Comparative analysis of beneficial effects of vancomycin treatment on Th1- and Th2-biased mice and the role of gut microbiota.

AIMS: The aim was to understand the time-dependent antibiotic-induced perturbation pattern of gut microbiota and its effect on the innate immune and metabolic profile of the host. METHODS AND RESULTS: Vancomycin was administered at 50 mg kg-1 of body weight twice daily for six consecutive days to perturb the gut microbiota of C57BL/6 (Th1-biased) and BALB/c (Th2-biased) mice. Following treatment with vancomycin, we observed a reduction in the abundance of phyla Firmicutes and Bacteroides and an increase in Proteobacteria in the gut for both strains of mice following treatment with vancomycin till day 4. Abundance of Akkermansia muciniphila of Verrucomicrobia phylum also increased, from day 5 onwards following vancomycin treatment. The time-dependent variation of gut microbiota was associated with increased (i) expression of toll-like receptors and inflammatory genes such as TNF-α, IL-6, and IL-17, (ii) gut barrier permeability and (iii) blood glucose level of the host. The results also showed that (i) transplantation of cecal microbiota from vancomycin-treated day 6 mice to day 3 vancomycin-treated mice helped in restoring blood glucose level in C57BL/6 mice and (ii) short-chain fatty acids like acetate, butyrate and propionate changed with the alteration of gut microbiota to induce differential regulation of host immune response. CONCLUSIONS: The current results revealed that an increase in A. muciniphila led to decreased inflammation and increased rate of glucose tolerance in the host. The treatment, with vancomycin till day 4, increased expression of inflammatory genes. The continuation of vancomycin for two more days reversed the effects. The effects were significantly more in C57BL/6 than BALB/c mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study established that the treatment with vancomycin till day 4 increased pathogenic bacteria but day 5 onwards provided significant health-related benefits to the host by increasing A. muciniphila more in C57BL/6 than BALB/c mice.

Ultra-high redox enzyme signal transduction using highly ordered carbon nanotube array electrodes.

We report on a highly ordered array of carbon nanotubes (CNTs) that serves as a universally direct nanoelectrode interface for redox proteins and provides an efficient conduit for electron transfer. The site-selective, covalent docking of the enzyme glucose oxidase (GO(x)) on the CNT tips is found to have a marked effect on enhancing electron transfer properties, as measured by cyclic voltammetry. A unimolecular electron transfer rate of 1500 s(-1) has been measured for this system, a value exceeding the rate of oxygen reduction by glucose oxidase. Furthermore, the redox enzyme-CNT array conjugate can be utilized as a quantitative, substrate-specific biosensor.

Local structure of M-DNA at the Nitrogen K-edge: evidence towards a metal ion induced conduction band in DNA.

M-DNA is an engineered duplex DNA that is synthesized from high doping of transition metal ions (Ni2+, Co2+ and Zn2+) into B-DNA at high pH. X-ray Absorption Near Edge Structure (XANES) of the Nitrogen K-edge shows remarkable differences between the doped and undoped DNA. A metal ion induced conduction band in the M-DNA is used to explain the XANES results.

Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate inflammation.

The severity of bovine respiratory infections has been linked to a variety of factors, including environmental and nutritional changes, transportation, and social reorganization of weaned calves. Fatal respiratory infections, however, usually occur when a primary viral infection compromises host defences and enhances the severity of a secondary bacterial infection. This viral-bacterial synergy can occur by a number of different mechanisms and disease challenge models have been developed to analyse host responses during these respiratory infections. A primary bovine herpesvirus-1 (BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolytica results in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model to demonstrate that stress significantly altered the viral-bacterial synergy resulting in fatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-like receptors (TLR) expression and increased pro-inflammatory responses which contribute to the severity of a Mannheimia haemolytica infection. TLRs play a critical role in detecting bacterial infections and inducing pro-inflammatory responses. It is difficult to understand, however, how stress-induced corticosteroids could enhance this form of viral-bacterial synergy. Nuclear translocation of the glucocorticoid receptor activates cell signalling pathways which inhibit both TLR signalling and pro-inflammatory responses. The apparent conundrum between stress-induced corticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease.

Metallic conduction through engineered DNA: DNA nanoelectronic building blocks.

A novel way of engineering DNA molecules involves substituting the imino proton of each base pair with a metal ion to obtain M-DNA with altered electronic properties. We report the first direct evidence of metalliclike conduction through 15 microm long M-DNA. In contrast, measurements on B-DNA give evidence of semiconducting behavior with a few hundred meV band gap at room temperature. The drastic change of M-DNA conductivity points to a new degree of freedom in the development of future molecular electronics utilizing DNA, such as creating all-DNA junction devices for use as nanoelectronic building blocks.

Role of Mg2+ in the interaction of anticancer antibiotic, chromomycin A3 with DNA: does neutral antibiotic bind DNA in absence of the metal ion?

