Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16.

The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.

Effects of dexamethasone on thyrotrophin and prolactin plasma levels in rats.

In the present study the influence of dexamethasone treatment of rats on the basal values of thyrotrophin (TSH) and prolactin (PRL) and the response of both hormones to thyrotrophin releasing hormone (TRH) has been investigated. Male rats were given 100 mug of dexamethasone/rat for 8 days at the same time of day. Four hours after the last administration of dexamethasone 200 ng or 100 mug of TRH/rat was injected ip. Blood was collected 10 min later by decapitation. TSH and PRL were estimated by radioimmunoassay (RIA) using the NIAMD kits. The basal and TRH stimulated values of PRL in plasma were significantly lower in dexamethasone treated rats than in controls (P less than 0.01). The basal TSH levels in the treated animals were also lowered (P less than 0.05). After 200 ng TRH/rat the increase in TSH was not as high in both groups than after the administration of 100 mug/rat. There was no significant difference between the response of TSH to TRH in the dexamethasone treated and the control rats. The different effects of dexamethasone on PRL and TSH release after TRH may give a further insight into the different regulating mechanisms of both hormones in rats.

[Calculation of radioimmunochemical determinations by "spline approximation" (author's transl)]. / Auswertung radioimmunochemischer Bestimmungsmethoden durch "Spline-Approximation"

A simplified method, based on the "spline approximation", is reported for the calculation of the standard curves of radioimmunochemical determinations. It is possible to manipulate the mathematical function with a pocket calculator, thus making it available for a large number of users. It was shown that, in contrast to the usual procedures, it is possible to achieve optimal quality control in the preparation of the standard curves and in the interpolation of unknown plasma samples. The recaluculation of interpolated values from their own standard curve revealed an error of 4.9% which would normally be an error of interpolation. The new method was compared with two established methods for 8 different radioimmunochemical determinations. The measured values of the standard curve showed a weighting, and there was a resulting quality control of these values, which, according to their statistical evalution, were more accurate than those of the others models (Ekins et al., Yalow et al., (1968), in: Radioisotopes in Medicine: in vitro studies (Hayes, R. L., Goswitz, F.A. & Murphy, B. E. P., eds) USA EC, Oak Ridge) and Rodbard et al. (1971), in: Competitive protein Binding Assys(Odell, W. D. & Danghedy, W. H., eds.) Lipincott, Philadelphia and Toronto). In contrast with these other models, the described method makes no mathematical or kinetic preconditions with respect to the dose-response relationship. To achieve optimal reaction conditions, experimentally determined reaction data are preferable to model theories.