Bone Disease in the Common Marmoset: Radiographic and Histological Findings.

The common marmoset (Callithrix jacchus) is a New World primate that is used in biomedical research due to its small size and relative ease of handling compared with larger primates. Although bone disease in common marmosets is well recognized, there are very few detailed descriptions in the literature that cover the range of lesions seen in these animals. For all animals used to model human disease, it is important to be aware of background lesions that may affect the interpretation of study findings. This retrospective study details bone diseases encountered in marmoset breeding colonies at 2 different institutions. Affected marmosets at Johns Hopkins University had lesions compatible with diagnoses of rickets, fibrous osteodystrophy and osteopenia. Affected marmosets at the Wisconsin National Primate Research Center exhibited severe lesions of osteoclastic bone resorption and remodeling that had an unusual distribution and were not easily categorized into a known disease entity. The purpose of this report is to document these naturally occurring skeletal lesions of common marmosets and suggest an approach to evaluating skeletal disease in prospective studies of these animals that will allow the most accurate diagnoses.

Identification and characterization of the non-PTS fru locus of Bacillus megaterium ATCC 14581.

A genetic locus that is adjacent to the gene encoding the small acid-soluble protein SASP C-4 of Bacillus megaterium has been identified. This locus, designated fru, contains a beta-fructosidase gene (fruA), a gene encoding a hydrophobic protein that is closely related to non-PTS sugar permeases of the proton symport type (fruP), and a gene coding for a transcriptional regulator of the LacI/GalR family (fruR). The FruA protein can hydrolyze sucrose and raffinose, but not maltose, isomaltose, trehalose, melibiose or lactose. The transcription initiation site of fruP has been mapped and the fruP promoter identified. Gel mobility shift assays showed that the FruR protein can bind specifically to a DNA fragment containing the fruP promoter region. DNase I footprinting analysis has defined the FruR binding site. Disruption of fruR led to high-level constitutive expression of fruPA, but had no effect on expression from the fruR promoter itself, indicating that FruR acts as a repressor of fruPA expression, but does not autoregulate its own synthesis. Interestingly, expression of fruPA in B. megaterium was not induced by sucrose, raffinose, fructose or inulin, whereas the constitutive expression of fruPA in a fruR mutant was repressed by both glucose and sucrose. Possible physiological implications of these findings are discussed.

Transcriptional regulation of the Bacillus subtilis bscR-CYP102A3 operon by the BscR repressor and differential induction of cytochrome CYP102A3 expression by oleic acid and palmitate.

The adjacent yrhI and yrhJ genes were identified by the Bacillus subtilis genome sequencing project. We now report that yrhJ (renamed CYP102A3) encodes a cytochrome P450 and that yrhI (renamed bscR) encodes a repressor that negatively regulates the transcription of the bscR-CYP102A3 operon. The transcriptional initiation site of bscR has been mapped by primer extension analysis. An 18-bp perfect palindromic sequence centered 65.5 bp downstream from the transcriptional initiation site of bscR has been identified as the binding site for BscR by gel mobility shift assays. Base substitutions in the 18-bp inverted repeat resulted in derepression of the bscR-xylE transcriptional fusion in vivo. bscR-xylE fusion studies and Northern blot analysis revealed that oleic acid and palmitate could induce the expression of the bscR-CYP102A3 operon to a considerable extent. However, only oleic acid was capable of preventing the binding of BscR to its operator DNA in vitro, suggesting that the induction of CYP102A3 expression by oleic acid and palmitate in B. subtilis might be mediated through different mechanisms.

Negative regulation of expression of the Bacillus megaterium bmlP1 gene by the bmlP1 3' flanking region.

We report that the expression of the Bacillus megaterium bmlP1 gene is subject to negative regulation by the bmlP1 3' flanking region. This repression occurred both in B. megaterium and in Escherichia coli. When the bmlP1 promoter was replaced with a heterologous promoter or when the orientation of the bmlP1 3' flanking region was reversed, the inhibitory effect was still observed. However, the bmlP1 3' flanking region was unable to exert repression on a heterologous gene when fused downstream in either orientation, and it was incapable of acting in trans. Dot blot and Northern blot analyses revealed that the repression occurred at the RNA level. Deletion analysis showed that the regulatory site responsible for the repression is located within a 116-bp region immediately following the bmlP1 gene. Possible mechanisms for this repression are discussed.

