Effect of DNA gyrase inhibitors pefloxacin, five other quinolones, novobiocin, and clorobiocin on Escherichia coli topoisomerase I.

Two coumarins, inhibitors of the B subunit of DNA gyrase, and six quinolones, inhibitors of the A subunit, were tested against Escherichia coli topoisomerase I-catalyzed DNA relaxation. Coumarins had no effect, whereas quinolones were inhibitors of the enzyme. This inhibition was compared with that of DNA gyrase and calf thymus topoisomerase I. The 50% inhibitory concentrations for E. coli topoisomerase I were about one order of magnitude higher than the corresponding values for E. coli DNA gyrase but were far lower than the known values for calf thymus topoisomerase I. There was a good relationship between inhibition of the two prokaryotic topoisomerases and MICs for E. coli, and the quinolones could be ranked in the same order in the three cases.     

Dual inhibitory activity of sitafloxacin (DU-6859a) against DNA gyrase and topoisomerase IV of Streptococcus pneumoniae

The in-vitro inhibitory activities of sitafloxacin (DU-6859a) and other quinolones against Streptococcus pneumoniae DNA gyrase and topoisomerase IV were measured. IC50s of levofloxacin, ciprofloxacin, sparfloxacin and tosufloxacin against DNA gyrase were almost three to 12 times higher than those against topoisomerase IV. On the other hand, sitafloxacin showed dual inhibitory activity against both enzymes and its IC50s were the lowest among those of the quinolones tested. These results suggest that sitafloxacin is an effective agent against pneumococcal infections and that the incidence of drug-resistant mutants is low.     

ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels.     

Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro.

The MICs of trovafloxacin, ciprofloxacin, ofloxacin, and sparfloxacin at which 90% of isolates are inhibited for 55 isolates of pneumococci were 0.125, 1, 4, and 0.5 microgram/ml, respectively. Resistant mutants of two susceptible isolates were selected in a stepwise fashion on agar containing ciprofloxacin at 2 to 10 times the MIC. While no mutants were obtained at the highest concentration tested, mutants were obtained at four times the MIC of ciprofloxacin (4 micrograms/ml) at a frequency of 1.0 x 10(-9). Ciprofloxacin MICs for these first-step mutants ranged from 4 to 8 micrograms/ml, whereas trovafloxacin MICs were 0.25 to 0.5 microgram/ml. Amplification of the quinolone resistance-determining region of the grlA (parC; topoisomerase IV) and gyrA (DNA gyrase) genes of the parents and mutants revealed that changes of the serine at position 80 (Ser80) to Phe or Tyr (Staphylococcus aureus coordinates) in GrlA were associated with resistance to ciprofloxacin. Second-step mutants of these isolates were selected by plating the isolates on medium containing ciprofloxacin at 32 micrograms/ml. Mutants for which ciprofloxacin MICs were 32 to 256 micrograms/ml and trovafloxacin MICs were 4 to 16 micrograms/ml were obtained at a frequency of 1.0 x 10(-9). Second-step mutants also had a change in GyrA corresponding to a substitution in Ser84 to Tyr or Phe or in Glu88 to Lys. Trovafloxacin protected from infection mice whose lungs were inoculated with lethal doses of either the parent strain or the first-step mutant. These results indicate that resistance to fluoroquinolones in S. pneumoniae occurs in vitro at a low frequency, involving sequential mutations in topoisomerase IV and DNA gyrase. Trovafloxacin MICs for wild-type and first-step mutants are within clinically achievable levels in the blood and lungs of humans.     

Quinolone-resistant Neisseria gonorrhoeae: correlation of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV with antimicrobial susceptibility profiles.

Fifty-five clinical strains of Neisseria gonorrhoeae were examined for mutations in the gyrA and parC genes and for antimicrobial susceptibility profiles. The MICs of quinolones for 31 strains with alterations in GyrA were significantly higher than the MICs for 24 strains without such alterations. Eleven strains with alterations in both GyrA and ParC were significantly more resistant to fluoroquinolones than those with alterations in GyrA alone. The MICs of cephalosporins for these strains were also significantly higher than those for other strains.     

