Specific immobilization of firefly luciferase through a biotin carboxyl carrier protein domain.

Firefly luciferase (Photinus pyralis) was fused with a histidine tag and a biotin carboxyl carrier protein (BCCP) domain at its amino terminus. Highly purified recombinant luciferase was obtained by a one-step purification protocol, utilizing immobilized metal affinity chromatography. The novel BCCP-luciferase had properties, stability, and activity similar to those of native luciferase. The biotin molecule on the BCCP domain allowed specific immobilization of BCCP-luciferase on avidin-coated surfaces via the biotin-avidin interaction.

Opiate withdrawal increases ProTRH gene expression in the ventrolateral column of the midbrain periaqueductal gray.

The midbrain periaqueductal gray matter (PAG) has a critical role in the modulation of behavioral and autonomic manifestations of the opiate withdrawal syndrome. We report a nearly 5-fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive proTRH-derived peptides, but not the mature TRH tripeptide was concomitantly observed in these cells. These findings indicate that proTRH-derived peptides synthesized in neurons of the ventrolateral PAG may function as modifiers of opiate withdrawal responses.

Multiple effects on peripheral hematology following administration of recombinant human interleukin 12 to nonhuman primates.

Interleukin 12 has demonstrated a wide spectrum of bioactivity on T and NK cells both in vitro and in vivo. Therapeutic potential of these activities has been suggested by studies in murine tumor, viral and microbial models of disease. We have investigated the in vivo effect of rhlL-12 in cynomolgus monkeys treated with 1 micrograms/kg/day by bolus i.v. or s.c. injection for 5 days. Treated group transient decreases in total WBC counts as compared to controls, with reversable decreases seen mainly in the lymphocyte and monocyte subsets. Phenotypic analysis showed a decrease in the number of CD4+ and CD8+ cells/microL on Days 2 and 4. Reversible thrombocytopenia and anemia were noted in both treated groups as compared to controls. Plasma neopterin concentrations were increased in both treated groups as compared to controls. These data show that rhlL-12 has multiple effects on peripheral hematology and suggests that this model may be useful to investigate in vivo bioactivity of rhlL-12.

Identification of nodS and nodU, two inducible genes inserted between the Bradyrhizobium japonicum nodYABC and nodIJ genes.

The so-called common nodulation (nod) gene cluster of Bradyrhizobium japonicum is characterized by a unique composition of genes that are arranged in the following order: nodY, nodA, nodB, nodC, nodS, nodU, nodI, nodJ. As reported here, the identification of the two new genes nodS and nodU resulted from the DNA sequencing of a 4.5-kilobase nodC-downstream region covering nodS, nodU, nodI, and nodJ. The predicted NodS, NodU, NodI, and NodJ proteins had the following respective amino acid (aa) lengths and molecular weights (Mr): 209 aa, Mr 23,405; 569 aa, Mr 62,068; 306 aa, Mr 34,127; and 262 aa, Mr 28,194. The 3' end of nodC overlapped the 5' end of nodS by 71 nucleotides. Using translational fusions of lacZ to nodC, nodS, and nodU, the expression of these genes was shown to be inducible by the isoflavone daidzein and depended on transcription from a DNA region farther upstream. These data and the adjacent location of all genes suggested the existence of a nodYABCSUIJ operon. The nodI and nodJ gene products shared about 70% sequence similarity with the corresponding Rhizobium leguminosarum bv. viciae proteins; NodI belongs to the family of ATP-binding proteins that are constituents of bacterial binding protein-dependent transport systems. By interspecies hybridization, DNA regions homologous to nodSU were detected in other strains of Bradyrhizobium. Likewise, nodS- and nodU-like genes were identified in Rhizobium sp. strain NGR234 (A. Lewin, E. Cervantes, W. Chee-Hoong, and W. J. Broughton, Molecular Plant-Microbe Interactions 3:317-326, 1990) in which nodS confers host specificity for Leucaena leucocephala.(ABSTRACT TRUNCATED AT 250 WORDS)