Loop Domain Peptides from the SOS ras-Guanine Nucleotide Exchange Protein, Identified from Molecular Dynamics Calculations, Strongly Inhibit ras Signaling

In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.     

An Effector Peptide from Glutathione-S-Transferase-pi Strongly and Selectively Blocks Mitotic Signaling by Oncogenic ras-p21

Oncogenic ras-p21 directly activates jun-N-terminal kinase (JNK) and its substrate, jun as a unique step on its mitogenic signal transduction pathway. This activation is blocked by the specific JNK-jun inhibitor, glutathione-S-transferase-pi (GST-pi). Four domains of GST-pi have been implicated in this regulatory function: 34-50, 99-121, 165-182, and 194-201. The 34-50 domain is unique in that it does not affect GST-pi binding to JNK-jun but blocks jun phosphorylation by JNK. We now find that it completely blocks oncogenic (Val 12-) ras-p21-induced oocyte maturation but has no effect on insulin-induced oocyte maturation. Because the latter process requires activation of wild-type ras-p21, this peptide appears to be specific for inhibiting only the oncogenic form of ras-p21, suggesting its use in treating ras-induced tumors.     

Optimized expression and refolding of human keratoepithelin in BL21 (DE3)

Keratoepithelin (KE) is an extracellular protein participating in cell adhesion and differentiation. Mutations of the KE gene (on 5q31 in humans) cause deposition of abnormal proteins (amyloid and non-amyloid) in corneal stroma and lead to several corneal dystrophies in humans. However, further studies on the KE protein have been limited by the intrinsic difficulty of purifying this protein. A high-expression plasmid containing human KE gene was constructed to generate recombinant KE proteins in Escherichia coli. The plasmid was transformed into E. coli BL21 (DE3) and the recombinant protein was expressed as an insoluble His-tagged fusion protein and purified by nickel chelation affinity chromatography under denaturing conditions. On average, 12 mg of purified KE was routinely obtained from 1L of culture media. The recombinant KE was refolded in arginine-containing dialysis solutions and the recovery of bioactive KE typically was approximately 70%. The procedures developed in this report should enable reproducible production of KE and related mutant proteins in large quantities and facilitate future studies on biochemical and biophysical properties of KE and the pathogenesis of related corneal dystrophies.     

Identification of the site of inhibition of mitogenic signaling by oncogenic ras-p21 by a ras effector peptide

We have previously found that a ras switch 1 domain peptide (PNC-7, residues 35–47) selectively blocks oocyte maturation induced by oncogenic (Val 12–containing) ras-p21 protein and also blocks c-raf–induced oocyte maturation. We now find that oncogenic ras-p21 does not inhibit oocyte maturation of a constitutively activated raf protein (raf BXB) that is lacking most of the first 301 amino terminal amino acids, including the major ras binding domain and accessory ras-binding regions. We also find that a dominant negative raf that completely blocks c-raf–induced maturation likewise does not block raf-BXB–induced maturation. We conclude that PNC-7 blocks ras by binding to the amino-terminal domain of raf and that raf BXB must initiate signal transduction in the cytosol.     

The Discovery of orally available thrombin inhibitors : Studies towards the optimisation of CGH1668

The chemical optimisation of CGH1668 1 is described employing an in vivo model of absorption to determine the influence on bioavailability of single point modifications to five key molecular templates. The discovery of an orally bioavailable and selective thrombin inhibitor, 24, highlights the utility of this approach.     

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer

Introduction

Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.

Methods

Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.

Results

The ISL1 genes’ promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).

Conclusions

In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.     

Screening Marine Fungi for Inhibitors of the C4 Plant Enzyme Pyruvate Phosphate Dikinase: Unguinol as a Potential Novel Herbicide Candidate

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C₄ plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 ± 0.8 μM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C₄ plant but no effect on a model C₃ plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.     

