ObjectiveTo assess the utility of erythrocyte methotrexate-polyglutamate (MTX-PG) concentrations in determining the safety and efficacy of MTX in patients with rheumatoid arthritis (RA).Methods79 MTX-naïve patients with RA were enrolled in this prospective 76-week cohort study. MTX was initiated, and a predefined dose-escalation protocol was followed. Erythrocyte MTX-PG concentrations were measured using liquid chromatography. The associations of MTX-PG concentrations with disease activity and adverse events were analysed.ResultsDose escalation of MTX resulted in increased MTX-PG concentrations and a decrease in the mean Disease Activity Score in 28 joints (DAS28). A significant association was observed between total MTX-PG concentrations and ΔDAS28 at week 12 (β=−0.013, p=0.003) and at week 24 (β=−0.014, p=0.003). The maximum MTX-PG levels were significantly higher in patients presenting with elevated transaminases (≥100 IU/L) than in those without (146 vs 106 nmol/L, p=0.009). Receiver operating characteristic curve analysis revealed that a total MTX-PG concentrations of 83 nmol/L at week 12 was the threshold for a DAS28 improvement of ≥1.2 at week 24, and 105 nmol/L was the threshold for transaminases of ≥50 IU/L and 131 nmol/L for transaminases of ≥100 IU/L. MTX-PG concentrations were strongly influenced by body mass index and a serum albumin level.ConclusionsMTX-PG concentrations are a useful biomarker in MTX therapy, in terms of efficacy and safety.

Glucocorticoid induction of the tyrosine aminotransferase gene deviates from that of many glucocorticoidresponsive genes by having a lower EC 50 and displaying more agonist activity with a given antiglucocorticoid. A cis-acting element, located 3646 base pairs upstream of the start of tyrosine aminotransferase gene transcription, has been found to be sufficient to reproduce these variations with heterologous genes and promoters (Oshima, H., and Simons, S. S., Jr. (1992) Mol. Endocrinol. 6, 416 -428). This element has been called a glucocorticoid modulatory element, or GME. Others have called this sequence a cyclic AMP-responsive element (CRE) due to the binding of the cyclic AMP response element binding protein (CREB). We now report the partial purification and characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa that bind to the GME/ CRE as a heteromeric complex. This purification was followed by the formation of a previously characterized, biologically relevant band in gel shift assays. By several biochemical criteria, the GMEBs differed from many of the previously described CREB/CREM/ATF family members. Partial peptide sequencing revealed that the sequences of these two proteins have not yet been described. Size exclusion chromatography and molecular weight measurements of the gel-shifted band demonstrated that the GMEBs bound to the GME as a macromolecular complex of about 550 kDa that could be dissociated by deoxycholate. Similar experiments showed that CREB bound to the GME as heteromeric complexes of about 310 and 360 kDa. As determined from gel shift assays, GMEB1 and -2 are not restricted to rat liver cells but appear to be ubiquitous. Thus, these novel GMEBs may participate in a similar modulation of other glucocorticoid-inducible genes in a variety of cells.

Variations in the biological activity of antisteroids, as determined by their percent agonist activity, is a well known but poorly understood phenomenon. For example, in tyrosine aminotransferase (TAT) induction by the antiglucocorticoid dexamethasone 21-mesylate in rat hepatoma tissue culture cells, the percent agonist activity varies with the density of cultured cells. A 21-basepair sequence of the rat TAT gene has now been isolated which confers all of the induction properties of the endogenous TAT gene to homologous and heterologous promoters and genes. We call this 21-basepair sequence, which acts in concert with a trans-acting factor identified by gel shift experiments, a glucocorticoid modulatory element. The changes in induction properties were found to be independent of the fold induction by dexamethasone, thus arguing that the GME does not synergize with the glucocorticoid response element. A model incorporating this new element is advanced which can explain the observed variations of TAT induction and may be generally applicable for the mechanism of action of other steroid hormones.

Background: Recent studies have suggested that cardiac troponin T (cTnT) and troponin I may detect ongoing myocardial damage involved in the progression of chronic heart failure (CHF). This study was prospectively designed to examine whether the combination of cTnT, a marker for ongoing myocardial damage, and B-type natriuretic peptide (BNP), a marker for left ventricular overload, would effectively stratify patients with CHF after initiation of treatment.

