Combined Strategy of Radioactive <Superscript>125</Superscript>I Seeds and Salinomycin for Enhanced Glioma Chemo-radiotherapy: Evidences for ROS-Mediated Apoptosis and Signaling Crosstalk

Radioactive ¹²⁵I seeds-based radiotherapy has achieved great success in treatment of human cancers. However, radioresistance and severe side effects badly limited its clinic application. Recently, chemoradiotherapy as a superior strategy has been rapidly developed and widely used in clinic. However, the underlying mechanism remains elusive. Herein, in the present study, a combined chemoradiation model of ¹²⁵I seeds and salinomycin (SAL) in vitro and in vivo was designed, and the enhanced anticancer efficiency and mechanism were also evaluated in human glioma. The results showed that combined treatment of ¹²⁵I seeds and SAL induced enhanced growth inhibition against human glioma cells through induction of cell apoptosis. Further investigation revealed that combined treatment of ¹²⁵I seeds and SAL triggered enhanced DNA damage through inducing reactive oxide species (ROS) generation. Additionally, enhanced dysfunction of MAPKs and AKT pathways both contributed to combined treatment-induced growth inhibition against human glioma cells. Importantly, the U251 human glioma xenograft growth was effectively inhibited by combined treatment of ¹²⁵I seeds and SAL by induction of cell apoptosis with involvement of inhibiting cell proliferation and angiogenesis. Taken together, our results indicated that combined treatment of ¹²⁵I seeds and SAL achieved enhanced growth inhibition and apoptosis in human glioma in vitro and in vivo through triggering ROS-mediated DNA damage and regulation of MAPKs and AKT pathways, which validated that the combined strategy of using ¹²⁵I seeds and SAL could be a highly efficient way to achieve enhanced glioma chemo-radiotherapy.     

Simulation of DNA fragment distributions after irradiation with photons

The Monte Carlo track structure code PARTRAC has been further improved by implementing electron scattering cross-sections for liquid water and by explicitly modelling the interaction of water radicals with DNA. The model of the genome inside a human cell nucleus in its interphase is based on the atomic coordinates of the DNA double helix with an additional volume for the water shell. The DNA helix is wound around histone complexes, and these nucleosomes are folded into chromatin fibres and further to fibre loops, which are interconnected to build chromosomes with a territorial organisation. Simulations have been performed for the irradiation of human fibroblast cells with carbon K and aluminium K ultrasoft x-rays, 220 kVp x-rays and ⁶⁰Co γ-rays. The ratio single-strand breaks to double-strand breaks (ssb/dsb) for both types of ultrasoft x-rays is lower than for γ-rays by a factor of 2. The contributions of direct and indirect effects to strand break induction are almost independent of photon energy. Strand break patterns from indirect effects reflect differences in the susceptibility of the DNA helix to OH^• attack inside the chromatin fibre. Distributions of small DNA fragments (<3 kbp) are determined by the chromatin fibre structure irrespective of whether direct or indirect effects are causing the breaks. In the calculated fragment size distributions for larger DNA fragments (>30 kbp), a substantial deviation from random breakage is found only for carbon K irradiation, and is attributed to its inhomogeneous dose distribution inside the cell nucleus. For the other radiation qualities, the results for larger fragments can be approximated by random breakage distributions calculated for a yield of dsb which is about 10% lower than the average for the whole genome. The excess of DNA fragments detected experimentally in the 8–300 kbp region after x-ray irradiation is not seen in our simulation results. Received: 19 October 1998 / Accepted in revised form: 14 January 1999     

125Iodine decay in DNA: A discussion of its effectiveness for the breaking of DNA strands

