Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase.

BACKGROUND: Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers. However, the optimal patient population who benefit from this treatment is unknown. METHODS: Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20. RESULTS: Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment. Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS. Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS-), giving CBR rates of 52.9 vs 10%, P=0.041. Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive. The survival of ASS- patients receiving at least four doses at 320 IU m(-2) was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024. CONCLUSION: ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression. Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.

Dimerization of the human cytomegalovirus protease: kinetic and biochemical characterization of the catalytic homodimer.

The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized. The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species. Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration. The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated. The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp. The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form. Monomeric HCMVp is inactive. This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer. This protease dimerization may have regulatory significance during viral replication.

Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein.

The structure of human rhinovirus-14 3C protease (3Cpro) has been determined at 2.3 A resolution and refined to an R factor of 0.22. This cysteine protease folds into two topologically equivalent six-stranded beta barrels and in this sense is similar to trypsin-like serine proteases. However, there are differences in the lengths and positioning of individual beta strands as well as in loops connecting elements of secondary structure. The catalytic residues Cys-146, His-40, and Glu-71 are positioned as in serine proteases, but the oxyanion hole is moved 1-1.2 A away. Residues that bind to the 5' noncoding region of rhinovirus genomic RNA are located on the opposite side of the molecule from the active site. Interactions between individual 3Cpro molecules in the crystal lattice suggest a model for intermolecular proteolytic cleavage of the 3CD polyprotein.

Photosynthesis, Growth, and Ultraviolet Irradiance Absorbance of Cucurbita pepo L. Leaves Exposed to Ultraviolet-B Radiation (280-315 nm).

Net photosynthesis, growth, and ultraviolet (UV) radiation absorbance were determined for the first leaf of Cucurbita pepo L. exposed to two levels of UV-B irradiation and a UV-B radiation-free control treatment. Absorbance by extracted flavonoid pigments and other UV-B radiation-absorbing compounds from the first leaves increased with time and level of UV-B radiation impinging on leaf surfaces. Although absorbance of UV-B radiation by extracted pigments increased substantially, UV-B radiation attenuation apparently was insufficient to protect completely the photosynthetic apparatus or leaf growth processes. Leaf expansion was repressed by daily exposure to 1365 Joules per meter per day of biologically effective UV-B radiation but not by exposure to 660 Joules per meter per day. Photosynthesis measured through ontogenesis of the first leaf was depressed by both UV-B radiation treatments. Repression of photosynthesis by UV-B radiation was especially evident during the ontogenetic period of maximum photosynthetic activity.

Net Photosynthesis, Electron Transport Capacity, and Ultrastructure of Pisum sativum L. Exposed to Ultraviolet-B Radiation.

Pisum sativum L. was exposed to ultraviolet-B (UV-B) radiation (280-315 nm) in greenhouse and controlled environment chambers to examine the effect of this radiation on photosynthetic processes. Net photosynthetic rates of intact leaves were reduced by UV-B irradiation. Stable leaf diffusion resistances indicated that the impairment of photosynthesis did not involve the simple limitation of CO(2) diffusion into the leaf. Dark respiration rates were increased by previous exposure to this radiation. Electron transport capacity as indicated by methylviologen reduction was also sensitive to UV-B irradiation. The ability of ascorbate-reduced 2,6-dichlorophenolindophenol to restore much of the electron transport capacity of the UV-B-irradiated plant material suggested that inhibition by this radiation was more closely associated with photosystem II than with photosystem I. Electron micrographs indicated structural damage to chloroplasts as well as other organelles. Plant tissue irradiated for only 15 minutes exhibited dilation of thylakoid membranes of the chloroplast in some cells. Some reduction in Hill reaction activity was also evidenced in these plant materials which had been irradiated for periods as short as 15 minutes.

Photosynthesis, Dark Respiration, and Growth of Rumex patientia L. Exposed to Ultraviolet Irradiance (288 to 315 Nanometers) Simulating a Reduced Atmospheric Ozone Column.

Net photosynthesis, dark respiration, and growth of Rumex patientia L. exposed to a ultraviolet irradiance (288-315 nanometers) simulating a 0.18 atm.cm stratospheric ozone column were determined. The ultraviolet irradiance corresponding to this 38% ozone decrease from normal was shown to be an effective inhibitor of photosynthesis and leaf growth. The repressive action on photosynthesis accumulated through time whereas leaf growth was retarded only during the initial few days of exposure. Small increases in dark respiration rates occurred but did not continue to increase with longer exposure periods. A reduction in total plant dry weight and leaf area of approximately 50% occurred after 22 days of treatment, whereas chlorophyll concentrations remained unaltered.