Antimicrobial activity of a new fluoroquinolone, DU-6859a, against quinolone-resistant clinical isolates ofPseudomonas aeruginosa with genetic alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV

The in vitro antimicrobial activity of DU-6859a, a new fluoroquinolone, was tested against 22 clinical isolates ofPseudomonas aeruginosa, including strains with fluoroquinolone resistance-associated alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV. The MICs of DU-6859a for 10 isolates with both altered GyrA and ParC and for 7 isolates with altered GyrA alone ranged from 1.56 to 25 μg/mL and from 0.78 to 6.25 μg/mL, respectively. The MIC of DU-6859a at which 90% of the strains were inhibited (MIC₉₀) of these 17 isolates was 12.5 μg/mL. However, there was no significant difference between DU-6859a and the other quinolones tested against 5 strains which did not have any alterations of either GyrA or ParC. Based on the MIC₉₀s, DU-6859a exhibited 8- to 32-fold greater activity than the currently available fluoroquinolones against strains having alterations in DNA gyrase and topoisomerase IV.     

Infrequent occurrence of single mutations in topoisomerase IV and DNA gyrase genes among US levofloxacin-susceptible clinical isolates of Streptococcus pneumoniae from nine institutions (1999-2003)

OBJECTIVES: Prevalence of single quinolone-resistance determining region (QRDR) mutations in Streptococcus pneumoniae was studied from nine institutions over 5 years to track the incidence of single QRDR mutations. METHODS: All 1106 levofloxacin-susceptible pneumococci (MICs < or = 2.0 mg/L) identified from 1112 total isolates (99.5% susceptibility) in TRUST 3 (1999), TRUST 5 (2001) and TRUST 7 (2003) surveillance studies from the same nine hospitals in nine states were screened for QRDR mutations. Using pyrosequencing, the strains were screened for mutations corresponding to hot spots Asp-78, Ser-79 and Asp-83 in ParC; Asp-80, Ser-81 and Glu-85 in GyrA; Asp-435 in ParE and Asp-435 in GyrB. DNA sequencing of QRDRs was performed to confirm mutations. RESULTS: No QRDR mutations were found in any of the isolates with levofloxacin MICs < or = 0.5 mg/L and no gyrA or gyrB QRDR mutations were found in any of the screened isolates (MICs < or = 2 mg/L). Four single-step QRDR mutants with the following amino acid substitutions were found: ParE Asp-435 to Asn (isolated in 1999 in Colorado); ParC Asp-83 to Asn (isolated in 2001 in Kentucky); ParC Ser-79 to Phe (isolated in 2003 in Indiana) and ParC Ser-79 to Tyr (isolated in 2003 in California). These non-clonal strains had levofloxacin MICs of 1 mg/L and were non-susceptible to ciprofloxacin (MIC 2-4 mg/L). CONCLUSIONS: Overall prevalence of single QRDR mutations in levofloxacin-susceptible S. pneumoniae with MICs of < or = 2 mg/L was 0.4% (4/1106) and has remained <1% within nine institutions over 5 years (1999-2003).     

Quinolone-resistant Haemophilus influenzae in a long-term-care facility: nucleotide sequence characterization of alterations in the genes encoding DNA gyrase and DNA topoisomerase IV

Fluoroquinolone-resistant isolates of Haemophilus influenzae, obtained from a long-term care facility, were examined for nucleotide sequence differences in the quinolone-resistance-determining regions of gyrA, gyrB, parC, and parE. Similarities among the resistant isolates, plus multiple differences with susceptible isolates, suggest clonal dissemination involving two resistant subclones.     

Dual targeting of DNA gyrase and topoisomerase IV: target interactions of garenoxacin (BMS-284756, T-3811ME), a new desfluoroquinolone

We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 x 10(-12) to 4.0 x 10(-11)) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 micro g/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5' terminus of grlB and the 3' terminus of gyrA.     

STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies. METHODS: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA. RESULTS: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta, delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4 variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1, beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains. CONCLUSIONS: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.     

G-protein sensitivity of ligand binding to human dopamine D(2) and D(3) receptors expressed in Escherichia coli: clues for a constrained D(3) receptor structure

Human dopamine D(2) and D(3) receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with G(i/o)-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [(125)I]iodosulpride and [(3)H]7-OH-DPAT, were similar for hD(3) receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD(2) receptors in E. coli versus CHO cell membranes (measured with [(3)H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([(3)H]7-OH-DPAT) binding at hD(2) receptors but not at hD(3) receptors. Also, [(3)H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD(2) but clearly measurable at hD(3) receptors in E. coli membranes. Addition of a G(i/o)-protein mix to E. coli membranes increased high affinity [(3)H]7-OH-DPAT binding in a concentration-dependent manner at hD(2) and hD(3) receptors; this effect was reversed by addition of GppNHp. The potency of the G(i/o)-protein mix to reconstitute high affinity binding was similar for hD(2) and hD(3) receptors. Thus, agonist binding to D(3) receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D(3) receptors (versus D(2) receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.     

Asthma and COPD: differences and similarities. With special reference to the usefulness of budesonide/formoterol in a single inhaler (Symbicort) in both diseases

Asthma and chronic obstructive pulmonary disease (COPD) both have a high prevalence worldwide and yet each condition remains underdiagnosed. Despite a number of common features, these inflammatory respiratory syndromes have distinct clinical outcomes. COPD represents a greater economic burden than asthma because it has a less favourable prognosis and is associated with greater morbidity and mortality. Therefore, it is important to distinguish between these two diseases at an early stage, so that appropriate therapy can be prescribed to prevent deterioration. However, effective treatments that may be used in both conditions can minimise the effects of misdiagnosis and maximise the impact of treatment without the associated complexity when both conditions occur together. The current review summarises the differences and similarities of asthma and COPD, in terms of risk factors, pathophysiology, symptoms and diagnosis, to provide greater understanding of the role of budesonide/formoterol in a single inhaler in both diseases.     

Crystal structure of a DNA duplex cross-linked by 6-thioguanine–6-thioguanine disulfides: reversible formation and cleavage catalyzed by Cu(ii) ions and glutathione

Herein, we determined the crystal structure of a DNA duplex containing consecutive 6-thioguanine–6-thioguanine disulfides. The disulfide bonds were reversibly formed and cleaved in the presence of Cu(ii) ions and glutathione. To our knowledge, this is the first reaction in which metal ions efficiently accelerated disulfide bond formation between thio-bases in duplexes.

The crystal structure of a DNA duplex cross-linked by 6-thioguanine–6-thioguanine disulfides has been solved.