Summary ¹²⁵I incorporated in DNA is known to be exceptionally toxic. Values of D₀ range from about 40 to about 90 decays for survival of mammalian cells. The effectiveness of¹²⁵I in DNA with respect to the induction of breaks of the DNA strands, however, appears to be comparatively low. The numbers of strand breaks per energy deposited in subnuclear cellular structures such as DNA is smaller for a disintegration of¹²⁵I than for-rays. The difference in effectiveness diminishes with increasing mass of the considered sensitive volume. The apparent inefficiency of¹²⁵I-decay may, on one hand, result from a waste of local energy deposition. On the other hand, it may be caused by a multitude of local strand breaks (clusters) induced by¹²⁵I-decay which are measured as one break only by the conventionally applied techniques of strand break measurement. The apparent inefficiency of¹²⁵I may be evidence furthermore for the importance of considering not only the DNA as the sensitive target but with equal pertinence the gross sensitive volume, i.e. the whole cell nucleus [12]. Further, for drawing meaningful comparisons, it may be necessary to take into consideration the microdosimetric event size distributions for the critical targets [1].Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

DFT modeling on the suitable crown ether architecture for complexation with Cs<Superscript>+</Superscript> and Sr<Superscript>2+</Superscript> metal ions

Crown ether architectures were explored for the inclusion of Cs⁺ and Sr²⁺ ions within nano-cavity of macrocyclic crown ethers using density functional theory (DFT) modeling. The modeling was undertaken to gain insight into the mechanism of the complexation of Cs⁺ and Sr²⁺ ion with this ligand experimentally. The selectivity of Cs⁺ and Sr²⁺ ions for a particular size of crown ether has been explained based on the fitting and binding interaction of the guest ions in the narrow cavity of crown ethers. Although, Di-Benzo-18-Crown-6 (DB18C6) and Di-Benzo-21-Crown-7 (DB21C7) provide suitable host architecture for Sr²⁺ and Cs⁺ ions respectively as the ion size match with the cavity of the host, but consideration of binding interaction along with the cavity matching both DB18C6 and DB21C7 prefers Sr²⁺ ion. The calculated values of binding enthalpy of Cs metal ion with the crown ethers were found to be in good agreement with the experimental results. The gas phase binding enthalpy for Sr²⁺ ion with crown ether was higher than Cs metal ion. The ion exchange reaction between Sr and Cs always favors the selection of Sr metal ion both in the gas and in micro-solvated systems. The gas phase selectivity remains unchanged in micro-solvated phase. We have demonstrated the effect of micro-solvation on the binding interaction between the metal ions (Cs⁺ and Sr²⁺) and the macrocyclic crown ethers by considering micro-solvated metal ions up to eight water molecules directly attached to the metal ion and also by considering two water molecules attached to metal-ion-crown ether complexes. A metal ion exchange reaction involving the replacement of strontium ion in metal ion-crown ether complexes with cesium ion contained within a metal ion-water cluster serves as the basis for modeling binding preferences in solution. The calculated O-H stretching frequency of H₂O molecule in micro-solvated metal ion-crown complexes is more red-shifted in comparison to hydrated metal ions. The calculated IR spectra can be compared with an experimental spectrum to determine the presence of micro-solvated metal ion–crown ether complexes in extractant phase.     

Iodine-125 decay in a synthetic oligodeoxynucleotide. II. The role of auger electron irradiation compared to charge neutralization in DNA breakage

Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. II. The Role of Auger Electron Irradiation Compared to Charge Neutralization in DNA Breakage. The dramatic chemical and biological effects of the decay of DNA-incorporated (125)I stem from two consequences of the Auger electron cascades associated with the decay of the isotope: high local deposition of radiation energy from short-range Auger electrons, and neutralization of the multiply charged tellurium atom. We have analyzed the extensive data reported in the companion paper (Radiat. Res. 153, 000-000, 2000), in which DNA breakage was measured after (125)I decay in a 41-bp oligoDNA. The experimental data collected under scavenging conditions (2 M dimethylsulfoxide) were deconvoluted into two components denoted as radiation and nonradiation, the former being attributed to energy deposition by Auger electrons. The contribution of the components was estimated by adopting various assumptions, the principal one being that DNA breakage due to the radiation mechanism is dependent on the distance between the decaying (125)I atom and the cleaved deoxyribosyl unit, while the nonradiation mechanism, associated with neutralization of the multiply charged tellurium atom, contributes equally at corresponding nucleotides starting from the (125)I-incorporating nucleotide. Comparison of the experimental data sets collected under scavenging and nonscavenging (without dimethylsulfoxide) conditions was used to estimate the radiation-scavengeable component. Our analysis showed that the nonradiation component plays the major role in causing breakage within 4-5 nucleotides from the site of (125)I incorporation and produces about 50% of all single-stranded breaks. This overall result is consistent with the relative amounts of energy associated with Auger electrons and the charged tellurium atom. However, the nonradiation component accounts for almost four times more breaks in the top strand, to which the (125)I is bound covalently, than in the bottom strand, thus suggesting an important role of covalent bonds in the energy transfer from the charged tellurium atom. The radiation component dominates at the distances beyond 8-9 nucleotides, and 36% of the radiation-induced breaks are scavengeable.     

