Cloning and expression of the Epstein-Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme.

The gene encoding the Epstein-Barr virus (EBV)-specific dUTPase was amplified from virus DNA by PCR. The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein. The protein from E. coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs. Preliminary experiments yielded a Km value of about 0.8 microM for dUTP. MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated B cells harbouring either type 1 or type 2 EBV. The protein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression. We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein. The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from normal, healthy carriers and from patients with various diseases. While the sera of EBV-negative individuals (0/3) or healthy carriers (0/33) did not contain detectable levels of antibodies, patients with mononucleosis (5/18), chronic EBV infection (2/7), EBV reactivation (7/20) and human immunodeficiency virus infection (5/24) showed elevated antibody titres against the enzyme. This indicated that the dUTPase is expressed during EBV replication and reactivation. The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication.

Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase.

dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E. coli contains a total of six tyrosine residues per trimer. About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity. Deacetylation with N-hydroxylamine leads to full reactivation. Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification. In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP. These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme.

Disseminated intravascular coagulation associated with toxic epidermal necrolysis (Lyell's syndrome).

Evidence of disseminated intravascular coagulation was recorded in eight patients with toxic epidermal necrolysis (TEN)--Lyell's syndrome. Patients were treated with low doses of heparin in combination with the usual treatment of TEN, i.e. maintenance of fluid and electrolyte balance, systemic corticosteroids, antibiotics and aseptic dressings, in the Intensive Care Unit environment. It is suggested that the alteration of haemostasis and inter-related biological systems, such as activation of components of complement, kinins and immunoglobulins, may affect the outcome of TEN.

A hybrid modular set for on-line preprocessing of data in electrophysiology.

An introductory review of hardware aspects of on-line experimental data processing reveals that the combination of a specialized (hard-wired) preprocessing unit coupled with a programmable laboratory computer is an optimal set up for an electrophysiological laboratory. The paper deals with a proposed modular system, which makes the assembly of a large number of different preprocessing units possible. Some practical applications of the preprocessing units coupled with a LINC (D.E.C.) computer are presented in conclusion.

A low frequency active filter with plug-in tuning impedances.

A new approach to the construction of low frequency (1 Hz -- 20 kHz) active filters, based on the application of a pair of IC operational amplifiers forming a separate module and a plug-in unit containing RC tuning circuits, is described. The electronic circuits and the design of the filters are presented with all details.