Antitumor antibiotic, Chromomycin A3 (CHR), inhibits DNA replication and transcription via reversible interaction with double stranded DNA with GC-base specificity. The interaction, at and above physiological pH, requires the presence of bivalent metal ions, such as Mg2+. Anionic antibiotic does not bind DNA in the absence of Mg2+. In this paper we have examined the structural potential of neutral CHR at pH 5.2 to bind DNA in the absence of Mg2+. We have demonstrated the ability of the neutral antibiotic to bind DNA by means of different spectroscopic techniques and evaluated the necessary thermodynamic parameters for elucidation of the molecular basis of recognition. The results are compared with the scenario when Mg2+ is present in the system, because the ultimate aim of these studies is to elucidate the role of Mg2+ in CHR-DNA recognition. Neutral CHR binds to Mg2+ with lesser affinity than its anionic form. Spectroscopic features of the drug and its Mg2+ complex indicate self association of the antibiotic in the absence and presence of Mg2+. GC-base specificity of the drug and its Mg2+ complex are retained at pH 5.2, though the modes of recognition of DNA by the two ligands are different. Minor groove width of DNA plays a role in the accommodation of the ligand(s) during the GC base specific recognition while positive charge of Mg2+ in CHR:Mg2+ complex further facilitates the association. Relatively lower affinity of the neutral drug and its Mg2+ complex for DNA can be ascribed to the self association of these ligands in the absence of DNA.

The role of polyamines, Na(+) and K(+) in the formation of triple helices between purine oligonucleotides and the promoter region of the human c-src proto-oncogene.

Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine.purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na(+) and K(+). In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations all values tended to plateau in the 10(9)/M range. In contrast to previous reports, K(+) did not inhibit triplex formation and at 150 mM the binding constants were again in the 10(9)/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K(+) the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K(+) was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 microM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K(+) may not be detrimental. However, it may be difficult to achieve binding constants above approximately 10(9)/M even in the presence of polycations.

M-DNA: pH Stability, Nuclease Resistance and Signal Transmission.

Abstract In the presence of divalent metal ions (Zn(2)+, Co(2)+, and Ni(2)+) and at pHs above 8, duplex DNA forms a complex called M-DNA. M-DNA can be converted back to B-DNA by addition of EDTA or lowering the pH. The stability of M-DNA depends on the metal ion and/or the sequence of DNA. For calf thymus DNA the order of stability with decreasing pH is: Ni(2+)> Co(2+)>Zn(2++). The interconversion with B-DNA shows hysteresis; once formed Ni-M- DNA remains stable for more than one hour at pH 7, but conversion of B-DNA to M-DNA is slow at pHs below 8. Among synthetic sequences, poly[d(AT)] does not form M-DNA whereas the phosphorothioate analogues form only at pH 9.0. In contrast, the Ni-M-DNA form of poly[d(GC)] is stable even at pH 6.5. Ni-M-DNA is resistant to cleavage by DNase I whereas B-DNA is digested rapidly under identical conditions. The Co(2)+ and Ni(2+) forms of M-DNA were paramagnetic with increased mass susceptibilities (χ) compared to other metal complexes. Signal transmission in M-DNA was tested by constructing duplexes of 54 base pairs with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Quenching of fluorescein fluorescence was observed for the Zn(2+) form of M-DNA only when the DNA was labeled with both donor and acceptor. Therefore, the pathway of quenching maybe via electron transfer. Taken together, these results suggest that M-DNA is a distinct conformation with tightly bound metal ions, and certain forms may be stable under physiological conditions. Furthermore, M-DNA may be used as a molecular wire for signal transmission over long distances.

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire.

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau

Thermodynamic and kinetic studies of the formation of triple helices between purine-rich deoxyribo-oligonucleotides and the promoter region of the human c-src proto-oncogene.

The thermodynamic and kinetic parameters of triplex formation between four purine-rich oligonucleotides and a 22 bp pyrimidine. purine tract in the promoter region of the c-src gene were determined by fluorescence polarization studies. Three of these four oligonucleotides were 11 nt in length, corresponding to the left, central or right portion of the tract, while the fourth was a 22mer covering the whole tract. Binding constants ( Ka) were measured as a function of Mg2+ concentration (0-10 mM) and temperature (0-41 degrees C). In 10 mM Mg2+, K a for the left, central and right 11mers were 0.26, 0.75 and 1.4 x 10(8)/M, respectively, while for the 22mer the value was 1.8 x 10(8)/M at 22 degrees C. Under the same conditions, Ka was estimated by an electrophoretic band shift technique. The agreement between the two methods was acceptable for the 22mer but not for the 11mers. Kinetic measurements demonstrated that the rate of dissociation of the 22mer from the triplex was significantly slower than that of the 11mers, providing an explanation for the observed discrepancy. The entropy and enthalpy of triplex formation were calculated from van't Hoff plots. In all cases the entropy was favourable, especially for the 22mer and for the 11mer with the lowest guanine content. The enthalpy was unfavourable for the 22mer and most favourable for the 11mer with the highest guanine content. These results provide a thermodynamic explanation for length and sequence effects on the formation of purine.pyrimidine.purine triplexes.

Interactions of the antiviral quinoxaline derivative 9-OH-B220 [2, 3-dimethyl-6-(dimethylaminoethyl)- 9-hydroxy-6H-indolo-[2, 3-b]quinoxaline] with duplex and triplex forms of synthetic DNA and RNA.