Dosimetric comparison between realistic ocular model and other models for COMS plaque brachytherapy with <Superscript>103</Superscript>Pd, <Superscript>131</Superscript>Cs,and <Superscript>125</Superscript>I radioisotopes

Nowadays, Monte Carlo calculations are commonly used for the evaluation of dose distributions and dose volume histograms in eye brachytherapy. However, currently available eye models have simple geometries, and main substructures of the eye are either not defined in details or not distinguished at all. In this work absorbed doses of eye substructures have been estimated for eye plaque brachytherapy using the most realistic eye model available, and compared with absorbed doses obtained with other available eye models. For this, a medium-sized tumour on the left sides of the right eye was considered. Dosimetry calculations were performed for four different eye models developed based on a literature review, and using a 12 mm Collaborative Ocular Melanoma Study plaque containing ¹³¹Cs, ¹⁰³Pd, and ¹²⁵I sources. Obtained results illustrate that the estimated doses received by different eye substructures strongly depend on the model used to represent the eye. It is shown here that using a non-realistic eye model leads to a wrong estimation of doses for some eye substructures. For example, dose differences of up to 35% were observed between the models proposed by Nogueira and co-workers and Yoriyaz and co-workers, while doses obtained by use of the models proposed by Lesperance and co-workers, and Behrens and co-workers differed up to 100 and 63% as compared to the situation when a realistic model was used, respectively. Moreover, comparing different radionuclides showed that the most uniform dose distribution in the considered tumour region was that from ¹³¹Cs, with a coefficient of variation of 33%. In addition, considering the realistic eye model, it was found that the radiosensitive region of the lens received more than the threshold dose of cataract induction (0.5 Gy), for all investigated radionuclides.     

Iodine-125 decay in a synthetic oligodeoxynucleotide. I. Fragment size distribution and evaluation of breakage probability

Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. I. Fragment Size Distribution and Evaluation of Breakage Probability. Incorporation of (125)I-dC into a defined location of a double-stranded oligodeoxynucleotide was used to investigate DNA breaks arising from decay of the Auger electron-emitting isotope. Samples of the oligodeoxynucleotide were also labeled with (32)P at either the 5' or 3' end of either the (125)I-dC-containing (so-called top) or opposite (bottom) strand and incubated in 20 mM phosphate buffer or the same buffer plus 2 M dimethylsulfoxide at 4 degrees C during 18-20 days. The (32)P-end-labeled fragments produced by (125)I decays were separated on denaturing polyacrylamide gels, and the (32)P activity in each fragment was determined by scintillation counting after elution of fragments from the gel. The relative fragment size distributions were then normalized on a per decay basis and converted to a distribution of single-strand break probabilities as a function of distance from the (125)I-dC. The results of three to five experiments for each of eight possible combinations of labels and incubation conditions are presented as a table showing the relative numbers of (32)P counts in different fragments as well as graphs of normalized fragment size distributions and probabilities of breakage. The average numbers of single-strand breaks per (125)I decay are 3. 3 and 3.7 in the top strand and 1.3 and 1.5 in the bottom strand with and without dimethylsulfoxide, respectively. Every (125)I decay event produces a break in the top strand, and breakage of the bottom strand occurs in 75-80% of the events. Thus a double-strand break is produced by (125)I decay with a probability of approximately 0.8.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Binding of <Superscript>3</Superscript>H-WIN-35,428 and <Superscript>125</Superscript>I-RTI-121 to Human Platelet Membranes

