression level of greater mouse-eared bats (Myotis myotis) during agonistic interactions

Bats rely heavily on acoustic signals in order to communicate with each other in a variety of social contexts. Among those, agonistic interactions and accompanying vocalizations have received comparatively little study. Here, we studied the communicational behaviour between male greater mouse-eared bats (Myotis myotis) during agonistic encounters. Two randomly paired adult males were placed in a box that allowed us to record video and sound synchronously. We describe their vocal repertoire and compare the acoustic structure of vocalizations between two aggression levels, which we quantified via the bats’ behaviour. By inspecting thirty, one-minute long encounters, we identified a rich variety of social calls that can be described as two basic call types: echolocation-like, low-frequency sweeps and long, broadband squawks. Squawks, the most common vocalization, were often noisy, i.e. exhibited a chaotic spectral structure. We further provide evidence for individual signatures and the presence of nonlinear phenomena in this species’ vocal repertoire. As the usage and acoustic structure of vocalizations is known to encode the internal state of the caller, we had predicted that the spectral structure of squawks would be affected by the caller’s aggression level. Confirming our hypothesis, we found that increased aggression positively correlated with an increase in call frequency and tonality. We hypothesize that the extreme spectral variability between and within squawks can be explained by small fluctuations in vocal control parameters (e.g. subglottal pressure) that are caused by the elevated arousal, which is in turn influenced by the aggression level.     

Role of Cdc42-Cla4 interaction in the pheromone response of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4.     

Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR

Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.     

Grafting of thrombopoietin-mimetic peptides into cystine knot miniproteins yields high-affinity thrombopoietin antagonists and agonists

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high-affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin-mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c-Mpl receptor agonists with EC(50) values in the low nanomolar or picomolar range. One selected miniprotein-derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand-induced dimerization.     

Cerebrocortical beta activity in overweight humans responds to insulin detemir

Background

Insulin stimulates cerebrocortical beta and theta activity in lean humans. This effect is reduced in obese individuals indicating cerebrocortical insulin resistance. In the present study we tested whether insulin detemir is a suitable tool to restore the cerebral insulin response in overweight humans. This approach is based on studies in mice where we could recently demonstrate increased brain tissue concentrations of insulin and increased insulin signaling in the hypothalamus and cerebral cortex following peripheral injection of insulin detemir.

Methodology/Principal Findings

We studied activity of the cerebral cortex using magnetoencephalography in 12 lean and 34 overweight non-diabetic humans during a 2-step hyperinsulinemic euglycemic clamp (each step 90 min) with human insulin (HI) and saline infusion (S). In 10 overweight subjects we additionally performed the euglycemic clamp with insulin detemir (D). While human insulin administration did not change cerebrocortical activity relative to saline (p = 0.90) in overweight subjects, beta activity increased during D administration (basal 59±3 fT, 1^st step 62±3 fT, 2^nd step 66±5, p = 0.001, D vs. HI). As under this condition glucose infusion rates were lower with D than with HI (p = 0.003), it can be excluded that the cerebral effect is the consequence of a systemic effect. The total effect of insulin detemir on beta activity was not different from the human insulin effect in lean subjects (p = 0.78).

Conclusions/Significance

Despite cerebrocortical resistance to human insulin, insulin detemir increased beta activity in overweight human subjects similarly as human insulin in lean subjects. These data suggest that the decreased cerebral beta activity response in overweight subjects can be restored by insulin detemir.     

Intestinal metabolism of T-2 toxin in the pig cecum model

T-2 toxin, a toxic member of the group A trichothecenes, is produced by various Fusarium species that can potentially affect human health. As the intestine plays an important role in the metabolism of T-2 toxin for animals and humans, the degradation and metabolism of T-2 toxin was studied using the pig cecum in vitro model system developed in the author??s group. In order to study the intestinal degradation of T-2 toxin by pig microbiota, incubation was performed with the cecal chyme from four different pigs in repeat determinations. A large variation in the intestinal degradation of T-2 toxin was observed for individual pigs. T-2 toxin was degraded almost completely in one out of four pigs, in which only 3.0?±?0.1?% of T-2 toxin was left after 24?h incubation. However, in the other three incubations with pig cecal suspension, 54.1?±?11.7?C68.9?±?16.1?% of T-2 toxin were still detectable after 24?h incubation time. The amount of HT-2 toxin was increased along with the incubation time, and HT-2 toxin accounted for 85.2?±?0.7?% after 24?h in the most active cecum. HT-2 toxin was the only detectable metabolite formed by the intestinal bacteria. This study suggests that the toxicity of T-2 toxin for pigs is caused by the combination of T-2 and HT-2 toxins.     

The endocannabinoid system controls key epileptogenic circuits in the hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.     

Hybridisation between South polar skua (Catharacta maccormicki) and Brown skua (C. antarctica lonnbergi) in the Antarctic Peninsula region

Hybridisation between South polar skua (C. maccormicki) and Brown skua (C. antarctica lonnbergi) in the area of the Antarctic Peninsula is known at least since the beginning of the last century but no survey has been done so far. This paper reviews information on the species composition of skua colonies of more than 10 pairs in the Antarctic Peninsula region, and the incidence of mixed pairs. Morphometrics, population size and breeding success were examined in detail at King George Island. The northward distribution of South polar skuas extended to King George Island (62°11′ S 59°00′ W), with a small outlying population on Signy Island (60°45′ S 45°36′ W), whereas Brown skuas did not breed further south than Anvers Island archipelago (64°46′ S 64°03′ W). The proportion of mixed pairs was highest at the northern end of the ∼500-km-wide hybrid zone. Body size distribution of sympatric skuas from King George Island is clearly bimodal but overlaps considerably and hybrids cannot be identified. Skua population sizes at Potter Peninsula/King George Island remained stable between 1994 and 2004. Numbers of mixed breeding pairs fluctuated more strongly than those of pure species pairs. Breeding success of Brown skuas varied the least.     

Design and synthesis of novel N-sulfonyl-2-indole carboxamides as potent PPAR-gamma binding agents with potential application to the treatment of osteoporosis

The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.