USP7 (HAUSP) is a deubiquitinating enzyme, which plays a crucial role in regulating the levels of the p53 tumour suppressor protein, through its ability to prevent the proteasomal degradation of the Ubiquitin ligase for p53, Hdm2. Supporting evidence suggests that an inhibitor of USP7 would act to abrogate the action of Hdm2, and thereby elevate levels of the p53 protein, with associated therapeutic benefits in cancer and potentially other diseases. In this article, we describe the characterisation of differential enzyme activity of both the full length and putative catalytic domain of human USP7 expressed in both bacterial and insect cell expression systems. We also demonstrate the way in which variations in the reducing environment surrounding the enzyme can dramatically affect both the stability of the enzyme and the range of small molecules able to inhibit the catalytic activity of the enzyme. Furthermore, we describe the validation and use of this assay for a high-throughput screening approach, again highlighting the critical nature of the enzyme's environment. Taken together, these findings not only increase our understanding of the enzymatic activity of deubiquitinating enzymes, but also highlight several key considerations of importance in the development of therapeutic agents against this novel class of therapeutic targets.

Draining lymph node cells isolated from mice 24 h following topical exposure to a variety of contact-sensitizing chemicals, including the dinitrobenzene derivatives, 2,4-dinitrochlorobenzene and 2,4-dinitrothiocyanobenzene, contained increased numbers of dendritic cells (DCs). The increase in frequency of DCs was time-dependent and preceded significant changes in either lymph node cellularity or lymph node cell pro-liferative activity. The degree of DC accumulation was also influenced by the chemical used and the concentration employed for sensitization. In the context of contact allergy, the biological relevance of this phenomenon to the induction of hapten-specific responses is indicated by the fact that relatively small numbers of DC-enriched fractions of lymph node cells (comprising approximately 70% DCs), but not unfractionated or DC-depleted populations, transferred sensitization to naive animals. Moreover, using the skin-sensitizing fluorochrome, fluorescein isothiocyanate, it was observed that 24 h following exposure the majority of lymph node cells bearing high concentrations of antigen were within the DC-rich fraction.

Immunoradiometric assays using antibodies to spleen and heart ferritin were combined with Mössbauer studies on normal and pathological erythrocytes. All erythrocytes examined were found to contain greater amounts of heart type than spleen type ferritin. The ferritin concentration in erythrocytes from patients with beta thalassaemia, sickle cell disease and sideroblastic anaemia is much higher than in normal cells. When the concentration of ferritin-like iron in the pathological erythrocytes measured by Mössbauer spectroscopy is compared to the total amount of ferritin assayed by the two antibodies in the same haemolysates the iron/protein ratio ranges between 0.3 and 3.4. The iron/protein ratio in iron-filled ferritin molecules is about 0.56 and values in excess of this suggest that the iron detected in these cells is a mixture of ferritin molecules, partly denatured ferritin polymers and 'haemosiderin'. There is a possibility that erythrocytes contain an immunologically distinct type of ferritin that is not detected by existing assays, but we have no direct evidence for this.

DNA distribution in bone marrow cells from 10 normal subjects and 34 patients with MDS (24 with refractory anaemia (RA) and 10 with excess blasts (RAEB] was determined by flow cytometry using a FACS III. DNA histograms were resolved into G0/1, S and G2/M compartments by fitting Gaussian distributions and the DNA content of G0/1 cells, expressed as DNA index (DI), was determined. In 19 MDS patients the DI was outside the normal range, those with RA tending to be hyperdiploid. Two patients with RAEB had a second G0/1 peak. In five cases of RA the number of cells in G0/1 was below the normal range and the mean value was significantly lower than normal for this group as a whole (P = 0 X 018). Eleven (RA) patients had a higher percentage of cells in G2/M than normal (P = 0 001). In RAEB patients there is a suggestion of increased numbers in G0/1 and a decrease in S phase and G2/M cells. All three of the deaths amongst RA patients and four out of the five deaths in RAEB patients since the beginning of this study were associated with an abnormal DI at the initial investigation.

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