The development of DNA and protein microarrays represents a significant advance in transcriptomics and proteomics research. Such arrays allow the high-throughput, parallel analysis of protein occurrence and interactions and gene expression. However, this advance has not been matched by equivalent technology for analysis of glycomes. One reason for this is that compared to proteins, it is difficult to reliably immobilise populations of chemically and structurally diverse glycans. We describe the development of a new microarray slide surface to which diverse glycan structures can be directly immobilised without prior derivatisation of the slide surface or any modification of the arrayed samples. The slides can be used to produce comprehensive microarrays of carbohydrates, glycoproteins and proteoglycans using isolated samples or cell extracts. Using standard microarray equipment, a series of carbohydrate microarrays were generated and probed with a panel of monoclonal antibodies with specificities for glycan epitopes. The arrays were highly reproducible, stable, and could be stored dry for several months. Glycans play central roles in development, carcinogenesis, cell adhesion, and immunity and are increasingly the subject of therapeutic approaches. We anticipate that the development of carbohydrate microarrays will be important for the high-throughput analysis of glycans and their molecular interactions.

(t1 ⁄2 at 15°C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.The human cytosolic thymidine kinase, TK1 (EC 2.7.1.21), is a key enzyme in the salvage synthesis of TMP from thymidine. Intracellular TMP is quickly phosphorylated to TDP and TTP. Because TTP is an allosteric effector of ribonucleotide reductase, imbalances in the TTP pool disturb the supply of both purines and pyrimidines for DNA synthesis and repair. In turn, imbalanced deoxynucleoside triphosphate (dNTP) pools increase the mutation rate and probability of carcinogenesis (1-3).TK1 is a cell cycle-regulated enzyme. Its activity fluctuates with DNA synthesis, being high in dividing and malignant cells and low in quiescent cells (4, 5). The expression of TK1 is meticulously controlled on the transcriptional and post-transcriptional level (6, 7). At the enzymatic level, ATP, besides being a cosubstrate, has been shown to be a regulator of the catalytic activity of TK1 (8). Thus, exposure to ATP induces a reversible enzyme concentration-dependent transition from a low thymidine affinity dimer of about 50 kDa (K m ϭ 15 M) to a high affinity tetramer (K m ϭ 0.7 M). To further investigate the effect of ATP, we constructed a pET3a-TK1 plasmid (9) containing the amino acid coding region of TK1 cDNA from the pTK11 plasmid of Bradshaw and Deininger (10), who had used SV40 transformed human fibroblasts as the mRNA source. We expressed the resulting recombinant TK1 (rTFi-TK1) in Escherichia coli, and purified and characterized the enzyme. To our surprise we found that the enzymatic properties of rTFi-TK1 differed markedly from those of the endogenous Ly-TK1 with respect to the regulatory effect of ATP (9). Irrespective of preexposure to ATP, the recombinant rTFi-TK1 was a permanent tetramer of about 100 kDa with high affinity to thymidine with a K m value about 0.4 M (9). At that time, we assumed that the amino acid sequences of rTFi-TK1 and Ly-TK1 were identical and explained the divergent properties of rTFi-TK1 by the absence of post-translational modification of TK1 when expressed in E. coli (9). Because the pET3a-TK1 expression system was not satisfactory in terms of amount of TK1 protein produced, we constructed another expression plasmid, pGEX-2T-LyTK1. Here, the amino acid coding region of TK1 from normal human lymphocytes was cloned into the pGEX-2T vector, and the recombinant TK1 was expressed as an isopropyl-1-thio-␤-D-galactopyranoside-inducible glutathione S-transferase fusion protein. In contrast to the findings with rTFi-TK1, our preliminary kinetic experiments showed that the recombinant lymphocyte TK1 (rLy-TK1) 1 behaved essentially as the endogenous lymphocyte enzyme, Ly-TK1, regarding kinetic and oligomerization properties. Therefore, absence of posttranslational modification of rTFi-TK1 in E. coli cannot ex...

Neo-angiogenesis is a critical process for tumor growth and invasion and has become a promising target in cancer therapy. This manuscript reviews three currently relevant anti-angiogenic agents targeting the vascular endothelial growth factor system: bevacizumab, ramucirumab and sorafenib. The efficacy of anti-angiogenic drugs in adjuvant therapy or as neo-adjuvant treatment has been estimated in clinical trials of advanced breast cancer. To date, the overall observed clinical improvements are unconvincing, and further research is required to demonstrate the efficacy of anti-angiogenic drugs in breast cancer treatments. The outcomes of anti-angiogenic therapy have been highly variable in terms of tumor response. New methods are needed to identify patients who will benefit from this regimen. The development of biomarkers and molecular profiling are relevant research areas that may strengthen the ability to focus anti-angiogenic therapy towards suitable patients, thereby increase the cost-effectiveness, currently estimated to be inadequate.

No abstract

A series of sterically hindered 0-hydroxy aromatic ketones were synthesized, including benzeae, naphthalene, pbeoanthene and pyrene derivatives. Deuterium isotope effects on the 13C chemical shifts of 2-hydroxy-l-acenaphthone and other sterically hindered, intramolcularly hydrogen-bonded aromatic ketones (OH exchanged) are shown to he M U S U~~. The two-bond isotope effects are very large. Likewise are the istope eff& on C-0, C-1, C-3 a d C-4 carbon resonances and some show unltsual signs. These unusual effects 8re e x p l a i d by a higher degree of twist in the deuterio than the protio compound. Steric isotope effects are also observed on OH chemical shifts of sterically hindered o-hydroxy acetyl aromatic compounds deuteriated at the methyl group. These isotope effeets show non-additivity. For one-bond isotope effects, IA"C ( l a 0), hydrogen bonding leads to a decrease, whereas twisting of the carbonyl group leads to an increase. Two hydrogen bonds to the same acceptor has a reduced cumulative effect. Data for sterically hindered, hydrogen-bonded compounds are found to fall outside the correlation between 6(''O) and 1A13C(180).KEY WORDS NMR NMR Isotope effects on "C chemical shifts Deuterium isotope effects "0 isotope effects Intramolecular hydrogen bonding Steric strain Hydrogen bond strength