The basal-level expression of the cytochrome P450(BM-1) gene is negatively affected by the bm1P1 gene of Bacillus megaterium.

Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450(BM-1) in Bacillus megaterium has been provided in our previous report. In the present study, we show that the basal-level expression of the P450(BM-1) promoter-xylE transcriptional fusion is significantly reduced when the bm1P1 gene is present on the same plasmid. Moreover, disruption of the chromosomal bm1P1 gene results in enhanced expression of the P450(BM-1) promoter-xylE fusion on a multicopy plasmid. By using a binary vector system, we found that expression of the P450(BM-1) promoter-xylE fusion from a high-copy plasmid is substantially repressed by expression of bm1P1 from a low-copy plasmid. Taken together, these results suggest that the basal-level expression of P450(BM-1) is negatively affected by bm1P1. Possible mechanisms for this control are discussed.

Cloning, expression, and catabolite repression of a gene encoding beta-galactosidase of Bacillus megaterium ATCC 14581.

A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).

Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450BM-1 in bacillus megaterium.

The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbiturate-mediated induction of cytochrome P450BM-1 in Bacillus megaterium. We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously. In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450BM-1 gene. Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450BM-1 expression. A multicopy plasmid harboring only the P450BM-1 promoter region could increase expression of the chromosome-encoded P450BM-1. The level of expression is comparable with that shown by a multicopy plasmid harboring the P450BM-1 promoter region along with the bm1P1 gene. These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450BM-1 expression. Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450BM-1 promoter. This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450BM-1.

Iron and salicylate induction of cytochrome P450BM-1 in Bacillus megaterium.

The effects of iron and salicylate on the expression of cytochrome P450s in Bacillus megaterium were investigated in this report. Immunoblot analysis showed that the addition of 4 mM ferric iron or 10 mM salicylate to the culture medium resulted in a significant increase in the P450BM-1 level, while the same condition had little effect on P450BM-3 expression. Substantial induction of chloramphenicol acetyltransferase (CAT) activity by iron and salicylate in B. megaterium cells bearing a P450BM-1 promoter-cat transcriptional fusion vector suggests that the induction of P450BM-1 by iron and salicylate occurs at the transcriptional level. Unexpectedly, in contrast to the bm1P1-dependent induction of P450BM-1 by pentobarbital, disruption of bm1P1 gene did not affect induction of P450BM-1 by iron and salicylate. This result suggests that the induction of P450BM-1 by iron and salicylate occurs by a bm1P1-independent mechanism. To our knowledge, this is the first example of an iron-regulated cytochrome P450 gene in prokaryotes.

Changes in plasma levels of lipids and lipoprotein composition in patients with Kawasaki disease.

Studies on the response of plasma lipids and lipoproteins to Kawasaki disease are scarce so far. The purpose of this study was to investigate the changes in plasma levels of lipids and apolipoproteins as well as the composition of different lipoproteins in patients during the acute and convalescence phases of Kawasaki disease. The results showed that during the acute phase, the concentrations of plasma high density lipoprotein (HDL)-cholesterol, apolipoprotein A-I (apoA-I) and A-II (apoA-II) were significantly reduced. While the reduction of HDL-cholesterol was mainly related to the lowering of esterified and unesterified cholesterols in HDL2 during the acute stage of Kawasaki disease, most of which recovered during the subsequent convalescence phase. The plasma concentration of triglycerides was 46% higher in patients during the acute phase of Kawasaki disease than in the control subjects, which may be ascribed to the increase of triglycerides in very low density lipoprotein (VLDL), low density lipoprotein (LDL) and HDL2. The variables studied above did not appear to be independent parameters. The level of plasma apoA-I showed a stronger negative association with triglyceride concentration (r = -0.22) than apoA-II (r = -0.11) and HDL-cholesterol (r = -0.07). Furthermore, the levels of cholesterol, apoA-I and apoA-II in HDL2, but not in HDL3, were inversely correlated with the levels of triglyceride. We conclude that the temporary changes of lipid levels associated with Kawasaki disease results predominantly from alterations of lipoprotein composition.