The dopamine transporter (DAT) is a protein regulating dopamine concentration in the synaptic cleft through the re-uptake mechanism. The DAT is the main target of psychostimulants and seems to play a pivotal role in neuronal degeneration and different neuropsychiatric disorders involving the dopamine system. Exhaustive research, however, regarding the presence of this protein in human platelets is still inconclusive, although it is thought that it might provide a peripheral tool to serve as a mean of exploring the same structure present in the brain. Therefore, we assessed some binding assays in platelets derived from healthy human subjects by means of ³H-WIN 35,428, a compound which is considered a selective ligand for the labelling of this protein, and by means of ¹²⁵I-RTI-121, another compound with high specificity for DAT. The results showed that the binding of ³H-WIN-35,428 was too low to enable the detection of any structure; the binding of ¹²⁵I-RTI-121, on the other hand, revealed the presence of two binding sites with pharmacological profiles similar to that of the serotonin transporter (SERT). In conclusions, therefore, platelets would not seem to be a useful model for exploring the DAT, given the prevalence therein of the SERT and the difficulty of labelling the DAT with the currently available ligands.     

A simple method to adjust inconsistently referenced <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of proteins

Inconsistent ¹³C and ¹⁵N chemical shift referencing is a continuing problem associated with protein chemical shift assignments deposited in BioMagResBank (BMRB). Here we describe a simple and robust approach that can quantitatively determine the ¹³C and ¹⁵N referencing offsets solely from chemical shift assignment data and independently of 3D coordinate data. This novel structure-independent approach permitted the assessment and determination of ¹³C and ¹⁵N reference offsets for all protein entries deposited in the BMRB. Tests on 452 proteins with known 3D structures show that this structure-independent approach yields ¹³C and ¹⁵N referencing offsets that exhibit excellent agreement with those calculated on the basis of 3D structures. Furthermore, this protocol appears to improve the accuracy of chemical shift-derived secondary structural identification, and has been formally incorporated into a computer program called PSSI (http//www.pronmr.com).Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7441-3     

Simulation of exon deletion mutations induced by low-LET radiation at the HPRT locus

Friedland, W., Li, W. B., Jacob, P. and Paretzke, H. G. Simulation of Exon Deletion Mutations Induced by Low-LET Radiation at the HPRT Locus. Radiat. Res. 155, 703-715 (2001). The induction of HPRT mutants with exon deletions after irradiation with photons was simulated using the biophysical radiation track structure model PARTRAC. The exon-intron structure of the human HPRT gene was incorporated into the chromatin fiber model in PARTRAC. After gamma and X irradiation, simulated double-stranded DNA fragments that overlapped with exons were assumed to result in exon deletion mutations with a probability that depended on the genomic or the geometric distance between the breakpoints. The consequences of different assumptions about this probability of deletion formation were evaluated on the basis of the resulting fractions of total, terminal and intragenic deletions. Agreement with corresponding measurements was obtained assuming a constant probability of deletion formation for fragments smaller than about 0.1 Mbp, and a probability of deletion formation decreasing with increasing geometric or genomic distance between the end points for larger fragments. For these two assumptions, yields of mutants with exon deletions, size distributions of deletions, patterns of deleted exons, and patterns of deleted STS marker sites surrounding the gene were calculated and compared with experimental data. The yields, size distributions and exon deletion patterns were grossly consistent, whereas larger deviations were found for the STS marker deletion patterns in this comparison.     

