Identification and sequences of the Treponema pallidum flhA, flhF, and orf304 genes.

The recently identified fla operon of Treponema pallidum contains several genes that encode motility-related proteins. We have determined the nucleotide sequences of three genes, designated flhA, flhF, and orf304, that are located immediately downstream of the flhB gene in the fla operon. The flhA gene encodes a 707-amino acid protein that contains five putative membrane spanning domains. FlhA has strong homology with members of a family of proteins that are involved in flagellar biogenesis and regulation/secretion of virulence-related proteins. The flhF gene encodes a 437-amino acid protein that contains three consensus elements that are characteristic of a GTP-binding domain. The orf304 gene encodes a 304-amino acid protein that contains a consensus ATP-binding motif. The order of the flhA, flhF, and orf304 genes is identical to that of corresponding genes in the Bacillus subtilis che/fla operon. Due to the location of the flhA, flhF and orf304 genes in the T. pallidum fla operon, we hypothesize that the FlhA, FlhF, and Orf304 proteins are involved in the biogenesis/assembly of treponemal periplasmic flagella.

Nucleotide sequence of the Treponema pallidum eno gene.

We determined the nucleotide sequence of the enolase (eno) gene of Treponema pallidum, the noncultivable agent of syphilis. The deduced amino acid sequence of T. pallidum enolase (Eno) is 432 amino acids long with a predicted molecular mass of 46.7 kDa. The Eno amino acid sequence has a high degree of homology to the amino acid sequences of prokaryotic and eukaryotic Eno. This is the first eno sequence reported for a bacterium in the order Spirochaetales.

Identification, sequences, and expression of Treponema pallidum chemotaxis genes.

Treponema pallidum, the agent of syphilis, is a pathogenic spirochete that has no known mechanisms of genetic exchange and cannot be continuously cultivated in vitro. A probe based on the nucleotide sequence of the T. pallidum cheA gene was used to screen a T. pallidum genomic DNA library. A treponemal DNA region containing four open reading frames (orfs) was identified. The proteins encoded by these orfs have significant homology with proteins involved in bacterial chemotaxis. The orfs have been designated cheA, cheW, cheX, and cheY. The cheA, cheW, and cheY genes were individually-cloned and expressed in vitro. The observed molecular mass of each protein correlated well with its predicted molecular mass. Reverse transcriptase-PCR data indicate that cheA through cheY are co-transcribed. The organization of these genes suggests that they comprise an operon. We hypothesize that the ability to sense and respond to nutrient gradients is important for the survival and dissemination of T. pallidum in vivo. The presence of a putative che operon strongly suggests that T. pallidum has the potential for a chemotactic response.

Sequences of the Salmonella typhimurium mglA and mglC genes.

The nucleotide sequences of the mglA and mglC genes of Salmonella typhimurium (St) LT2 have been determined. The deduced amino acid (aa) sequences of MglA and MglC are 506 and 302 aa long with predicted molecular masses of 56,484 and 31,551 Da, respectively. The aa sequences of St MglA and MglC are homologous to the corresponding Mgl proteins of Escherichia coli, Haemophilus influenzae, Treponema pallidum and Mycoplasma genitalium. The order of the St mgl operon is mglBAC.

Do genetic factors explain associations between muscle strength, lean mass, and bone density? A twin study.

Are the associations between muscle strength, lean mass, and bone mineral density (BMD) genetically determined? Based on within-pair differences in 56 monozygotic (MZ) and 56 dizygotic (DZ) female twin pairs, mean age 45 yr (range 24-67), BMD was associated with lean mass, independent of fat mass and height (P < 0.05). A 10% increment in femoral neck (FN) BMD was associated with a 15% increment in lean mass (approximately 6 kg). BMD was associated with muscle strength (measured in 35 pairs) before, but not after, adjusting for lean mass. Based on age-adjusted cross-sectional analyses, same-trait correlations (+/- SE) in MZ pairs were double those in DZ pairs: FN BMD (0.62 +/- 0.08, 0.33 +/- 0.12) and lean mass (0.87 +/- 0.03, 0.30 +/- 0.11; all P < 0.001), consistent with a genetic hypothesis. The cross-trait correlation (r) between lean mass and FN BMD in the same individual was 0.43 +/- 0.06. The cross-trait cross-twin correlation between lean mass in one twin and FN BMD in the other was 0.31 +/- 0.07 in MZ pairs, approximately 75% of the cross-trait correlation (r) and 0.19 +/- 0.09 in DZ paris (P < 0.001). After adjusting for height and fat mass, the MZ and DZ cross-trait cross-twin correlations were no different (0.16 +/- 0.08 and 0.13 +/- 0.09, respectively). Therefore, genetic factors account for 60-80% of the individual variances of both FN BMD and lean mass, and > 50% of their covariance. The association between greater muscle mass and greater BMD is likely to be determined by genes regulating size.

Identification and sequences of the Treponema pallidum mglA and mglC genes.