The 21-base pair glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is the only cis-acting element known to modulate the transcriptional activity of receptors bound to glucocorticoid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoids, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear protein(s) that has been demonstrated to bind to the GME is of major interest as a possible transcription factor with hitherto undescribed properties. Subsequent studies indicated that not one but two proteins of 88 and 67 kDa ‫؍(‬ GMEB-1 and -2, respectively) formed a heteromeric complex with double-stranded GME oligonucleotides in gel shift assays and participated in the expression of GME activity (Oshima, H., Szapary, D., and Simons, S. S., Jr. (1995) J. Biol. Chem. 270, 21893-21910). Here, we report the use of polymerase chain reaction of degenerate oligonucleotides and 5-and 3-rapid amplification of cDNA ends to clone two cDNAs of 2.0 and 1.9 kilobase pairs that probably result from alternative splicing. Both cDNAs encoded open reading frames containing all four previously sequenced peptides. The longer 2.0-kilobase pair cDNA encoded an open reading frame for an acidic, 529-amino acid protein and afforded a major 67-kDa and a minor 58-kDa protein after in vitro transcription/translation. Both proteins were recognized by a mono-epitopic antibody raised against a peptide of GMEB-2. The in vitro translated protein bound to GME DNA in gel shift assays. However, the binding to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived from HTC cell cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derived from diverse organisms as follows: Drosophila (DEAF-I), rat (Suppressin), and Caenorhabditis elegans (three unknown open reading frames). Collectively, these data suggest that the 67-kDa GMEB-2 not only is an important factor for the modulation of glucocorticoid receptor bound to glucocorticoid response elements but also may belong to a novel family of transcription factors.

Background: Heart-type fatty acid-binding protein (H-FABP) is proposed as an early biomarker for acute myocardial infarction (AMI), but its prognostic value is unclear in acute coronary syndrome (ACS). We evaluated the prognostic value of the H-FABP concentration relative to cardiac troponin T (cTnT) in the early hours of ACS. Methods: Serum concentrations of H-FABP and cTnT were measured on admission in 328 consecutive patients hospitalized for ACS within 6 h after the onset of chest pain [AMI, 241 (73.5%) patients; ST-segment elevation myocardial infarction, 154 (47.0%) patients; and emergent coronary angiography within 24 h after admission, 287 (87.5%) patients]. Cardiac events, which were defined as cardiac death or subsequent nonfatal AMI, were monitored for 6 months after admission. Results: During the 6-month follow-up period, there were 25 cardiac events, including 15 cardiac deaths and 10 subsequent nonfatal AMIs. Stepwise multivariate analyses including clinical, electrocardiographic, and biochemical variables revealed that increased H-FABP (above the median of 9.8 g/L), but not increased cTnT (above the median of 0.02 g/L), was independently associated with

No abstract

A total of 12 VanA-type vancomycin-resistant enterococci, consisting of 10 Enterococcus faecium isolates and two Enterococcus avium isolates, were examined in detail. The vancomycin resistance conjugative plasmids pHT␣ (65.9 kbp), pHT␤ (63.7 kbp), and pHT␥ (66.5 kbp) were isolated from each of three different E. faecium strains. The plasmids transferred highly efficiently between enterococcus strains during broth mating and were homologous with pMG1 (Gm r ; 65.1 kb).Gene transfer systems are an essential requirement for the spread of drug resistance in microorganisms. In general, the systems of efficient plasmid transfer have not been well characterized for the gram-positive bacteria. However, enterococci possess potent and unique capabilities of transferring plasmids among themselves and to other genera (4,5,21,35). One type of enterococcal plasmid consists of the group of narrow-hostrange and pheromone-responsive plasmids (4,5,9). The other type consists of the broad-host-range pAM␤1 and pIP501 plasmids, which were originally isolated from Enterococcus faecalis (8,24) and Streptococcus agalactiae (13, 18), respectively, and transfer on a solid surface at low frequency (8,13,18,24,27,40).We have described the isolation of the pheromone-independent gentamicin resistance conjugative plasmid pMG1 (Gm r ; 65.1 kb) from an Enterococcus faecium clinical isolate in Japan (20). pMG1 transfers efficiently among enterococcus strains during broth mating. pMG1-like plasmids are widely disseminated in vancomycin-resistant E. faecium clinical isolates obtained from a hospital in the United States (39).In this report, we show that the VanA resistance encoded on a Tn1546-like transposon was mediated by a pMG1-like plasmid and that this vancomycin resistance pMG1-like plasmid was capable of highly efficient transfer among the enterococci.Drug resistance of VRE isolates and isolation of vancomycin resistance conjugative plasmids. The laboratory strains and plasmids used in this study are listed in Table 1. A total of 12 isolates of vancomycin-resistant enterococci (VRE) were used in this study ( Table 2). The vancomycin resistance of each strain transferred to E. faecium BM4105RF at a frequency of about 10 Ϫ5 per donor cell by mating in broth for 4 h at 37°C.The transconjugants of each strain acquired only vancomycin and teicoplanin resistance, indicating that the glycopeptide resistance was transferred during broth mating. Analysis of agarose gel electrophoresis of restriction fragments of plasmid DNAs of each strain showed many DNA bands, indicating that each of the strains harbored several plasmids (Fig. 1, A1). The conjugative vancomycin resistance plasmid pHT␣ was identified from the transconjugant of E. faecium FH1 by repeated transfer experiments between E. faecium BM4105 strains. The plasmids isolated from each of the strains were classified into three types, ␣, ␤, and ␥, with respect to the restriction profiles that hybridized to the type ␣ plasmid pHT␣ (Fig. 1, A2) ( Table 2). The pHT␤ and pHT␥ plasmids, which were typ...