Density functional calculations of chemical shielding of backbone <Superscript>15</Superscript>N in helical residues of protein G

We performed density functional calculations of backbone ¹⁵N chemical shielding tensors in selected helical residues of protein G. Here we describe a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone ¹⁵N. We analyzed the role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field. Our results show that the dipole moment of the α-helix can cause significant deshielding of ¹⁵N; therefore, it needs to be considered when calculating ¹⁵N chemical shielding. We found that it is important to include interactions with the side chains that are close in space when the charged form for ionizable side chains is adopted in the calculation. We also illustrate how the ionization state of these side chains can affect the chemical shielding tensor elements. Chemical shielding calculations using a 8-residue fragment model in vacuum and adopting the charged form of ionizable side chains yield a generally good agreement with experimental data.     

<Superscript>31</Superscript>P NMR correlation maps of <Superscript>18</Superscript>O/<Superscript>16</Superscript>O chemical shift isotopic effects for phosphometabolite labeling studies

Intramolecular correlations among the ¹⁸O-labels of metabolic oligophosphates, mapped by J-decoupled ³¹P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the ¹⁸O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of ¹⁸O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological usefulness of the J-decoupled ³¹P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally ¹⁸O labeled in perfused mammalian hearts.     

Simulation of DNA damage after proton irradiation

The biophysical radiation track simulation model PARTRAC was improved by implementing new interaction cross sections for protons in water. Computer-simulated tracks of energy deposition events from protons and their secondary electrons were superimposed on a higher-order DNA target model describing the spatial coordinates of the whole genome inside a human cell. Induction of DNA double-strand breaks was simulated for proton irradiation with LET values between 1.6 and 70 keV/microm and various reference radiation qualities. The yield of DSBs after proton irradiation was found to rise continuously with increasing LET up to about 20 DSBs per Gbp and Gy, corresponding to an RBE up to 2.2. About half of this increase resulted from a higher yield of DSB clusters associated with small fragments below 10 kbp. Exclusion of experimentally unresolved multiple DSBs reduced the maximum DSB yield by 30% and shifted it to an LET of about 40 keV/microm. Simulated fragment size distributions deviated significantly from random breakage distributions over the whole size range after irradiation with protons with an LET above 10 keV/microm. Determination of DSB yields using equations derived for random breakage resulted in an underestimation by up to 20%. The inclusion of background fragments had only a minor influence on the distribution of the DNA fragments induced by radiation. Despite limited numerical agreement, the simulations reproduced the trends in proton-induced DNA DSBs and fragment induction found in recent experiments.     

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3<Superscript>+</Superscript>, CD14<Superscript>+</Superscript> and CD19<Superscript>+</Superscript>

Background

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3⁺, CD14⁺ and CD19⁺. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.

Methods

PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3⁺, CD14⁺, CD19⁺ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.

Results

Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3⁺ cells then in 8/26 (30.8%) of CD14⁺ and CD19⁺ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3⁺ (2.4%), then in CD19⁺ (1.2%), and much lower in CD14⁺ (0.4%) cells.

Conclusions

Our results indicate that CD3⁺ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C backbone resonance assignment of the C-terminal domain of enzyme I from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone ¹H, ¹⁵N and ¹³C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the ¹H–¹⁵N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.     

An enhanced sensitivity methyl <Superscript>1</Superscript>H triple-quantum pulse scheme for measuring diffusion constants of macromolecules

We present a pulse scheme that exploits methyl ¹H triple-quantum (TQ) coherences for the measurement of diffusion rates of slowly diffusing molecules in solution. It is based on the well-known stimulated echo experiment, with encoding and decoding of TQ coherences. The size of quantifiable diffusion coefficients is thus lowered by an order of magnitude with respect to single-quantum (SQ) approaches. Notably, the sensitivity of the scheme is high, approximately ¾ that of the corresponding single quantum experiment, neglecting relaxation losses, and on the order of a factor of 4 more sensitive than a previously published sequence for AX₃ spin systems (Zheng et al. in JMR 198:271–274, 2009) for molecules that are only ¹³C labeled at the methyl carbon position. Diffusion coefficients measured from TQ- and SQ-based experiments recorded on a range of protein samples are in excellent agreement. We present an application of this technique to the study of phase-separated proteins where protein concentrations in the condensed phase can exceed 400 mg/mL, diffusion coefficients can be as low as ~10^?9 cm²s^?1 and traditional SQ experiments fail.     