A 53-base-pair inverted repeat negatively regulates expression of the adjacent and divergently oriented cytochrome P450(BM-1) gene and its regulatory gene, bm1P1, in Bacillus megaterium.

To study the role of the cis-acting element(s) in controlling the expression of the cytochrome P450(BM-1) gene and its upstream regulatory gene, bm1P1, in Bacillus megaterium, various deletion derivatives were constructed. A 53-bp inverted repeat located midway between the P450(BM-1) gene and bm1P1 gene was found in vivo to negatively regulate the expression of both genes, the regulation of which may occur at the transcriptional level. The promoter of the P450(BM-1), gene was also identified and found to be similar to those recognized by the sigmaA RNA polymerase of Bacillus subtilis. Possible mechanisms by which the 53-bp inverted repeat regulates the gene expression are discussed.

Repetitive elements in the third intron of murine apolipoprotein A-I gene.

Genomic DNAs containing the gene encoding apolipoprotein A-I (apoA-I) from four strains of mice and three strains of rats have been sequenced. Some peculiar repetitive sequences were found in the third intron of apoA-I of the murine species. The striking features include regular tandem repeats of C(A)4 and C(A)6 in mice and long A-tracts in rats. Not completely identical but very similar motifs were found in mice or rats belonging to the same species while repetitive elements from different species show some variation from their species-specific consensus sequences. These repetitive motifs are very similar to the sequences flanking human Alu and rodent B1 repetitive elements, although no evidence for the existence of Alu or B1 was found near the peculiar repetitive DNA sequences in apoA-I gene.

Purification and characterization of 6xHis-tagged Bm3R1 repressor of Bacillus megaterium.

The bm3R1 gene-encoded repressor controls the expression of cytochrome P450BM-3 gene as well as its own expression in Bacillus megaterium. We have developed an efficient system for overexpression and purification of Bm3R1 protein by nickel ion affinity chromatography. Adding six histidine residues at either N-terminus or C-terminus of Bm3R1 repressor caused no loss of its operator DNA binding ability. We have investigated the interaction between Bm3R1(His)6 and its operator DNA by equilibrium and kinetic methods. The results showed that the apparent dissociation constant is around 1.8 x 10(-9) M and the half-life of Bm3R1(His)6-operator complex is about 50 minutes. We have also found by gel filtration chromatography that Bm3R1(His)6 may exist in low oligomeric state(s) in solution, which is competent for binding to its operator DNA.

Inhibition by barbiturates of the binding of Bm3R1 repressor to its operator site on the barbiturate-inducible cytochrome P450BM-3 gene of Bacillus megaterium.

In our previous publication (Shaw, G.-C., and Fulco, A. J. (1992) J. Biol. Chem. 267, 5515-5526), we reported that Bm3R1, a protein encoded in an open reading frame just upstream from the cytochrome P450BM-3 gene, is a repressor critically involved in the barbiturate-inducible expression of this gene in Bacillus megaterium. We now describe the purification of the Bm3R1 protein from an overproducing Escherichia coli strain harboring a bm3R1 gene-carrying plasmid and report the effect of barbiturate inducers on the interaction of Bm3R1 with a fragment of B. megaterium DNA containing the bicistronic operator and promoter sequences. Gel filtration analysis revealed that, under our experimental conditions, most of the Bm3R1 protein exists in highly aggregated forms. Gel mobility shift assays showed that Bm3R1 protein bound specifically to a segment of DNA containing the promoter-operator region of the bm3R1 gene while DNase I footprinting experiments established that Bm3R1 protected a region of DNA that covers and flanks the palindromic operator sequence. The interaction between Bm3R1 repressor and its operator, in vitro, was strongly inhibited by the addition of 2 mM pentobarbital or 2 mM methohexital (strong in vivo inducers of P450BM-3) but not by the same concentration of phenobarbital (a relatively weak inducer) or by mephobarbital (a non-inducer). A detailed comparison of pentobarbital and methohexital at concentrations lower than 2 mM indicated that methohexital was 5-10 times more effective as an inhibitor of Bm3R1 binding in vitro, as compared with its 7-fold greater inducer potency in vivo. The observation that the in vitro inhibition effects of barbiturates on the interaction of Bm3R1 repressor and its operator correlate strongly with their in vivo potency as inducers of cytochrome P450BM-3 suggests a mechanism for the induction process. It seems plausible that the barbiturate inducers might bear a conformational resemblance to and mimic the mode of action of an as yet unidentified endogenous inducer(s) in B. megaterium that functions by releasing the binding of Bm3R1 repressor from its operator site.

