Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P-DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 microM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10(6) bp versus 3.7 lesions/10(6) bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3-10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mbeta16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P-DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Nucleoside diphosphate kinase from Mycobacterium tuberculosis cleaves single strand DNA within the human c-myc promoter in an enzyme-catalyzed reaction

The reason for secretion of nucleoside diphosphate kinase (NdK), an enzyme involved in maintaining the cellular pool of nucleoside triphosphates in both prokaryotes and eukaryotes, by Mycobacterium tuberculosis is intriguing. We recently observed that NdK from M.tuberculosis (mNdK) localizes within nuclei of HeLa and COS-1 cells and also nicks chromosomal DNA in situ (A. K. Saini, K. Maithal, P. Chand, S. Chowdhury, R. Vohra, A. Goyal, G. P. Dubey, P. Chopra, R. Chandra, A. K. Tyagi, Y. Singh and V. Tandon (2004) J. Biol. Chem., 279, 50142–50149). In the current study, using a molecular beacon approach, we demonstrate that the mNdK catalyzes the cleavage of single strand DNA. It displays Michaelis–Menten kinetics with a k_cat/K_M of 9.65 (±0.88) × 10⁶ M⁻¹ s⁻¹. High affinity (K_d ≈ K_M of ~66 nM) and sequence-specific binding to the sense strand of the nuclease hypersensitive region in the c-myc promoter was observed. This is the first study demonstrating that the cleavage reaction is also enzyme-catalyzed in addition to the enzymatic kinase activity of multifunctional NdK. Using our approach, we demonstrate that GDP competitively inhibits the nuclease activity with a K_I of ~1.9 mM. Recent evidence implicates mNdK as a potent virulence factor in tuberculosis owing to its DNase-like activity. In this context, our results demonstrate a molecular mechanism that could be the basis for assessing in situ DNA damage by secretory mNdK.     

Novel role of sphingosine kinase 1 as a mediator of neurotrophin-3 action in oligodendrocyte progenitors

We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.     

Functional segregation of the TCR and antigen-MHC complexes on the surface of CTL

As CTL adhere to and lyze their targets, they extract cognate Ag-MHC and represent this on their own cell surface. Whether such self-presented cognate Ag stimulate the TCR of a CTL is uncertain. To analyze this, we examined TCR capping in response to self-presented Ag. We found that OVA peptide-specific OT-1 CTL that were pulsed with cognate peptide Ag did not cap their TCR, implying that the autologously presented MHC-Ag complex does not normally stimulate the TCR. However, this functional separation of the TCR and its ligand on the cell surface was not absolute. Treatment of Ag-pulsed OT-1 CTL with agents that alter cell surface charge, including trypsin, papain, tunicamycin, neuraminidase, and polybrene, allowed Ag-specific TCR capping. The TCR capped together with the restricting MHC molecule on the surface of the cell, implying an interaction between the TCR and cell-associated Ag. Further, the treated CTL underwent a time- and dose-dependent suicidal death that was both Fas- and perforin-dependent. Therefore, our results indicate that the association of the TCR with its MHC-peptide ligand on the surface of a CTL is normally proscribed by biophysical properties of the plasma membrane. Overcoming this restriction allows TCR stimulation and induces CTL effector functions and cell suicide.     

Passive immunization against beta-amyloid peptide protects central nervous system (CNS) neurons from increased vulnerability associated with an Alzheimer's disease-causing mutation

To characterize the effects of the familial Alzheimer's disease-causing Swedish mutations of amyloid precursor protein (SwAPP) on the vulnerability of central nervous system neurons, we induced epileptic seizures in transgenic mice expressing SwAPP. The transgene expression did not change the seizure threshold, but consistently more neurons degenerated in brains of SwAPP mice as compared with wild-type littermates. The degenerating neurons were stained both by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and by Gallyas silver impregnation. A susceptible population of neurons accumulated intracellular Abeta and immunoreacted with antibodies against activated caspase-3. To demonstrate that increased Abeta levels mediated the increased vulnerability, we infused antibodies against Abeta and found a significant reduction in neuronal loss that was paralleled by decreased brain levels of Abeta. Because the SwAPP mice exhibited no amyloid plaques at the age of these experiments, transgenic overproduction of Abeta in brain rendered neurons susceptible to damage much earlier than the onset of amyloid plaque formation. Our data underscore the possibility that Abeta is toxic, that it increases the vulnerability of neurons to excitotoxic events produced by seizures, and that lowering Abeta by passive immunization can protect neurons from Abeta-related toxicity.     