The binding of an antiviral quinoxaline derivative, 2,3-dimethyl- 6 - (dimethylaminoethyl) - 9 - hydroxy - 6H - indolo - [2,3 - b]quinoxaline (9-OH-B220), to synthetic double and triple helical DNA (poly(dA).poly(dT) and poly(dA).2poly(dT)) and RNA (poly(rA). poly(rU) and poly (rA).2poly(rU)) has been characterized using flow linear dichroism (LD), circular dichroism (CD), fluorescence spectroscopy, and thermal denaturation. When either of the DNA structures or the RNA duplex serve as host polymers a strongly negative LD is displayed, consistent with intercalation of the chromophoric ring system between the base-pairs/triplets of the nucleic acid structures. Evidence for this geometry also includes weak induced CD signals and strong increments of the fluorescence emission intensities upon binding of the drug to each of these polymer structures. In agreement with intercalative binding, 9-OH-B220 is found to effectively enhance the thermal stability of both the double and triple helical states of DNA as well as the RNA duplex. In the case of poly(dA).2poly(dT), the drug provides an unusually large stabilization of its triple helical state; upon binding of 9-OH-B220 the triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5 deg. C in a 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. When triplex RNA serves as host structure, LD indicates that the average orientation angle between the drug chromophore plane and the helix axis of the triple helical RNA is only about 60 to 65 degrees. Moreover, the thermal stabilizing capability, as well as the fluorescence increment, CD inducing power and perturbations of the absorption envelope, of 9-OH-B220 in complex with the RNA triplex are all less pronounced than those observed for the complexes with DNA and duplex RNA. These features indicate binding of 9-OH-B220 in the wide and shallow minor groove of poly(rA).2poly(rU). Based on the present results, some implications for the applications of this low-toxic, antiviral and easily administered drug in an antigene strategy, as well as its potential use as an antiretroviral agent, are discussed.

Role of magnesium ion in mithramycin-DNA interaction: binding of mithramycin-Mg2+ complexes with DNA.

Mithramycin is an anticancer drug that blocks macromolecular synthesis via reversible interaction with the DNA template in the presence of bivalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not clear. We approached the problem in two steps via studies on the interactions between (i) mithramycin and Mg2+ and (ii) mithramycin-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for the purpose. From equilibrium and kinetic studies, we earlier reported that MTR forms two different types of complexes with Mg2+ [Aich, P., & Dasgupta, D. (1990) Biochem. Biophys. Res. Commun. 173, 689]. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of mithramycin: Mg2+) and complex II (with 2:1 stoichiometry in terms of mithramycin: Mg2+). In this report, we have further characterized these complexes by fluorescence spectroscopy. Interactions of these complexes with calf thymus DNA were examined to elucidate their binding. Evaluation of binding parameters (intrinsic binding constant and stoichiometry) from spectrophotometric and fluorimetric titrations suggests that the complexes bind differently to the same DNA. Measurement of van't Hoff enthalpies for the interaction of the two ligands and DNA shows that the complex I-DNA interaction is exothermic, in contrast to the endothermic nature of the complex II-DNA interaction. This could originate from a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. Our studies to detect the nature of the groove via which these complexes bind to DNA suggest that both complexes approach via the minor groove of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between antitumor antibiotic chromomycin A3 and Mg2+. I. Evidence for the formation of two types of chromomycin A3-Mg2+ complexes.

Chromomycin A3 (CHRA3) is an antitumor antibiotic which binds to Mg2+. In the present communication, we show, by means of equilibrium spectroscopic studies (such as absorption, fluorescence and circular dichroism), that two types of CHRA3-Mg2+ complexes (of 1:1 and 1.9:1 stoichiometries in terms of CHRA3:Mg2+, respectively) are formed depending on the concentrations of CHRA3 and Mg2+. The rate constant and activation energy for the formation of two complexes are different, thereby reinforcing the proposition that they are different molecular species. This observation is novel and significant in order to understand the anticancer property of the drug. It also provides explanation for earlier observations that site, affinity parameters and mode of interaction of the drug with DNA in the presence of Mg2+ depend on the relative concentration of Mg2+.

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.

Role of Mg++ in the mithramycin-DNA interaction: evidence for two types of mithramycin-Mg++ complex.

Mithramycin(MTR, structure shown in Figure 1) [and the related compound Chromomycin A3(CHRA3)] are antitumor antibiotics which inhibit DNA dependent RNA polymerase activity via reversible interaction with DNA only in the presence of divalent metal ion such as Mg++. In order to understand the role of Mg++ in MTR-DNA interaction, absorbance and CD spectroscopic techniques are employed to study the binding of MTR to Mg++. These studies show: i) the drug alone binds to Mg++ and ii) two different types of drug-Mg++ complexes are formed at low(Complex I) and high(Complex II) ratios of the concentration of Mg++ and MTR. We propose that these two complexes would bind to the same DNA with different affinities and rates. This result suggests that the relative concentration of Mg++ is an important factor to be taken into account to understand the molecular basis of MTR-DNA interaction.