Barbiturate-mediated regulation of expression of the cytochrome P450BM-3 gene of Bacillus megaterium by Bm3R1 protein.

In a previous publication (Wen, L.-P., Ruettinger, R. T., and Fulco, A.J. (1989) J. Biol. Chem. 264, 10996-11003), we reported that about 1 kilobase of 5' flanking sequence was required for barbiturate-inducible expression of the cytochrome P450BM-3 gene in Bacillus megaterium. We have now found, by analysis of various deletion and frameshift derivatives of this region, that an open reading frame immediately upstream of the B. megaterium cytochrome P450BM-3 structural gene encodes a protein, designated Bm3R1, which negatively controls the expression of the P450BM-3 gene at the transcriptional level. A helix-turn-helix DNA binding motif was found near the N-terminal portion of Bm3R1 protein. The 5' terminus of the bm3R1 transcript generated in vivo was determined by nuclease S1 mapping and primer extension analysis to be 44 base pairs upstream of the translation initiation sequence GTG of bm3R1. A putative promoter sequence with a high degree of similarity to the -35 and -10 consensus sequence recognized by the Bacillus subtilis sigma-43 factor was located at an appropriate distance from the transcription start site. A B. megaterium mutant which highly constitutively produced P450BM-3 protein was isolated and complementation of this cytochrome P450BM-3-constitutive mutant by a DNA fragment containing the wild-type bm3R1 gene indicated that the mutation in this locus was trans-dominant. Sequence analysis of the bm3R1 gene and its upstream region from this mutant, after amplification by the polymerase chain reaction, identified a single base change that resulted in a glycine to glutamate substitution in the beta-turn region of the DNA binding motif. By placing the bm3R1 gene under the control of a tac promoter and changing the translation initiation sequence from GTG to ATG, we succeeded in overproducing the Bm3R1 protein in Escherichia coli. A 20-bp perfect palindromic putative operator site, located between the presumed promoter sequences and the bm3R1 structural gene, was defined both by in vivo titration of Bm3R1 repressor and by gel mobility shift assays using the cell-free extracts containing the overproduced wild-type or mutant Bm3R1 protein. The barbiturate effect in mediating the induction of cytochrome P450BM-3 appears to be indirect but probably involves, in part, the release of inhibition by Bm3R1 repressor protein by interfering with its binding to the palindromic putative operator sequence and perhaps to other sites on the regulatory region of the gene encoding cytochrome P450BM-3.

Studies on outbreak of viral hepatitis at Calcutta with special reference to serological investigations.

Sixty cases of acute viral hepatitis were studied from clinical, biochemical and in particular serological point of view. Majority of the patients had significant pre-icteric and icteric phase with moderate elevations of bilirubin, SGOT and SGPT and marginal elevations of serum alkaline phosphatase. Cholestatic features were observed only in 6.7% of cases. All subjects improved and there was no death in this series. Serological marker studies revealed hepatitis A in 8 (13.3%) cases and hepatitis B in 3 (5.0%) cases. Rest 49 cases were possibly due to non-A, non-B hepatitis. As there was no evidence of parenteral transmission, it was concluded that this epidemic was water borne from contaminated municipal water supply.