Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.     

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.     

Comparative avian phylogeography of Cameroon and equatorial Guinea mountains: implications for conservation

We illustrate the use of Faith's 'Phylogenetic Diversity' measure to compare the phylogeographic structure of two bird species with patterns of avian endemism across six mountains in Cameroon and Equatorial Guinea. The Mountain Greenbul and Cameroon Blue-headed Sunbird showed phylogeographic patterns that together defined three biogeographic regions: Bioko, Mt. Cameroon, and the northern mountains of Cameroon. In contrast, the distributions of endemic species were largely a function of geographical distance, with close mountains sharing more endemic species than distant mountains. Moreover, for both species, populations on Mt. Cameroon were distinctive with respect to the ecologically relevant character bill size. Our results, while preliminary, illustrate the utility of a comparative approach for identifying geographical regions that harbour evolutionarily distinct populations and caution against using only the distributional patterns of endemics to prioritize regions for conservation. Results show that patterns of endemism may not be concordant with patterns of phylogenetic diversity nor morphological variation in a character important in fitness. While incorporation of additional species from unrelated taxa will be necessary to draw definitive conclusions about evolutionarily distinct regions, our preliminary results suggest a conservation approach for the Afromontane region of the Gulf of Guinea that would: (i) emphasize protection of both Bioko and Mt. Cameroon, thereby maximizing preservation of within-species phylogenetic and morphologic diversity; (ii) emphasize protection within the northern mountains to further conserve intraspecific phylogenetic diversity and maximize protection of endemic species.     

Synthesis and pharmacology of new enantiopure delta(3)-4-arylkainoids

Seven delta(3)-4-arylkainoids possessing various 4-position aromatic and heteroaromatic groups were synthesized and their apparent affinities were measured in order to explore the influences of 4-position electron density and stereochemistry on receptor affinity and specificity. Kainoids 1a-f were shown to be selective agonists at the NMDA receptor and the electron rich furanyl and thienyl analogues exhibited the highest affinities. Naphthylkainoid 1g proved to be a nonselective antagonist at the iGluRs.     

Substituted 1,6-naphthyridines as human cytomegalovirus inhibitors: conformational requirements

Substituted 1,6-naphthyridine derivatives, a new class of human cytomegalovirus inhibitors, were prepared to demonstrate the role of intramolecular hydrogen bonds to maintain the compounds in their active conformation.     

Semiquantitative immunoblots of membrane protein-epidermal growth factor

Membrane proteins or cytokines are sometimes difficult to isolate and purify. Our group recently concentrated on epidermal growth factor (EGF) protein expression studies. Mature EGF was initially identified from mouse submaxillary gland extract as a stimulator of eyelid opening and incisor eruption when injected into newborn mice and rats. The EGF precursor is a transmembrane protein with eight additional EGF-like repeats. Our previous study has shown that the EGF precursor without these eight EGF-like repeats (hEGF) was biologically active. Here, we introduce a modified method for rapid detection of hEGF. The membranous protein was directly extracted from various organs of transgenic mice (including the submandibular gland, kidney, liver, heart, and testis) with two different buffers and easily detected by semiquantitative immunoblotting.     

Dynamics of flow, resistance, and intramural vascular volume in canine coronary circulation

Varying coronary volume will vary vascular resistance and thereby have an effect on coronary hemodynamics. Six ventricular septa were isolated from anesthetized dogs, dispersed in a biaxial stretch apparatus at diastolic stress, and perfused artificially with an oxygenated perfluorochemical emulsion at maximal vasodilation. Flow and thickness were measured continuously by an electromagnetic flow probe and sonomicrometer. Pressure was varied sinusoidally around 30, 50, and 70 mmHg with an amplitude of 7.5 mmHg; frequencies ranged between 0.015 and 7 Hz. Bode plots of admittance (flow/pressure) and capacitance (scaled thickness/pressure) were constructed. A two-compartment model was used in which the resistances vary with volume. Realistic values of microvascular compliance ( approximately 0.3 ml x mmHg(-1) x 100 g(-1)) were found. Values 10 times higher were then found when resistances were forced to be constant. We concluded that volume dependence of resistances have to be taken into account when dynamic or static pressure-flow relations are studied and conceal the effect of a large intramyocardial compliance on arterial hemodynamics.     

Evolution of the prohormone convertases: identification of a homologue of PC6 in the protochordate amphioxus

Many of the protein precursors traversing the secretory pathway undergo cleavage at multibasic sites to generate their bioactive forms. The proprotein convertases (PCs), a family of subtilisin-like proteases, are the major endoproteases that serve this function. Genes encoding seven distinct members of this family have so far been characterized in vertebrates: furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6 and PC7/PC8/LPC. Multiple PC genes have also been cloned from a number of invertebrates, including Drosophila melanogaster and Caenorhabditis elegans. These findings suggest that gene duplication and diversification of the PCs have occurred throughout metazoan evolution. To investigate the structural and functional changes which have occurred during vertebrate development, we have analyzed the expression of PC genes in the protochordate amphioxus. We have previously shown that amphioxus express homologous PC2 and PC1/PC3 genes [Proc. Natl. Acad. Sci. USA 92 (1995) 3591]. Here we report the characterization of amphioxus cDNAs encoding proteases with a high degree of similarity to mammalian PC6. Three cDNAs encoding three PC6 isoforms differing only in their carboxy-terminal sequences were found, derived by alternative splicing. Two isoforms appear to be soluble enzymes, whereas the third contains a transmembrane hydrophobic segment and thus is likely to be membrane-bound. All three variants contain many repeats of a cysteine-rich motif that is found in several other PC family members. Thus, amphioxus, like the vertebrates, expresses two types of PCs, e.g., PC2 and PC1/PC3 which function in the regulated secretory pathway in neuroendocrine cells, and the more widely expressed PC6 which functions mainly in the constitutive pathway.