Immune reconstitution inflammatory syndrome in a HIV-infected patient with disseminated tuberculosis.

Immune reconstitution inflammatory syndrome (IRIS) is the paradoxical worsening of pre-existing infectious processes after commencement of anti-retroviral therapy (ART) in HIV-infected patients. Its manifestations are dependent on the underlying opportunistic infections. We report a case of an HIV-infected patient with disseminated tuberculosis, who responded to anti-tuberculosis therapy but suffered from paradoxical worsening after commencement of ART.

Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C.

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-(14)C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2, 6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic m/z of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of approximately 0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O(2), but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP(+). Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The m/z 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the m/z 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.

Affinity selection of DNA-binding proteins displayed on bacteriophage lambda.

Two transcription factors, human ATF1, its DNA-binding domain (ATF1BD), and the DNA-binding domain (GAL4BD) of the yeast GAL4 protein, were displayed on the surface of bacteriophage lambda vectors and efficiently selected by DNA fragments immobilized in microtiter wells. The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, lambdafoo and lambdafooDc, respectively. After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4,000-fold over the vector phages. Further, the GAL4BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on lambdafooDc and lambdafoo, respectively. The ATF1BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on lambdafoo. These results suggest that the lambdafoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.

A symbiont-produced protein and bacterial symbiosis in Amoeba proteus.

Gram symbiotic X-bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the host's nucleus as detected by confocal and immunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N-terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoeba's nucleus, S29x is suspected to be involved in affecting the expression of amoeba's nuclear gene(s).

The s29x gene of symbiotic bacteria in Amoeba proteus with a novel promoter.

Gram-symbiotic bacteria (called X-bacteria), present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein, S29x. S29x is exported into the host's cytoplasm across the bacterial membranes and the symbiosome membrane. The complete nucleotide (nt) sequence of the s29x gene of X-bacteria has been determined, and the promoter sequence and tsp have also been identified. The gene has a nonconventional promoter with putative nt sequences different from the known consensus sequences. When Escherichia coli cells are transformed with s29x, the gene is expressed and the product is secreted into the culture medium. Functions of S29x are not fully known, but it is suspected that S29x plays an important role in the symbiotic relationship between amoebae and X-bacteria.