Design and synthesis of 3-(2-pyridyl)pyrazolo[1,5-a]pyrimidines as potent CRF1 receptor antagonists

A series of 3-(2-pyridyl)pyrazolo[1,5-a]pyrimidines was designed and synthesized as antagonists for the corticotrophin-releasing factor-1 (CRF(1)) receptor. Several compounds such as 20c (K(i)=10 nM) exhibited good binding affinities at the CRF(1) receptor. In addition, 20c had adequate solubility in water.     

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNF in the lung. TNF in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNF in chronic hypoxia we exposed TNF null (TNF–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O₂). Equivalent erythocytosis (Hematocrit increased by 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice ( 75%), yet was unaltered in the TNF–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNF–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNF is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNF signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNF.     

The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers

We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA(0) and alpha alpha-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes-Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a 'blood substitute' based on mimicking O(2) transport by RBCs to prevent autoregulatory changes in blood flow and pressure.     

Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold

BACKGROUND: Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. RESULTS: The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. CONCLUSIONS: The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.     

Anthropoid cranial base architecture and scaling relationships

This paper examines how various measures of basicranial length and cranial base angulation affect the relationship between basicranial flexion and relative brain size in anthropoids, including Homo sapiens. Most recent studies support the "spatial packing" hypothesis, that basicranial flexion in haplorhines maximizes braincase volume relative to basicranial length. However, a few studies find the basicranium is less flexed in H. sapiens than expected for other anthropoids, suggesting that other factors contribute to variation in hominin basicranial flexion. The measure of relative brain size used to test the spatial packing hypothesis, the Index of Relative Encephalization (IRE), is calculated with basicranial length (BL) in its denominator, so that shorter BL and larger brain size potentially inflate H. sapiens IREs. To investigate this problem, the lengths of midline cranial floor sections were scaled relative to the cube root of endocranial volume in 157 specimens from 18 anthropoid species. Results indicate that the posterior cranial base and planum sphenoideum are significantly shorter in H. sapiens than in other anthropoids, accounting for higher IREs. Including the cribriform plate in BL, advisable in studies using anthropoids, affects whether H. sapiens differs from other anthropoids for basicranial flexion vs. IRE. However, despite a shorter BL and elevated IRE, H. sapiens does not deviate significantly from the anthropoid relationship between basicranial flexion and relative brain size for two cranial base angles. Because different measures of cranial base angulation change how H. sapiens falls along the anthropoid regression line, it remains equivocal whether the basicranium is less flexed in H. sapiens than in other anthropoids when compared to relative brain size.     

Calcium-dependent adhesion is necessary for the maintenance of prosomeres

Cell adhesion has been suggested to function in the establishment and maintenance of the segmental organization of the central nervous system. Here we tested the role of different classes of adhesion molecules in prosencephalic segmentation. Specifically, we examined the ability of progenitors from different prosomeres to reintegrate and differentiate within various brain regions after selective maintenance or removal of different classes of calcium-dependent versus -independent surface molecules. This analysis implicates calcium-dependent adhesion molecules as central to the maintenance of prosomeres. Only conditions that spared calcium-dependent adhesion systems but ablated more general (calcium-independent) adhesion systems resulted in prosomere-specific integration after transplantation. Among the members of this class of adhesion molecules, R-cadherin shows a striking pattern of prosomeric expression during development. To test whether expression of this molecule was sufficient to direct progenitor integration to prosomeres expressing R-cadherin, we used a retroviral-mediated gain-of-function approach. We found that progenitors originally isolated from prosomere P2 (a region which does not express R-cadherin), when forced to express this molecule, can now integrate more readily into R-cadherin-expressing regions, such as the cortex, the ventral thalamus, and the hypothalamus. Nonetheless, our analysis suggests that while calcium-dependent molecules are able to direct prosomere-specific integration, they are not sufficient to induce progenitors to change their regional identity. While diencephalic progenitors from R-cadherin-expressing regions of prosomere 5 could integrate into R-cadherin-expressing regions of the cortex, they did not express the cortex-specific gene Emx1 or the telencephalic-specific gene Bf-1. Furthermore, diencephalic progenitors that integrate heterotopically into the cortex do not persist postnatally, whereas the same progenitors survive and differentiate when they integrate homotopically into the diencephalon. Together our results implicate calcium-dependent adhesion molecules as key mediators of prosomeric organization but suggest that they are not sufficient to bestow regional identities.     

