Discovery of 3,3-dimethyl-5-hydroxypipecolic hydroxamate-based inhibitors of aggrecanase and MMP-13

A series of pipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was discovered based on screening known inhibitors of TNF-alpha converting enzyme (TACE). Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group. Incorporation of geminal alkyl substitution at the 3-position of the piperidine ring improved metabolic stability, presumably by increasing steric hindrance around the metabolically labile hydroxamic acid. This modification also resulted in dramatic improvement of aggrecanase activity with a slight reduction in selectivity versus MMP-1. Synthesis, structure activity relationships, and strategies to reduce metabolic clearance are described.     

Evidence for multiple Pleistocene refugia in the postglacial expansion of the eastern tiger salamander,Ambystoma tigrinum tigrinum

Abstract Pleistocene glaciations were important determinants of historical migration and, hence, current levels of genetic diversity within and among populations. In many cases, these historical migrations led to the existence of disjunct populations of plants and animals. However, the origin and timing of arrival of these disjunct populations is often debated. In the current study, we identify potential refugia and estimate the timing of vicariance events of the eastern tiger salamander, Ambystoma tigrinum tigrinum , using mitochondrial sequence data. The results suggest a vicariant event 0.75–2 million years ago, separating the tiger salamanders to the east and west of the Apalachicola River Basin. East of the Appalachians, there appear to be multiple independent refugia with little migration among the remaining populations. In particular, populations along the Atlantic Coastal Plain were likely isolated in a coastal plain refugium in the Carolinas. Migrants from this refugium were the likely source of colonists for populations occupying previously glaciated areas along the northeastern Atlantic Coast. A second potential refugium occurs in the Blue Ridge Mountains of western Virginia. This refugium contains a disjunct population of the eastern tiger salamander, as well as a community of nearly 70 other disjunct plant and animal species. The tiger salamanders here have been isolated from other populations for 200,000–500,000 years. These results suggest that disjunct mountain populations of Coastal Plain species may have existed in situ throughout the Pleistocene in Appalachian refugia. Therefore, these disjunct populations are not of recent origin, but rather exist as relicts of a warmer, more widespread fauna and flora that is now restricted to the Coastal Plain.     

Adjuvanticity of an IL-12 fusion protein expressed by recombinant deltaG-vesicular stomatitis virus

The remarkable immunomodulatory and adjuvant properties of rIL-12 have been well described. Many early studies documenting the adjuvanticity of IL-12 were performed using the murine model of Listeria monocytogenes infection. In this report, we describe the construction of an attenuated recombinant vesicular stomatitis virus (VSV-deltaG) that encodes a single-chain IL-12 fusion protein (IL-12F), and the use of this virus as an expression vector to produce large quantities of IL-12F. VSV-expressed IL-12F (vIL-12F) was then co-administered to mice along with a poorly immunogenic listerial antigen preparation as a vaccine regimen and the resulting immune responses were monitored. The vIL-12F was found to have adjuvant properties similar to those observed for rIL-12. Co-administration of vIL-12F and listerial antigen elicited powerful cell-mediated immune responses that conferred long-lived protective listerial immunity. These studies demonstrated that VSVdeltaG-IL12F-infected cells secrete bioactive single-chain IL-12, and laid the foundation for studies using VSVdeltaG-IL12F as a vector for delivery of IL-12F in vivo.     

A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate beta -catenin-dependent signaling

Wnt proteins initiate the canonical (beta-catenin-regulated) signaling cascade by binding to seven-transmembrane spanning receptors of the Frizzled (Fz) family together with the coreceptors LRP5 and -6, members of the low density lipoprotein receptor-related protein family (LRP). Several reports have shown physical and functional associations between various Wnt, LRP, and Frizzled molecules; however, the underlying mechanisms for selectivity remain poorly understood. We present data on a novel set of Wnt-Fz fusion constructs that are useful for elucidating mechanisms of Wnt signal transduction specificity in both Xenopus embryos and 293T cells. In 293T cells, coexpression of several Wnt-Fz fusion proteins with LRP6, but not LRP5, significantly activated a Wnt-responsive promoter, Optimized TOPFlash. Interestingly, Wnt proteins from both the Wnt1 and Wnt5A classes, when fused to the same Frizzled, can synergize with LRP6 to activate signaling and induce secondary axes in Xenopus embryos. However, when several Wnt-Fz constructs containing different Frizzled molecules were tested, it was found that all Frizzled molecules are not equivalent in their ability to activate the canonical Wnt pathway in this context. The data suggest that the distinction between the two Wnt classes lies not in intrinsic differences in the molecules but via the Frizzled molecules with which they interact.     