Effects of Ketamine on the Balance of Ions Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>, Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> in the Ischemia-reperfusion Affected Spinal Cord Tissues in Rabbits

To test the effects of ketamine on metal ion balance in the spinal cord tissues after ischemic reperfusion (I/R), 24 white adult Japanese rabbits were randomly assigned to sham operation group, I/R group or ketamine-treated I/R group. Spinal cord injuries in I/R group and ketamine-treated I/R group were induced by aortic occlusions. Rabbits in ketamine-treated I/R group were intravenously infused 10 mg/kg ketamine twice: once at 10 min before aortic clamping and once at the onset of reperfusion. Post-operative neurological functions and concentrations of ions Ca²⁺, Mg²⁺, Cu²⁺ and Zn²⁺ in the spinal cord were assessed. Compared with the sham operation group, rabbits in the I/R group showed significantly worsened neurological functions as scored with the modified Tarlov criteria and altered concentrations of ions Ca²⁺, Mg²⁺, Cu²⁺ and Zn²⁺. These unfavorable changes were significantly reversed in the ketamine-treated I/R group, suggesting that the potent protective effects of ketamine against the I/R-induced spinal cord injuries may be due to its ability to maintain ion balance in the I/R affected tissues.     

Cyclophilin a increases CD68<Superscript>+</Superscript> cell infiltration in rat experimental periodontitis

Cyclophilin A (CyPA) is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. This study is to observe the expression and distribution of CyPA and CD68⁺ cells in the histopathogenesis of rat ligation-induced experimental periodontitis, and assess the role of CyPA in CD68⁺ cell infiltration in rat experimental periodontitis. Experimental periodontitis was induced by ligation according to our previous method. CyPA expression in gingival tissues was detected by western blotting. Immunohistochemistry was applied for CyPA and CD68 distribution. For further certifying the role of CyPA in CD68⁺ cell infiltration, the right mandibular first molar received 0.1 μM CyPA locally by gingival injection every 2 days (L?+?C group), while the left mandibular first molar received saline as a control group (L group). The number of CD68⁺ cells in the experimental periodontitis was observed by immunohistochemistry. Alveolar bone destruction was assessed by micro-computerized tomography (micro-CT). Osteoclast was observed through TRAP staining. Nuclear factor (NF)-κB phospho-p65 (p p65) and phosphor-IκBα (p IκBα) expressions were detected to investigate NF-κB activation. CyPA showed an increasing trend at 1–6 weeks after ligation. CyPA and CD68⁺ cells were present in the gingival inflammatory infiltration, and participated in alveolar bone destruction. In the L?+?C group, the number of CD68⁺ cells was increased compared with the L group, and greater alveolar bone destruction was observed. NF-κB p p65 and p IκBα expressions were upregulated in the L?+?C group compared with the L group indicating NF-κB activation. CyPA increases CD68⁺ cell infiltration in rat experimental periodontitis, suggesting CyPA might be an anti-inflammatory therapeutic target.     

Repairable and unrepairable DNA strand breaks induced by decay of3H and125I incorporated into DNA of mammalian cells

Summary Chinese hamster cells (Cl : 1) were labelled with³H-thymidine or¹²⁵Iododeoxyuridine for 18 h and after 3 h in non-radioactive medium they were stored at 0° C up to 6 h. The number of DNA strand breaks observed after the labelling period (37° C) or after treatment at 0° C was determined using the DNA-unwinding technique.¹²⁵I-decays in DNA were significantly more efficient than³H-decays in introducing unrepairable DNA strand breaks during the labelling period. 32% of¹²⁵I-induced and 3% of³H-induced DNA strand breaks were unrepaired after 21 h at 37° C. Comparison between the effects of¹²⁵I- or 3H-disintegrations in DNA in three different ways shows 7–12 times more pronounced effects for¹²⁵I-decays. For¹²⁵I-labelled cells 3–4 DNA strand breaks were found per decay and the corresponding value for³H- labelled cells was 2.