Increased IGF-I and IGF-II mRNA and IGF-I peptide in fusing rat cranial sutures suggest evidence for a paracrine role of insulin-like growth factors in suture fusion

Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (bone morphogenetic protein-4) protein in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, 10, 15, 20, 30, 50, and 80 days after birth (n = 110). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and 10 days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin protein were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce bone-promoting growth factors, such as osteocalcin.     

Synthesis of 1-methyl-3-phenylpyrazolo[4,3-b]pyridines via a methylation of 4-phthalimino-3-phenylpyrazoles and optimization toward highly potent corticotropin-releasing factor type-1 antagonists

1-Methyl-3-phenylpyrazolo[4,3-b]pyridines were synthesized via a cyclization reaction of 1-methyl-4-amino-3-phenylpyrazoles 8 with ethyl acetoacetate. Optimization of this series of compounds resulted in CRF(1) antagonists with subnanomolar binding affinity. Compounds bearing a polar group such as methoxy or hydroxy were also found to be very active.     

Activation of ryanodine receptor/Ca2+ release channels downregulates CD38 in the Namalwa B lymphoma

CD38 is a multifunctional ectoenzyme that catalyses formation of cyclic ADP ribose (cADPr), a second messenger that opens ryanodine receptor (RyR) Ca2+ channels. Despite its importance in signal transduction processes, little is known about the mechanisms regulating CD38 expression levels. In the current study, ryanodine stimulation of Ca2+ release in Namalwa cells decreased both CD38 protein abundance and cyclase activity. Reductions in cyclase activity were prevented by RyR antagonists, by lysosomal blockers, though not by calpain or proteasomal inhibitors. These findings indicate a novel negative feedback mechanism between RyR channel activity and CD38 abundance acts in cADPr signal transduction.     

Sedentary behavior during postnatal life is determined by the prenatal environment and exacerbated by postnatal hypercaloric nutrition

The discovery of a link between in utero experience and later metabolic and cardiovascular disease is one of the most important advances in epidemiology research of recent years. There is now increasing evidence that alterations in the fetal environment have long-term consequences on metabolic and endocrine pathophysiology in adult life. This process has been termed "fetal programming," and we have shown that undernutrition of the mother during gestation leads to obesity, hypertension, hyperphagia, hyperinsulinemia, and hyperleptinemia in offspring. Using this model of maternal undernutrition throughout pregnancy, we investigated whether prenatal influences may lead to alterations in postnatal locomotor behavior, independent of postnatal nutrition. Virgin Wistar rats were time mated and randomly assigned to receive food either ad libitum (ad libitum group) or at 30% of ad libitum intake (undernourished group). Offspring from UN mothers were significantly smaller at birth than AD offspring. At weaning, offspring were assigned to one of two diets [control or hypercaloric (30% fat)]. At ages of 35 days, 145 days, and 420 days, voluntary locomotor activity was assessed. At all ages studied, offspring from undernourished mothers were significantly less active than offspring born of normal birth weight for all parameters measured, independent of postnatal nutrition. Sedentary behavior in programmed offspring was exacerbated by postnatal hypercaloric nutrition. This work is the first to clearly separate prenatal from postnatal effects and shows that lifestyle choices themselves may have a prenatal origin. We have shown that predispositions to obesity, altered eating behavior, and sedentary activity are linked and occur independently of postnatal hypercaloric nutrition. Moreover, the prenatal influence may be permanent as offspring of undernourished mothers were still significantly less active compared with normal offspring at an advanced adult age, even in the presence of a healthy diet throughout postnatal life.