Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent^® and Nexavar^®, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.     

Colony size and not nest density drives reproductive output in the Common Tern Sterna hirundo

Density is known to be an important factor in population size regulation. Several mechanisms of density limitation have been identified in colonial birds. We studied competition in Common Terns Sterna hirundo to assess whether the factor limiting reproductive output was competition for nest‐sites, which is dependent on local nest density, or density‐dependent competition for food resources, which is dependent on overall colony size using the same foraging area. We found strong associations of both colony size and nest density with reproductive output in five colonies of Common Terns in three different habitats (one marine, two freshwater). Based on detailed long‐term datasets of six separate sub‐colonies of the Banter See colony that differed in nest density, we found that reproductive success was not related to nest density but to overall colony size, possibly a result of resource depletion and food competition. We also found carry‐over effects of colony size during rearing on post‐fledging return rate. These results have important implications for the conservation management plans aimed at recovering declining populations of Common Terns.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two-photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non-degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non-degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over approximately 0-480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4 degrees C) was identical over a range of culture medium osmolarities ( approximately 0-515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non-degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (>/=2,500 microm(3)) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non-degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180-515 mOsm).     

Neuronal microtubules: when the MAP is the roadblock

Recent studies shed new light on a potential cascade of events by which neurological diseases such as Alzheimer's lead to axonal degeneration. In this model, the pathology starts with an elevation in microtubule-associated proteins (MAPs) such as tau. This renders the microtubules less accessible to motor proteins, which impairs their capacity to sustain anterograde axonal transport of proteins and organelles. In response, the neuron hyperphosphorylates tau so that it dissociates from the microtubules. Unfortunately, the hyperphosphorylated tau forms abnormal filaments that are deleterious to the axon, and the tau-depleted microtubules become highly sensitive to microtubule-severing proteins such as katanin.     

ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion--Part II: shoulder, elbow, wrist and hand

In this communication, the Standardization and Terminology Committee (STC) of the International Society of Biomechanics proposes a definition of a joint coordinate system (JCS) for the shoulder, elbow, wrist, and hand. For each joint, a standard for the local axis system in each articulating segment or bone is generated. These axes then standardize the JCS. The STC is publishing these recommendations so as to encourage their use, to stimulate feedback and discussion, and to facilitate further revisions. Adopting these standards will lead to better communication among researchers and clinicians.     

Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein

Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5-7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5-7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was apparently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity.     

Diversity of methanotrophic bacteria in tropical upland soils under different land uses

Three upland soils from Thailand, a natural forest, a 16-year-old reforested site, and an agricultural field, were studied with regard to methane uptake and the community composition of methanotrophic bacteria (MB). The methane uptake rates were similar to rates described previously for forest and farmland soils of the temperate zone. The rates were lower at the agricultural site than at the native forest and reforested sites. The sites also differed in the MB community composition, which was characterized by denaturing gradient gel electrophoresis (DGGE) of pmoA gene fragments (coding for a subunit of particulate methane monooxygenase) that were PCR amplified from total soil DNA extracts. Cluster analysis based on the DGGE banding patterns indicated that the MB communities at the forested and reforested sites were similar to each other but different from that at the farmland site. Sequence analysis of excised DGGE bands indicated that Methylobacter spp. and Methylocystis spp. were present. Sequences of the "forest soil cluster" or "upland soil cluster alpha," which is postulated to represent organisms involved in atmospheric methane consumption in diverse soils, were detected only in samples from the native forest and reforested sites. Additional sequences that may represent uncultivated groups of MB in the Gammaproteobacteria were also detected.