The reversible cross-linking of nucleic acids has been investigated for use in biochemical and medicinal studies. For instance, a cross-linking reaction through imine bonds that reversibly formed at lower temperatures and dissociated at higher temperatures has been reported.¹ Metal ion-mediated base pairs have recently attracted interest. In DNA and RNA duplexes, metal ions are placed between bases, and coordination bonds between metal ions and bases stabilize the duplexes.² Metal ion-mediated base pairs are reversibly formed at lower temperatures and dissociated at higher temperatures. Oligonucleotides with thiol tethers have been used to cross-link duplexes and hairpin structures by forming disulfide bonds.³ Disulfide bonds are reversibly formed by oxidization and dissociated by reduction. Disulfide bond formation between 4-thiouracil (^4SU) and 6-thiohypoxanthine (^6SH), and between ^4SU and 6-thioguanine (^6SG), has been reported.⁴ Disulfide bond formation between ^4SU and ^6SG in duplexes has been investigated^4f and applied for mechanistic studies of flap endonucleases.^4h In the reports, I₂ (an oxidizing reagent) was used to accelerate disulfide bond formation. In this paper, we report a novel crystal structure of a DNA duplex containing two consecutive cross-linked ^6SG–^6SG pairs. Notably, disulfide bond formation between 6-thioguanine bases in a duplex was accelerated in the presence of Cu(ii) ions.A DNA dodecamer (ODN-I) with a pseudo-self-complementary sequence d(CGCGAXXBCGCG) (X = ^6SG, B = 5-bromouracil) formed a duplex (duplex-I) consisting of C–G and A–B Watson–Crick base pairs and X–X pairs (Fig. 1). The B residue was incorporated in ODN-I to apply single-wavelength anomalous dispersion (SAD) method for crystal structure analysis.Open in a separate windowFig. 1A scheme for preparation of a DNA duplex containing 6-thioguanine–6-thioguanine disulfides.Thiobases, including 2-thiothymine (^2ST), 4-thiothymine (^4ST), and 6-thioguanine (^6SG), form metallo-base pairs.⁵ Duplexes containing ^2ST pairs and ^4ST pairs are stabilized in the presence of Hg(ii) and Ag(i) ions. The formation of the ^4ST–Ag(i)₂–^4ST pair in which two Ag(i) ions are placed between ^4ST bases was revealed by a crystal structure.^5b Additionally, metal ion binding of ^6SG has been reported.^5c,d,e Consequently, it is expected that duplex-I, which contains ^6SG–M–^6SG metallo-base pairs, could be formed by mixing metal ions and ODN-I. However, in the presence of Cu(ii) ions, we observed crystals of duplex-Iss including cross-linked ^6SG–^6SG pairs.Prior to crystallization, 2 mM ODN-1 was mixed with 2 mM CuCl₂ at room temperature. Single crystals of ODN-1 were obtained for a few days in a droplet prepared by merging 1 μl of ODN-1/Cu(ii) mixed solution and 1 μl of crystallization solution containing 50 mM MOPS (pH 7.0), 10 mM spermine, 250 mM ammonium nitrate, and 10% 2-methyl-2,4-pentanediol, which was equilibrated against 250 μl of 40% 2-methyl-2,4-pentanediol. In the crystal, two DNA fragments formed an antiparallel right-handed helix, as expected (Fig. 2a). The DNA duplex contains seven canonical Watson–Crick G–C and two A–B base pairs. At one end, two complementary residues, 5′-end C1 and 3′-end G12′, do not form Watson–Crick G–C base pairs, bulge out from the helix, and are involved in crystal packing contact (Fig. 2b). At the center of the duplex, two contiguous 6-thioG residues, X6 and X7, form disulfide-bonded base pairs with the X7′ and X6′ residues on the opposite strand, respectively (Fig. 2b and d). As a result, the DNA duplex is largely kinked at the center (Fig. 2d) where the minor groove of 6-thioG residues is widely exposed (Fig. 2c). A similar bent structure of a duplex containing an artificial disulfide pair has been solved by NMR spectroscopy.