Use of Radiation Hybrid panels to map genetic loci

The mapping of genetic loci within organisms has been accelerated by the advent of Radiation Hybrid (RH) panels. These panels are available for humans and non-humans including mice, baboon, rat, and canine. This article contains a general protocol for the use of the Genebridge 4 whole genome RH panel to map a human locus. This protocol may also be adjusted to suit the other RH panels currently available.     

In vitro inhibition of vitamin K dependent carboxylation by tetrachloropyridinol and the imidazopyridines

The compounds 2,3,5,6-tetrachloro-4-pyridinol (TCP) and the structurally related imidazopyridines (IP) cause hemorrhage and lower the plasma prothrombin level in animals. In vitro, TCP and the IP are more potent inhibitors of both the vitamin K dependent carboxylase which catalyzes the posttranslational gamma-carboxylation of specific glutamyl residues in proteins and the related vitamin K epoxidase activity than they are either of vitamin K epoxide reductase or of NAD-(P)H-K oxidoreductase. TCP and IP, as is the case with the coumarin and indandione anticoagulants, are competitive inhibitors of NAD(P)H-K oxidoreductae with respect to NADH. The epoxide reductase from coumarin-resistant rats is quite resistant to inhibition not only by warfarin but also by the IP, and to a lesser extent by TCP. When interpreted in light of published in vivo experiments, the data suggest that the principal site of anticoagulant action of the IP, but not TCP, is the epoxide reductase. The anticoagulant effect of TCP may be inhibition of the carboxylase itself. TCP is a significantly more potent inhibitor of the carboxylase and epoxidase than the IP; it inhibits both the enzymatic activities to the same degree with 50% inhibition observed at about 10(-5) M. Inhibition of the carboxylase by TCP is not competitive with respect to the pentapeptide substrate phenylalanyl-leucylglutamylglutamylleucine nor with respect to the following components of the in vitro carboxylase assay: imidazole, pyridoxal 5'-phosphate, dithiothreitol, KCl, sodium bicarbonate, oxygen, and vitamin K. The order of addition of components of the assay relative to the addition of inhibitor did not affect the degree of inhibition. Inhibition is readily reversed in experiments designed to dissociate an enzyme-inhibitor complex. Analysis of double-inhibitor experiments suggests that TCP and IP have the same binding site on the carboxylase.     

Type I Antifreeze Proteins Enhance Ice Nucleation above Certain Concentrations

In this study, we examined the effects that antifreeze proteins have on the supercooling and ice-nucleating abilities of aqueous solutions. Very little information on such nucleation currently exists. Using an automated lag time apparatus and a new analysis, we show several dilution series of Type I antifreeze proteins. Our results indicate that, above a concentration of ∼8 mg/ml, ice nucleation is enhanced rather than hindered. We discuss this unexpected result and present a new hypothesis outlining three components of polar fish blood that we believe affect its solution properties in certain situations.     

Antagonism of kainic acid lesions in the mouse hippocampus by U-54494A and U-50488H.

A morphometric study of kainic acid- (KA) induced lesions was designed for the study of the interaction of the diamines U-5449A and U-50488H with excitatory amino acids, and the dose-response relationship thereof. IC50S determined for binding at the kappa receptor and other opioid receptors demonstrated the lack of kappa activity of U-54494A, a structurally related analog of U-50488H. Both opiate kappa receptor related anticonvulsant diamines were tested for their ability to protect the mouse hippocampus from the cytopathological changes induced by KA in neurons and glia. The damage observed with i.c.v. KA in mouse was restricted to neurons of the CA3 pyramidal region and glia of the hippocampus. It involved massive cell loss and shrunken neurons with dark cytoplasm and nuclei. Groups treated with combinations of KA and U-54494A or U-50488H showed scarce damage, but patches of necrotic changes were still observed. Control animals treated with saline (i.c.v.) and U-54494A (s.c.) or U-50488H (s.c.) did not suffer any noticeable alterations of the polymorphic layers of the hippocampal formation. Image analysis of the CA3 area of the hippocampus was used to quantitate the vacuolization induced by KA lesions in the control and treated groups. By this method, both U-54494A and U-50488H were shown to protect this area in a dose-related fashion as evidenced by reduced vacuolization. The anticonvulsant properties of these compounds may result in the antagonism of the excitotoxic lesions. More specifically, the ability of these diamines to block depolarization-induced influxes of Ca++ may protect the CA3 cells from the cytotoxic effects of persistent depolarization.     

Biosynthesis of man-β-G1cNAc-G1cNAc-pyrophosphoryl-polyprenol by a solubilized enzyme from aorta

The particulate enzyme fraction from pig aorta was treated with Triton X-100 or Nonidet P-40 to yield a soluble enzyme preparation. This solubilized enzyme catalyzed the transfer of mannose from GDP-[¹⁴C]mannose, but not from [¹⁴C]mannosyl-phosphoryl-polyprenol, to G1cNAc-G1cNAc-pyrophosphoryl-polyprenol to form the trisaccharide-lipid, Man-β-GlcNAc-GlcNAc-pyrophosphoryl-polyprenol. The trisaccharide-lipid formed in these reactions was isolated by solvent fractionation and was subjected to mild acid hydrolysis to release the [¹⁴C]trisaccharide. Essentially all of the radioactivity was released from this trisaccharide as mannose upon treatment with β-mannosidase while α-mannosidase had no effect.