Treponema pallidum, the agent of syphilis, cannot be continuously cultivated in vitro. To identify treponemal genes encoding exported proteins, we performed TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Clone 6D2 was chosen for further study based on partial nucleotide sequence obtained from p6D2 containing a TnphoA insertion. A complete open reading frame (orf1) and a truncated orf (orf2) were identified in the treponemal DNA of p6D2. Orf1 encodes a hydrophobic protein of 531 amino acids with a calculated M(r) of 57,882 Da. The deduced amino acid sequence of Orf1 has homology to the MglC proteins of E. coli, Haemophilus influenzae, and Salmonella typhimurium. T. pallidum Orf1 (MglC) contains a conserved motif that is found in integral cytoplasmic membrane proteins of ATP-binding cassette (ABC) transport systems. T. pallidum orf2 encodes a protein of 496 amino acids with a calculated M(r) of 55,547 Da. The deduced amino acid sequence of Orf2 has homology to the MglA proteins of S. typhimurium, E. coli, H. influenzae, and Mycoplasma genitalium. Orf2 (MglA) contains two consensus ATP-binding motifs. T. pallidum mglA and mglC are located downstream of mglB, consistent with the gene order of previously identified mgl operons. The putative T. pallidum mgl operon encodes the first high-affinity ABC transport system identified in this spirochete.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17 beta-estradiol 4-hydroxylase.

Rates of microsomal 17 beta-estradiol (E2) hydroxylation at the C-2, -4, -6 alpha, and -15 alpha positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 alpha and -15 alpha positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low Km 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E2 catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low Km hydroxylation of E2 at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE2) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE2 formation predominantly over 4-MeOE2. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E2 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.

Body composition and muscle strength in healthy men receiving testosterone enanthate for contraception.

To determine the effect of androgens on body composition and muscle strength, we measured fat-free mass (kg), fat mass (kg), and bone density (g/cm2) by dual x-ray absorptiometry, and muscle strength (Newton meters) by dynamometry in a controlled, prospective study involving 13 nonathletic men receiving testosterone enanthate 200 mg/week in for 6 months and 8 healthy controls. Biochemical markers of bone turnover were measured in the treated subjects at baseline and 6 months. In the treated subjects at 6 months, fat-free mass (mean +/- SEM) increased by 9.6 +/- 1.0% (P < or = 0.01) whereas fat mass decreased by 16.2 +/- 6.7% (P < or = 0.05). Changes in muscle strength ranged from -1.6-19.2%. Only hip adduction increased 19.2 +/- 9.5% (P < 0.05). Changes in bone density ranged from -1.3-5.2%, decreasing significantly at one site and increasing significantly at four of the nine sites measured (P < 0.05). Serum testosterone increased by 91.1 +/- 7.5% (P < 0.01), and testicular volume decreased by 24.0 +/- 3.2% (P < 0.01). Serum osteocalcin increased by 35.7 +/- 17.3% (P < 0.05), serum immunoreactive PTH (iPTH) increased by 41.4 +/- 15.1% (P < 0.05), serum calcium decreased by 2.3 +/- 1.0% (P < 0.05), and serum albumin decreased by 4.5 +/- 1.7% (P < 0.05). There were no detectable changes in fat-free mass, fat mass, muscle strength, or bone density in controls. The administration of testosterone enanthate in pharmacological doses for 6 months resulted in a modest reduction in fat mass and small increases in fat-free mass, muscle strength, and bone density. These changes do not support the use of androgens for enhancing athletic performance.

PIP: In Melbourne, Australia, physicians compared data on 13 healthy, nonathletic, 21-37 year old men receiving an intramuscular injection of 200 mg testosterone enanthate once a week for 6 months with data on 8 age-matched healthy controls to examine the effect of this androgen on body composition and muscle strength and to determine whether it may enhance athletic performance. Dual x-ray absorptiometry measured total body and regional bone density. Dynamometry measured muscle strength. Controls did not experience any detectable changes in fat-free mass, fat mass, muscle strength, or bone density. Between baseline and 6 months of testosterone enanthate treatment, the fat-free mass of cases increased 9.6% (about 6 kg; p .01), while fat mass fell by 16.2% (about 2 kg; p .05). When compared with the literature, however, these changes in body composition were modest. It is not sure whether an increase in lean muscle mass or fluid retention accounted for the increase in fat-free mass. Muscle strength changes varied from -1.6% to 19.2%. Body weight increased by about 5% (around 4 kg). Of the 6 movements, hip adduction was the only significant muscle strength change (increased 19.2%; p .05). Bone density decreased by 1.3% at the skull (p .05), while it increased significantly at the lumbar spine, ribs, pelvis, and femoral neck (p .05). It did not change significantly at the arms, Ward's triangle, trochanter, or legs. The increase in bone density were not enough to reduce the risk of bone fractures. Testosterone enanthate treatment increased serum testosterone levels by 91.1% (p .01), serum osteocalcin by 35.7% (p .05), and the serum immunoreactive parathyrin by 41.4% (p .05). It decreased testicular volume by 24% (p .01), serum calcium levels by 2.3% (p .05), and serum albumin levels by 4.5% (p .05). Based on these findings, androgens, at least in the administered dose range, should not be used to enhance athletic performance.