^3g Such structural disorders might be necessary for incorporating the disulfide pairs. Electron density maps clearly indicate the formation of a disulfide bond between the S6 atoms of the X6–X7′ and X7–X6′ base pairs (Fig. S1^†). In the X6–X7′ and X7–X6′ base pairs, two 6-thio-G residues align almost perpendicularly.Open in a separate windowFig. 2Secondary (a) and tertiary (b–d) structures of the DNA duplex containing two disulfide-bonded base pairs between 6-thio-G residues. X and B residues are 2′-deoxy-6-thioguanosine and 2′-deoxy-5-bromouridine, respectively. Views are from a phosphate-ribose backbone (b) and from the minor groove (c and d) of the two consecutive disulfide-bonded base pairs.To investigate disulfide bond formation in solution, solutions containing ODN-I′ in the presence of oxidation reagents, Cu(ii) ions and I₂, were analysed by high-performance liquid chromatography (HPLC) with a reverse-phase silica gel column. In ODN-I′, the B base in ODN-I was replaced by a T base. One minute after Cu(ii) ions were added to the ODN-I′ solution, a peak with a longer retention time was observed (Fig. 3B). The peak was separated and analysed by electron spray ionization time-of-flight mass spectrometry, and the result indicated the formation of duplex-I′ss. The addition of a large excess of I₂ did not induce the formation of duplex-I′ss (Fig. 3C), which differs from previous reports in which I₂ was successfully used for disulfide bond formation between 4-thiouracil and 6-thioguanine residues in DNA and RNA duplexes.^4f,g Also, duplex-I′ss was not generated by using KBrO₃ as an oxidation reagent in 24 h (Fig. S4^†).Open in a separate windowFig. 3HPLC profiles of solutions containing ODN-I′ and Cu(ii) ions and I₂. (A) A solution containing 20 μM ODN-I′ in 400 mM NH₄NO₃, 50 mM MOPS (pH 7.0). (B); 320 μM CuCl₂ was added to the solution. (C); 1 mM I₂ was added to the solution. Reactions were incubated at 4 °C.Glutathione have been used for cleave disulfide bonds and thioethers on nucleobases.⁶ Glutathione was added to the solution containing duplex-I′ss and the reaction was analyzed by HPLC. The peak for duplex-I′ss was immediately diminished and a peak for ODN-I′ was observed (Fig. S2c^†). In contrast, the addition of EDTA to a solution of duplex-I′ss did not alter the HPLC profile (Fig. S2b^†). Consequently, X–X pair formation (disulfide bond formation) was accelerated in the presence of Cu(ii) ions.As duplex-I′ss formed, the thiocarbonyl groups of the 6-thioguanine residues converted into disulfide groups; consequently, the absorbance at approximately 340 nm decreased (Fig. 4).⁷Open in a separate windowFig. 4Absorbance spectra of (A) duplex-I′ (4 μM), (B) duplex-I′ss (approximately 1 μM). (C) The spectra were overlapped. For easily compared, four times lager value of duplex-I′ss''s absorption is plotted.In conclusion, we determined the crystal structure of a DNA duplex containing consecutive 6-thioguanine–6-thioguanine disulfides. This is the first crystal structure of a nucleic acid duplex containing covalently linked bases through disulfide bonds. The DNA duplex is largely kinked at the disulfide base pairs where the minor groove of 6-thioG residues is widely exposed. The disulfide bonds were reversibly formed and cleaved in the presence of Cu(ii) ions and glutathione. Interestingly, oxidizing reagents such as I₂ and KBrO₃ did not accelerate disulfide bond formation. The arrangement of 6-thioguanine residues in the duplex structure may be related to their reactions. To our knowledge, this is the first reaction in which metal ions efficiently accelerated disulfide bond formation between thio-bases in DNA duplexes. Studies of disulfide bond formation of thio-bases (^2ST, ^4ST, ^6SG, etc.) in the presence of metal ions and metallo-base pair formations (interactions of thio–base pairs and metal ions) are currently in progress.     