Using miniaturized radiotelemetry to discover the breeding grounds of the endangered New Zealand Storm Petrel Fregetta maoriana

Identification of breeding sites remains a critical step in species conservation, particularly in procellariiform seabirds whose threat status is of global concern. We designed and conducted an integrative radiotelemetry approach to uncover the breeding grounds of the critically endangered New Zealand Storm Petrel Fregetta maoriana (NZSP), a species considered extinct before its rediscovery in 2003. Solar‐powered automated radio receivers and hand‐held telemetry were used to detect the presence of birds on three island groups in the Hauraki Gulf near Auckland, New Zealand. At least 11 NZSP captured and radiotagged at sea were detected at night near Te Hauturu‐o‐Toi/Little Barrier Island with the detection of an incubating bird leading to the discovery of the first known breeding site for this species. In total, four NZSP breeding burrows were detected under mature forest canopy and three adult NZSP and two NZSP chicks were ringed. Telemetry data indicated NZSP showed strong moonlight avoidance behaviour over the breeding site, had incubation shifts of approximately 5 days and had a breeding season extending from February to June/July, a different season from other Procellariiformes in the region. Radiotelemetry, in combination with rigorously collected field data on species distribution, offers a valuable technique for locating breeding grounds of procellariiform seabirds and gaining insights into breeding biology while minimizing disturbance to sensitive species or damage to fragile habitat. Our study suggests an avenue for other breeding ground searches in one of the most threatened avian Orders, and highlights the general need for information on the location of breeding sites and understanding the breeding biology in data‐deficient birds.     

What do changes in prematch vs. postmatch, 1, 2, and 3 days postmatch body weight tell us about fluid status in English Premiership rugby union players?

This study investigated changes in body weight pre and postmatch and 1, 2, and 3 days postmatch. Thirty-six players contracted to an English Premiership rugby union club had their pre and postmatch body weight and 1, 2, and 3 day postmatch body weight recorded across 14 matches played (10 at home and 4 away) during the official 2003-2004 professional rugby union season, representing a total of 262 player appearances. Body weight was recorded using a set of calibrated Seca digital scales with players wearing underwear only and toweled dry of all sweat (postmatch). Players were allowed to ingest fluid ad libitum throughout each match. A number of players recorded pre to postmatch reductions of body weight of >2% with some as high as 4.9%. Significant position-specific mean reductions in prematch to postmatch body weight (±SD) were found for both forwards (1.94 ± 0.14 kg) and backs (1.04 ± 0.17 kg). The mean gain in postmatch to 1-day postmatch body weight was significant for forwards (1.40 ± 0.27 kg) but not for backs (0.76 ± 0.30 kg). There were no significant mean differences between prematch and 2 or 3 days postmatch body weight for either forwards or backs. Forwards on average lost a significantly greater proportion of their weight pre to postmatch than backs (p = 0.005). Forwards were on average 99.5% of the prematch weight at 1 day postmatch, whereas backs were 99.7% (p = 0.598). Forwards were 99.6% of their prematch weight at 3 days postmatch, whereas backs were 100.4% (p = 0.035). Changes in fluid status can be effectively monitored by recording changes in body weight and is useful where players are undertaking training sessions within 1, 2, or 3 days after their last match as a measure of rehydration status.     

Amino acid sequence of a ferredoxin from Rhodymenia palmata, a red alga in the florideophyceae

The amino acid sequence of a [2Fe-2S] ferredoxin from a red alga, Rhodymenia palmata in the family Florideophyceae, was determined by conventional methods. The ferredoxin is composed of 97 amino acid residues having five cysteines, but lacking methionine and tryptophan. It possesses a number of structural features of particular interest. The amino acid sequence is compared with those previously determined for ferredoxins from two red algae in the family Bangiophyceae. Conclusions from a comparison of the structures, by noting features such as the presence of gaps in the sequences and by constructing a phylogenetic tree, were consistent with the proposed taxonomic relationship among these algae.