Synthesis and evaluation of prodrugs for anti-angiogenic pyrrolylmethylidenyl oxindoles

Potential prodrugs of inhibitors of VEGF-induced angiogenesis have been investigated. The prodrug systems studied were the 4-nitrobenzyl, 2-nitrophenylacetyl and 3-methyl-3-(3,6-dimethylbenzo-1,4-quinon-2-yl)butanoyl groups, readily attached to acidic OH or NH groups in drug molecules, and released upon bioreductive activation. The anti-angiogenic compounds studied were the pyrrolylmethylidenyl oxindole SU5416 (semaxanib) and its novel 6-hydroxy derivative. The potentially pro-anti-angiogenic compounds were assayed for their ability to block VEGF-induced angiogenesis in HUVECS in comparison to the free agents.     

Synthesis, antifungal and antimicrobial activity of alkylphospholipids

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.     

Assessing core manipulative skills in a large, first-year laboratory

Responding to the concern from our faculty that undergraduate students do not have robust laboratory skills, we designed and implemented a strategy to individually teach and assess the manipulative skills of students in first-year laboratories. Five core laboratory skills were selected for the course entitled Human Biology, a large, first-year class of students, most of whom were enrolled in Bachelor of Pharmacy and Human Movement Studies. Here, we report details for the 365 students enrolled primarily in Pharmacy and Human Movement Studies bachelor degree programs in semester 1 of 2006. We designed a specific strategy to assess five core laboratory skills: 1) accurate and precise use of a micropipette, 2) calculation of dilutions and preparation of diluted samples of saline, 3) accurate representation of data using a graph, 4) use of a light microscope, and 5) acquisition of digital data by measuring the latent period for the Achilles reflex. Graduate tutors were trained to teach and assess each student on each skill. The development of competency was tracked for all students across all five skills. Most students demonstrated proficiency on their first attempt. The development of proficiency across the core skills depended on both the skill and degree program. In semester 2 of 2006, 854 students mostly enrolled in the Bachelor of Science degree program and were similarly taught and assessed on the same five core skills. This approach was an effective teaching and assessment strategy that, when applied beyond first year, should increase the level of laboratory skills across undergraduate programs in physiology.     

SOCS-1 is a central mediator of steroid-increased thymocyte apoptosis and decreased survival following sepsis

Suppressor of Cytokine Signaling (SOCS) proteins are recently identified inhibitors/regulators of cytokine/growth factor signaling pathways. We have previously shown that SOCS-3 is upregulated in mice after sepsis induced by cecal ligation and puncture; however, the contribution of SOCS-1 to septic morbidity and mortality is unclear. In the present study, we characterized SOCS-1 expression in different tissues and delineated putative mechanisms effecting SOCS-1 expression in thymus from septic mice. We observed no difference in SOCS-1 expression in blood, peritoneal leukocytes, lung, and spleen taken from sham or septic animals at 24 h after surgery. In contrast, SOCS-1 expression in thymus declined significantly after sepsis and this down-regulation of SOCS-1 was associated with increased thymocyte apoptosis as well as augmented Bax recruitment to the mitochondria. Administration of RU-38486, a steroid receptor antagonist, reversed the above effects in the septic thymus. Furthermore, SOCS-1+/− mice showed a significant higher mortality when compared to SOCS-1+/+ mice after sepsis. Together, these results show that sepsis increases steroid-induced thymic lymphoid cell apoptosis, which is associated with reduced SOCS-1 expression and increased Bax translocation to mitochondria. Survival data suggests that SOCS-1 protein may play an important role in sepsis.     

Dealing with uncertain absences in habitat modelling: a case study of a rare ground-dwelling parrot

In the development of a species distribution model based on regression techniques such as generalized linear or additive modelling (GLM/GAM), a basic assumption is that records of species presence and absence are real. However, a common concern in many studies examining species distributions is that absences cannot be inferred with certainty. This is particularly the case where the species is rare, difficult to detect and/or does not occupy all available habitat considered suitable. The western ground parrot ( Pezoporus wallicus flaviventris ) of southern Western Australia, Australia, is a case in point, as not only is it rare and difficult to detect, but it is also unlikely to occupy all available suitable habitat. A recent survey of ground parrots provided the opportunity to develop a predictive distribution model. As the data were susceptible to false absences, these were replaced with randomly selected 'pseudo' absences and modelled using GLM. As a comparison, presence-only information was modelled using a relatively new approach, MAXENT, a machine-learning technique that has been shown to perform comparatively well. The predictive performance of both models, as assessed by the receiver operating characteristic plot (ROC) was high (AUC > 0.8), with MAXENT performing only marginally better than the GLM. These approaches both indicated that the ground parrot prefers areas relatively high in altitude, distant from rivers, gently sloping to level habitat, with an intermediate cover of vegetation and where there is a mosaic of vegetation ages. In this case, the use of presence-only information resulted in the identification of important environmental attributes defining the occurrence of the ground parrot, but additional factors that account for the inability of the bird to occupy all suitable habitat should be a component of model refinement.     

RNase MRP and the RNA processing cascade in the eukaryotic ancestor

Background

Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. An example is RNase MRP processing ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simpler network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, herein called the Eukaryotic Ancestor.

Results

We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches, find previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of RNAs for RNase MRP, along with analysis of the target substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution.

Conclusion

We conclude that RNase MRP can now be placed in the RNA-processing cascade of the Eukaryotic Ancestor, highlighting the complexity of RNA-processing in early eukaryotes. Promoter analyses of MRP-RNA suggest that regulation of the critical processes of rRNA cleavage can vary, showing that even these key cellular processes (for which we expect high conservation) show some species-specific variability. We present our consensus MRP-RNA secondary structure as a useful model for further searches.

     

Slowmo is required for Drosophila germline proliferation

Null mutations in the Drosophila gene, slowmo (slmo), result in reduced mobility and lethality in first-instar larvae. Slowmo encodes a mitochondrial protein of unknown function, as do the two other homologs found in Drosophila. Here, we have studied a hypomorphic P-element allele of slmo demonstrating its effects on germline divisions in both testes and ovaries. Using in situ studies, enhancer-trap activity, and promoter fusions, we have shown that slmo expression in testes is found in the somatic cyst cells (SCC). The hypomorphic allele for Slmo revealed apoptotic loss of germline cells in the larval germline, culminating in a complete absence of the germline in adult flies. In females, a similar degeneration of the germarium is observed, while reporter gene expression is found in both germline and somatic cells. Using a null mutation in female germline clones, we find slmo is dispensable from the germline cells. Our results suggest that Slowmo is not required in germline cells directly, but is required in SCCs responsible for maintaining germline survival in both sexes.     

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.