High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles

The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system. We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli. The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E. coli. The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure. The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides. This resulted in a single amino acid change at the N-terminus of the protein. The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E. coli. The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E. coli to form VLPs.     

Conserved eukaryotic histone-fold residues substituted into an archaeal histone increase DNA affinity but reduce complex flexibility

Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.     

Glycine 38 is crucial for the ribonucleolytic activity of human pancreatic ribonuclease on double-stranded RNA

Evidence is now in favor of protein-facilitated mechanisms for the intestinal cholesterol absorption. Here we report that the unesterified cholesterol uptake by rat jejunal brush border membrane vesicles (BBMVs) is efficient, saturable, and protein-mediated. The human apolipoproteins biliary anionic peptide factor (APF) and A-I (apoA-I) up-regulate micellar cholesterol uptake in a dose-dependent manner, but for all tested concentrations (0.1-20 microM), the lipid-free APF was more efficient than apoA-I. This uptake stimulation was suppressed after addition of Pabs directed to the external lipid-binding domain of the CLA-1/SR-BI and reduced by Pabs directed to the external loop of CD36. Thus, CLA-1/SR-BI and to a lesser extent CD36 are involved in the regulation of intestinal cholesterol uptake. APF, the main protein bound to biliary lipids, is likely one of their physiological effectors. As APF is an unesterified cholesterol carrier, it could facilitate the intestinal absorption of biliary cholesterol.     

Cloning,overexpression, purification,and matrix-assisted refolding of DevS (Rv 3132c) histidine protein kinase of Mycobacterium tuberculosis

The devR-devS (Rv 3133c-Rv 3132c) two-component system of Mycobacterium tuberculosis was identified in our laboratory by RNA subtractive hybridization. This genetic system was predicted to encode a response regulator and histidine protein kinase, respectively. The putative histidine kinase protein DevS was overexpressed to high levels in Escherichia coli as a fusion protein with a hexahistidine tag, His(6)-DevS201, in the form of inclusion bodies. Here we report a "redox-based" method of matrix-bound renaturation of DevS protein. The refolded protein was biochemically active in an autophosphorylation reaction characteristic of histidine kinases and was suitable for the generation of polyclonal antibodies and as an antigen in ELISA.     

TNF-alpha as a potential mediator of cardiac dysfunction due to intracellular Ca2+-overload

TNF-alpha has been shown to be involved in cardiac dysfunction during ischemia/reperfusion injury; however, no information regarding the status of TNF-alpha production in myocardial injury due to intracellular Ca2+-overload is available in the literature. The intracellular Ca2+-overload was induced in the isolated rat hearts subjected to 5 min Ca2+-depletion and 30 min Ca2+-repletion (Ca2+-paradox). The Ca2+-paradox hearts exhibited a dramatic depression in left ventricular developed pressure, a marked elevation in left ventricular end diastolic pressure, and more than a 4-fold increase in TNF-alpha content. The ratio of cytosolic to homogenate nuclear factor-kappaB (NFkappaB) was decreased whereas the ratio of phospho-NFkappaB to total NFkappaB was increased in the Ca2+-paradox hearts. All these changes due to Ca2+-paradox were significantly attenuated upon treating the hearts with 100 microM pentoxifylline. These results suggest that activation of NFkappaB and increased production of TNF-alpha may play an important role in cardiac injury due to intracellular Ca2+-overload.