Temperature effects in hydrophobic interaction chromatography.

The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.

Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease.

Poly(ADP-ribosyl)ation is catalyzed by NAD+: protein(ADP-ribosyl) transferase (ADPRT), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD+ to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following gamma-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as approximately 30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.

On the biological role of the nuclear polymerizing NAD+: protein(ADP-ribosyl) transferase (ADPRT): ADPRT from Dictyostelium discoideum and inactivation of the ADPRT gene in the mouse.

Two approaches have been used to elucidate the role of the nuclear polymerizing NAD+:protein(ADP-ribosyl)-transferase (ADPRT): i) comparison of the primary structure of Dictyostelium discoideum ADPRT derived from a 2 kb, partial cDNA sequence with the mammalian, fish, amphibian and insect counterparts revealed an overall homology of 25%. Whereas the automodification domain was not conserved at all, the NAD+ binding domain (aa 859-908) showed more than 70% identical amino acids in all species. Together with the similar enzymatic properties of the ADPRTs the genetic conservation underlined the notion that ADPRT plays a major role in various cellular processes; and ii) inactivation of the ADPRT gene in murine embryonic stem cells by homologous recombination led to mouse strains with a complete lack of nuclear poly(ADP-ribosyl)ation. These ADPRT mutant mice were viable and fertile indicating that ADPRT is dispensable in mouse development. Moreover, repair of UV and MNNG induced DNA damage was not affected in ADPRT/3T3 like fibroblasts, as measured by reactivation of in vitro damaged reporter plasmids and unscheduled DNA synthesis. However, about 30% of the ADPRT mutant mice developed pathological skin aberrations on a mixed 129/Sv x C57B1/6 genetic background. These mice will be extremely useful to define the precise biological role of poly(ADP-ribosyl)ation.

Thin-layer electrophoresis of hydroxyethyl starches on a modified silica gel support.

The recent developed thin-layer electrophoresis on modified silanized silica gel was applied to the separation of hydroxyethyl starches (HES) and glycogen and of HES with different degrees of substitution. This method permits a rapid qualitative and semi-quantitative determination of HES in animal tissues such as liver, lung, heart and kidney after their disintegration with alkali and precipitation with ethanol.