6-Mercaptopurine is used in the treatment of pediatric leukemia and other cancers (1). The drug is transformed enzymically to 6-mercapto-IMP and then into other derivatives such as 6-thio-GTP, 6-thio-ITP and 6-thiomethyl-IMP. 6-Thio-GTP can be incorporated into DNA, increasing the susceptibility of DNA to damage. 6-Thiomethyl-IMP is a potent inhibitor of phosphoribosylpyrophosphate amidotransferase, suppressing de novo purine biosynthesis, and 6-mercapto-ITP is a potent inhibitor of RNA polymerase. Depletion of the adenine nucleotide pool (specifically ATP) by the action of 6-thiomethyl-IMP reduces levels of S-adenosyl-L-methionine, which in turn impedes methylation of DNA and RNA. Although mechanisms for the cytotoxic effects of 6-mercapto-IMP are known, the metabolic pathways by which 6-mercapto-IMP is inactivated as a drug are unclear.Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDPforming), EC 6.3.4.4) is likely to play a role in the metabolic effects of 6-mercapto-IMP. The synthetase governs the first committed step in the de novo biosynthesis of AMP (2), GTP ϩ IMP ϩ L-aspartate º GDP ϩ P i ϩ adenylosuccinate. 6-Mercapto-IMP is a competitive inhibitor with respect to IMP (3-5) and likely inhibits the enzyme in vivo.

Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.

Background:Vascular endothelial growth factor (VEGF)-targeting drugs normalise the tumour vasculature and improve access for chemotherapy. However, excessive VEGF inhibition fails to improve clinical outcome, and successive treatment cycles lead to incremental extracellular matrix (ECM) deposition, which limits perfusion and drug delivery. We show here, that low-dose VEGF inhibition augmented with PDGF-R inhibition leads to superior vascular normalisation without incremental ECM deposition thus maintaining access for therapy.Methods:Collagen IV expression was analysed in response to VEGF inhibition in liver metastasis of colorectal cancer (CRC) patients, in syngeneic (Panc02) and xenograft tumours of human colorectal cancer cells (LS174T). The xenograft tumours were treated with low (0.5 mg kg−1 body weight) or high (5 mg kg−1 body weight) doses of the anti-VEGF antibody bevacizumab with or without the tyrosine kinase inhibitor imatinib. Changes in tumour growth, and vascular parameters, including microvessel density, pericyte coverage, leakiness, hypoxia, perfusion, fraction of vessels with an open lumen, and type IV collagen deposition were compared.Results:ECM deposition was increased after standard VEGF inhibition in patients and tumour models. In contrast, treatment with low-dose bevacizumab and imatinib produced similar growth inhibition without inducing detrimental collagen IV deposition, leading to superior vascular normalisation, reduced leakiness, improved oxygenation, more open vessels that permit perfusion and access for therapy.Conclusions:Low-dose bevacizumab augmented by imatinib selects a mature, highly normalised and well perfused tumour vasculature without inducing incremental ECM deposition that normally limits the effectiveness of VEGF targeting drugs.

Using digital technology, we have assembled a virtual laboratory manual (VLM) that is a Web-based copy of our traditional laboratory manual. The VLM is used to enhance traditional laboratory instruction in histology. For each reference in the VLM to either a histological slide or an electron micrograph (EM), hyperlinks are included that download digital images derived from the students' glass slide sets or scanned EMs. The VLM serves as an atlas of digital images for concurrent study of similar sections by light microscopy during laboratory sessions. In addition, students can review the images from the VLM at remote locations. We have encouraged continued use of light microscopes in laboratories by basing the majority of practical examination identifications on analysis of marked histological slides that require students to use their own microscopes. The VLM provides the convenience of a Web-based resource with high-quality images, yet allows retention of the many excellent traditional aspects of our course. An example of a VLM laboratory on epithelium is available online (http://users.von.uc.edu/michaeje/VLM-Epithelium/exLab4.pdf).