The analgesic effects of interferential (IF) current on cold-pressor pain in healthy subjects: a single blind trial of three IF currents against sham IF and control

Interferential (IF) therapy is well documented as a therapeutic medium for pain relief. Despite widespread use by physiotherapists, there remains very limited laboratory or clinical evidence for the analgesic effect of IF.Fifty volunteers were randomly allocated to one of five groups ( n =10): control group (no treatment throughout); IF 100 Hz applied to the ipsilateral limb; IF 100 Hz applied to the contralateral limb; IF 30 Hz applied to the ipsilateral limb; Sham IF applied to the ipsilateral limb. Subjects were given repeated (six) cycles of cold-pressor pain and pain threshold scores were recorded for each cycle and compared using repeated measure ANOVA. Mean pain threshold values for all groups increased with the exception of the control group: where pain threshold values decreased across the cycles. Pain thresholds within IF treatment groups repeatedly increased, but no more than sham IF. IF at 30 Hz and 100 Hz applied to the contralateral limb offered more consistency in the rise in mean values, with the sham group demonstrating more variation in performance. However, statistical analysis failed to show any significant differences for the treatment effects either within groups across repeated cycles (P=0.123) or between the groups across cycles (P=0.552). IF does not significantly alter pain threshold.     

CD19 has a potential CD77 (globotriaosyl ceramide)-binding site with sequence similarity to verotoxin B-subunits: implications of molecular mimicry for B cell adhesion and enterohemorrhagic Escherichia coli pathogenesis

The glycosphingolipid globotriaosyl ceramide (CD77) and other globo- series glycolipids containing terminal galactose (Gal)alpha 1-4Gal residues function as receptors for the verotoxin (Shiga-like toxin) family of Escherichia coli-elaborated toxins. CD77 is also a marker for germinal center B lymphocytes and Burkitt's lymphoma cells. The pan B cell marker CD19 is a 95-kD membrane protein that appears early in B cell differentiation and is only lost upon terminal differentiation to plasma cells. CD19 is involved in signal transduction and has a regulatory role in B cell proliferation and differentiation in response to activation in vitro. However, an endogenous ligand for CD19 has not yet been identified. We report herein that the extracellular domain of CD19 has a potential CD77-binding site with extensive sequence similarity to the verotoxin B-subunits. These B-subunit-like sequences on CD19 are in close proximity following the organization of intervening amino acids into disulfide-linked domains. Cocapping of CD19 and CD77 on Burkitt's lymphoma-derived Daudi cells with anti-CD19 antibodies indicates that CD19 and CD77 are associated on the B cell surface. Cell surface binding of anti-CD19 antibodies is decreased on CD77-deficient mutant Daudi cells, suggesting that CD77 expression influences the surface expression of CD19. Wild-type Daudi cells, but not the CD19/CD77-deficient mutants, bind to matrices expressing the carbohydrate moiety of CD77 or other Gal alpha 1-4Gal containing glycolipids. This binding can be inhibited by anti-CD77 antibodies, the CD77-binding verotoxin B-subunit or anti-CD19 antibodies. Daudi cells exhibit a degree of spontaneous homotypic adhesion in culture while the CD77/CD19-deficient Daudi mutants grow as single cells. The stronger homotypic adhesion that occurs in B cells after antibody ligation of CD19 and that involves, to some extent, the integrin system, is also dramatically lower in the mutant cells relative to the parent cell line. However, reconstitution of mutant cells with CD77 restores the anti-CD19 mAb-induced adhesion to wild-type Daudi cell levels. These studies represent the first time that CD19-mediated signaling has been reconstituted in a low-responder B cell line. These convergent observations provide compelling evidence that CD19/CD77 interactions function in adhesion and signal transduction at a specific stage in B cell development and suggest that such interactions have a role in B lymphocyte homing and germinal center formation in vivo. By targeting CD77+ B cells, verotoxins may suppress the humoral arm of the immune response during infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efficacy of a potentized homeopathic drug (Arsenicum-Aalbum-30) in reducing cytotoxic effects produced by arsenic trioxide in mice: IV. Pathological changes, protein profiles, and content of DNA and RNA

OBJECTIVE: To examine if the potentized homeopathic drug Arsenicum Album-30 can help restore the damage produced in protein profiles, DNA and RNA contents in liver and testis as a result of arsenic treatment in mice. DESIGN: Sets of mice were injected with arsenic trioxide, one set was fed with Ars. Alb-30, another with Alcohol-30 and the final set was fed neither. The gel electrophoretic protein profiles and DNA and RNA contents in these three sets were studied. METHODS: Protein profiles were studied by SDS-PAGE method; the DNA and RNA contents were assayed by the standard methods through diphenylamine and orcinol reactions respectively. RESULTS: arsenic trioxide injection produced some pathological conditions, drastic changes (mainly reduction of protein bands) in protein sub-fractions, reduced DNA and RNA contents in both liver and testis; Ars. Alb-30-fed arsenic-intoxicated mice showed revival and restoration in both liver and testis as revealed by gel patterns and quantitative assay of DNA and RNA. CONCLUSION: Efficacy of the homeopathic drug Ars. Alb-30 in reducing arsenic-induced damage to protein and nucleic acids is substantiated and the mechanism of action of the homeopathic drug through expression of regulatory genes inferred.