Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-Methylcytosine

A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-([²H₃]-Methyl)cytosine and [methyl-²H₃]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02–1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and ΦX-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.     

Identification of nucleosides in hydrolysates of transfer RNA by high-resolution mass spectrometry

High-resolution mass spectrometry has been investigated as a technique for identification of modified nucleosides in unfractionated hydrolysates of transfer RNA. The method is based on recognition of predetermined sets of exact mass values which are characteristic of individual nucleoside components. Mass spectra are photographically recorded in a non-time-resolved fashion, so that differing rates of nucleoside vaporization are of no consequence. Experimental parameters of sensitivity, spectrometer resolution and rates of vaporization were studied. Under typical conditions, 1-4 micrograms of tRNA are hydrolyzed, converted to volatile trimethylsilyl derivatives, and mass spectra of the resulting mixture recorded during 3 min at resolution 20 000; 75-100% of the minor nucleosides are usually identified in a single recording. The method is complementary to conventional methods of identification which rely on chromatographic mobilities, and can in principle be generally applied to recognition of biologically or chemically modified bases in nucleic acid.     

O-acylation of hydroxyproline residues: effect on peptide-bond isomerization and collagen stability

In collagen, strands of the sequence XaaYaaGly form a triple-helical structure. The Yaa residue is often (2S,4R)-4-hydroxyproline (Hyp). The inductive effect of the hydroxyl group of Hyp residues greatly increases collagen stability. Here, electron withdrawal by the hydroxyl group in Hyp and its 4S diastereomer (hyp) is increased by the addition of an acetyl group or trifluoroacetyl group. The crystalline structures of AcHyp[C(O)CH3]OMe and Achyp[C(O)CH3]OMe are similar to those of AcHypOMe and AcProOMe, respectively. The O-acylation of AcHypOMe and AchypOMe increases the 13C chemical shift of its Cgamma atom: AcHyp[C(O)CF3]OMe congruent with Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe congruent with Achyp[C(O)CH3]OMe > or = AcHypOMe congruent with AchypOMe. This increased inductive effect is not apparent in the thermodynamics or kinetics of amide bond isomerization. Despite apparently unfavorable steric interactions, (ProHypGly)(10), which is O-acylated with 10 acetyl groups, forms a triple helix that has intermediate stability: (ProHypGly)(10) > {ProHyp[C(O)CH3]Gly}(10) > (ProProGly)(10). Thus, the benefit to collagen stability endowed by the hydroxyl group of Hyp residues is largely retained by an acetoxyl group.     

TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.     

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.     

Tumour necrosis factor alpha (TNF-alpha) interferes with Fas-mediated apoptotic cell death on rheumatoid arthritis (RA) synovial cells: a possible mechanism of rheumatoid synovial hyperplasia and a clinical benefit of anti-TNF-alpha therapy for RA

To investigate the mechanism of rheumatoid synovial hyperplasia (RASH), the influence of tumour necrosis factor alpha (TNF-alpha) on Fas-mediated apoptotic cell death (Fas-ACD) was examined on cultured rheumatoid synovial cells (RASCs). RASCs were obtained from the synovial tissues of eight patients with rheumatoid arthritis (RA) and SCs from eight patients with osteoarthritis (OA) were used as a control. To examine the influence of TNF-alpha on Fas-ACD, SCs were cultured with anti-Fas antibody (CH11) for 16 h in the absence or presence of different doses of recombinant TNF-alpha. ACD was determined by electron microscopic analysis and the percentage of apoptotic cells was calculated by trypan blue staining. In addition, the expression of Fas and Bcl-2 on RASCs was examined by flow cytometry. As a result, RASCs were more susceptible to Fas-ACD in vitro than OASCs. TNF-alpha interfered with Fas-ACD on RASCs in a dose-dependent manner. Moreover, removal of TNF-alpha activity by a neutralizing anti-TNF-alpha antibody (cA2) restored Fas-ACD. Flow cytometric analysis showed no significant changes in either Fas or Bcl-2 expression on RASCs after the culture with TNFalpha.These results suggest the following: (1) Fas-ACD might be diminished in vivo by local excessive TNF-alpha and this might contribute in part to RASH. (2) The inhibition of Fas-ACD on RASCs by TNF-alpha might not be associated with changes in the expression of Fas or Bcl-2. (3) In addition, considering a magnetic resonance imaging (MRI) finding of marked reduction in the RASH after cA2 treatment, blockade of TNF-alpha activity could restore Fas-ACD in RA synovium, implicating a clinical benefit of anti-TNF-alpha therapy for RA.     

Demonstration of autoimmunity in the tight skin-2 mouse: a model for scleroderma

The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.     

Role of IP(3) in modulation of spontaneous activity in pacemaker cells of rabbit urethra

Isolated interstitial ("pacemaker") cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at -60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging -860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, "fast" (<100 ms in duration) and "slow" (>1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca(2+) release. When D-myo-inositol 1,4,5-trisphosphate (IP(3))-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP(3). These results support the hypothesis that pacemaker activity in the urethra is driven by the IP(3)-sensitive store.     

Structure and biological activity of ascamycin, a new nucleoside antibiotic

A newly isolated strain of Streptomyces sp. produces a new nucleoside antibiotic, ascamycin and the corresponding dealanyl derivative. The structure of ascamycin was determined to be 2-chloro-9-beta-[5-O-(N-L-alanyl)sulfamoyl-D-ribofuranosyl]-adenine. Remarkable selective toxicity of ascamycin compared to the dealanyl derivative was accounted for on the basis of a dealanylating enzyme present in the envelope of sensitive bacteria. After dealanylation, it becomes permeable to cell membrane.