Wnt-independent activation of beta-catenin mediated by a Dkk1-Fz5 fusion protein

An XWnt8-Fz5 fusion protein synergizes with LRP6 to potently activate beta-catenin-dependent signaling. Here, we generated a fusion in which XWnt8 was fused to the N-terminus of LRP6 and show it synergizes with both Fz4 and Fz5 to potently transactivate beta-catenin-dependent Wnt signaling. Based on this, we hypothesized that the main function of Wnt is to nucleate the formation of a physical complex between LRP6 and a Frizzled. Dkk1, but not the related Dkk3, binds LRP6 and inhibits canonical Wnt signaling by blocking the interaction of Wnt and LRP6. Therefore, we reasoned that a covalent fusion of Dkk1 to Fz5 (Dkk1-Fz5) would mimic Wnt ligand by nucleating the formation of a complex containing Fz5 and LRP6, while Dkk3 (Dkk3-Fz5) would not. We found that Dkk1-Fz5, but not Dkk3-Fz5, potently synergized with LRP6 to activate signaling in a dishevelled-dependent manner.     

Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway

Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.     

The ability of a gonadotropin-releasing hormone antagonist, acyline, to prevent irreversible infertility induced by the indenopyridine, CDB-4022, in adult male rats: the role of testosterone

Intratesticular testosterone (ITT) is known to play a critical role in the maintenance of spermatogenesis. We have used acyline, a GnRH antagonist, to suppress testosterone (T) production, and acyline and T implants to study the prevention of irreversible infertility induced by CDB-4022. Vehicle or acyline was administered to proven fertile male rats (n = 5/group) at a dose (210 microg/day) that completely suppressed (P < 0.05) T production, as measured by serum T, and testicular function, either before, concurrent with, or after vehicle or a single oral dose of 2.5 mg CDB-4022/kg (Week 0). Vehicle-treated males remained fertile, whereas acyline-treated males exhibited transitory infertility. CDB-4022 alone caused irreversible infertility in all males. Importantly, CDB-4022-treated males recovered fertility when acyline was started before CDB-4022 (Weeks -4 to 0; Weeks -4-9), but not when acyline was administered concurrently with or after CDB-4022 (Weeks 0-9; Weeks 10-19). At the end of this study (Week 34), testes weights, spermatid head counts (SHC), and tubule differentiation indices (TDI) were suppressed (P < 0.05) in infertile CDB-4022-treated males, but in rats that recovered fertility, these parameters were similar (P > 0.05) to those in vehicle-treated males. In addition, serum inhibin B and epididymal androgen-binding protein levels were nondetectable in infertile CDB-4022-treated rats. To test whether suppression of ITT was critical for prevention of CDB-4022-induced infertility, proven fertile rats (n = 7-8/group) received vehicle, acyline alone, or acyline and a T implant for 4 wk before CDB-4022 (Week 0). The T implant increased ITT in acyline-treated rats. Although ITT was lower (P < 0.05) in the T-implanted males than in untreated rats, it was sufficient to sustain spermiogenesis. Serum rFSH levels were also elevated in rats treated with acyline + T as compared with acyline alone during the treatment interval, but rFSH was still lower than in vehicle-treated rats. Rats in all treatment groups were rendered infertile initially, but the acyline + CDB-4022-treated rats recovered fertility by Week 10. In contrast, rats treated with CDB-4022 alone or acyline + T + CDB-4022 remained infertile until at least Week 16. Testes weights, SHC, and TDI were within normal ranges for acyline + CDB-4022-treated rats, but were decreased (P < 0.05) in CDB-4022- or acyline + T + CDB-4022-treated rats. Serum inhibin B levels were nondetectable by Week 1 in males rendered irreversibly infertile by CDB-4022; levels increased transiently and returned to baseline in rats protected by acyline pretreatment. These data indicate that pretreatment with acyline was able to prevent irreversible infertility in CDB-4022-treated rats, whereas posttreatment with acyline did not promote spermatogonial differentiation, as has been observed by others in rats that received GnRH analogs and various other testicular toxicants. Suppression of ITT and possibly rFSH by acyline appeared to be crucial in preventing irreversible infertility induced by CDB-4022. In this regard, our results are similar to those of investigators who have studied other testicular toxicants. Continued development of CDB-4022 as a potential male contraceptive will depend largely on its safety profile and whether its antispermatogenic activity is reversible in primates.     

Parallel and interrelated neural systems underlying adaptive navigation

The ability to process in parallel multiple forms of sensoryinformation, and link sensory-sensory associations to behavior,presumably allows for the opportunistic use of the most reliableand predictive sensory modalities in diverse behavioral contexts.Evolutionary considerations indicate that such processing mayrepresent a fundamental operating principle underlying complexsensory associations and sensory-motor integration. Here, wesuggest that animal navigation is a particularly useful modelof such opportunistic use of sensory and motor information becauseit is possible to study directly the effects of memory on neuralsystem functions. First, comparative evidence for parallel processingacross multiple brain structures during navigation is providedfrom the literatures on fish and rodent navigation. Then, basedon neurophysiological evidence of coordinated, multiregionalprocessing, we provide a neurobiological explanation of learningand memory effects on neural circuitry mediating navigation.     

Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.