Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster.

Activator-dependent preinduction binding of sigma-70 RNA polymerase at the metal-regulated mer promoter.

Expression of the Tn21 mercury-resistance (mer) locus is controlled by the merR gene product, which represses mer structural gene (merTPCAD) transcription in the absence of mercuric ion [Hg(II)] and activates it in the presence of Hg(II). In vivo DNA methylation of the mer regulatory region (merOP) shows that, with or without the inducer Hg(II), MerR strongly protects four guanine residues in a dyadic region located between the -10 and -35 hexamers of the structural gene promoter (PTPCAD). Prior to induction by Hg(II), RNA polymerase is also bound at PTPCAD; occupancy of the uninduced promoter by RNA polymerase is dependent on MerR. Methylation and permanganate footprinting demonstrate that induction by Hg(II) results in MerR/Hg(II)-dependent promoter DNA melting in the -10 region of PTPCAD and in additional DNA structural distortions within the region of dyad symmetry. Thus, MerR fosters the binding of RNA polymerase to an inactive promoter, and upon induction, MerR/Hg(II) facilitates DNA distortions suitable for efficient formation of the active transcription complex.

[Ritalin: a stimulating drug in the treatment of children with minimal brain dysfunction (MBD)]. / Ritalin: een stimulans bij de behandeling van kinderen met minimal brain dysfunction (MBD).

Children with Minimal Brain Dysfunction (MBD) are vulnerable in society. Therapy consists of a combination of behaviour modification techniques, function training, psychotherapy, family therapy and counseling of parents and teachers. This way of treatment is not appropriate for all children with MBD. Some of them need additional therapy with a psychotropic drug e.g. methylphenidate (Ritalin). We describe a procedure to establish, whether Ritalin improves the therapeutic impact in a specific child. Eight children enrolled the program. In seven Ritalin improved the therapeutic possibilities for a longer period of time. The dose for an optimal therapeutic effect varied individually, and was relatively low (0.4-0.8 kg/kg/dg). The hypothesis concerning the effect of psychotropic drugs in MBD and the pharmacological properties of Ritalin are discussed. We believe this procedure to be applicable in any multidisciplinary setting, experienced with behavioural and learning problems in childhood.

Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21).

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.