High-Performance Liquid Chromatographic (HPLC) Assay Using Fluorescence Detection for the Simultaneous Determination of Gallopamil and Norgallopamil in Human Plasma

Gallopamil is a calcium-channel antagonist with reported activity in experimental animals three to five times higher than that of verapamil. An automated high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the simultaneous determination of gallopamil and its metabolite norgallopamil in plasma. Gallopamil was well resolved from norgallopamil and other metabolites, allowing simultaneous quantitation of both drugs. The detection limit for both gallopamil and norgallopamil was 0.9 ng/ml. This method has been successfully used for the determination of gallopamil and norgallopamil following the administration of 25-, 37.5-, and 50-mg oral doses of drug.     

A study of galactose intolerance in human and rat liver in vivo by 31P magnetic resonance spectroscopy.

An oral load of 20 mg/kg galactose produces significant changes in the 31P magnetic resonance spectrum of the liver of a galactosemic patient. The peak at 5.2 ppm (which includes inorganic phosphate and galactose-1-phosphate) increased on two occasions to about twice its original size 60 min after galactose administration. An oral load of 10 mg/kg galactose given to a second patient produced no discernible changes at 30 min. We have also used an animal model of galactose intolerance, in which galactose metabolism in rats was blocked by the acute administration of ethanol. Studies in vivo and in vitro showed that the increase in the peak at 5.2 ppm was largely due to galactose-1-phosphate. We have shown in this preliminary study that small amounts of galactose can produce significant elevation of hepatic galactose-1-phosphate, which can be detected by 31P magnetic resonance spectroscopy.     

Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor

Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.     

Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines.

Brucella abortus may be useful as a component of vaccines. This is because it possesses several unique properties as a carrier that enable it to stimulate human B cells even in the relative absence of T cells. Human immunodeficiency virus type 1 proteins conjugated to B. abortus could induce neutralizing antibodies against human immunodeficiency virus type 1. Recently we showed that the characteristics of lipopolysaccharide (LPS) derived from B. abortus are similar to those of the whole bacterium in that the LPS acts as a T-independent type 1 carrier in mice. In this study we wanted to determine whether LPS derived from B. abortus is associated with the adverse effects seen with other bacterial endotoxins. LPS purified from B. abortus by butanol extraction was shown to have less than 2% (wt/wt) contamination by protein and less than 1% (wt/wt) contamination by nucleic acids and to contain 1% (wt/wt) ketodeoxyoctanic acid. Compared with LPS derived from Escherichia coli, B. abortus LPS was 10,000-fold less potent in eliciting fever in rabbits, 268-fold less potent in killing D-galactosamine-sensitized mice, and 1,400-fold and 400-fold less potent in inducing interleukin-1 beta and tumor necrosis factor alpha production, respectively. These results suggest that B. abortus LPS is much less likely than the LPS from E. coli to evoke endotoxic shock; therefore, it may be feasible to incorporate B. abortus as a component of vaccines.     

Characterization of Brucella abortus Soluble Antigen Employed in Immunoassay

A soluble antigen extract of Brucella abortus (BASA) has been prepared by the National Veterinary Services Laboratories and furnished to a number of workers who are examining antibody-mediated and cell-mediated immune responses of cattle infected with B. abortus. Three lots of BASA were examined. There were quantitative but not qualitative differences among lots by content of protein, total carbohydrate, hexose, fatty acid, and 2-keto-3-deoxyoctonic acid. The presence of smooth lipopolysaccharide was demonstrated by the presence of 2-keto-3-deoxyoctonic acid and lipid, by Limulus lysate gelation activity, and by formation of characteristic lipopolysaccharide precipitates in immunoelectrophoresis. A polysaccharide antigen as well as two nonsurface antigens, A2 and C, were also identified. BASA is a satisfactory antigen for use in the enzyme-linked immunosorbent assay since the smooth lipopolysaccharide component bound to polystyrene and functioned in the test. Normal murine spleen cells showed a mitogenic response to BASA similar to that produced by purified smooth lipopolysaccharide. BASA has been used in other laboratories to stimulate peripheral blood leukocytes from cattle infected with B. abortus. Because BASA is a mixture of antigenic components shown to have mitogenic effects in the mouse system, questions on the nature of its stimulatory effect on bovine cells are raised.