Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c.

The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution. This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair. The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme. The crystal structure at 2.8 A of a complex between yeast cytochrome c peroxidase and horse heart cytochrome c was also determined. Although crystals of the two complexes (one with cytochrome c from yeast and the other with cytochrome c from horse) grew under very different conditions and belong to different space groups, the two complex structures are closely similar, suggesting that cytochrome c interacts with its redox partners in a highly specific manner.

Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP.

Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.

Mechanisms of resistance of confluent human and rat colon cancer cells to anthracyclines: alteration of drug passive diffusion.

Two colon cancer cell lines, HT-29 (human) and DHD/K12/TRb (rat), were grown as monolayer cultures to various confluence degrees. The cytotoxic efficacies of doxorubicin and 4'-deoxydoxorubicin, evaluated by a survival assay, and the nuclear drug concentrations, measured by microspectrofluorometry, were shown to progressively decrease with the augmentation of confluence. This confluence dependent resistance (CDR) to anthracyclines was demonstrated independent of the multidrug resistance drug efflux mechanism. The cellular uptake of three compounds (sodium [51Cr]chromate, D-[14C]alanine, L-[14C]glucose) known to passively diffuse across the cell membrane as anthracyclines do was also reduced in confluent cells. After trypsin cell detachment, the kinetics of reversion of the sodium [51Cr]chromate uptake decrease and that of CDR were similar. Therefore, CDR may be attributed to a reduction of anthracycline cell intake due to a general alteration of passive diffusion across the cell membrane. However, CDR is only partly explained by this phenomenon since a reduced sensitivity of confluent cells was observed compared with nonconfluent cells for a similar amount of drug in their nuclei. CDR could explain the high resistance to anthracyclines of some solid tumors, such as colon tumors, in which cancer cells are tightly aggregated.

Pullout characteristics of percutaneous pedicle screws with different cement augmentation methods in elderly spines: An in vitro biomechanical study.

BACKGROUND: Vertebroplasty prefilling or fenestrated pedicle screw augmentation can be used to enhance pullout resistance in elderly patients. It is not clear which method offers the most reliable fixation strength if axial pullout and a bending moment is applied. The purpose of this study is to validate a new in vitro model aimed to reproduce a cut out mechanism of lumbar pedicle screws, to compare fixation strength in elderly spines with different cement augmentation techniques and to analyze factors that might influence the failure pattern. MATERIALS AND METHODS: Six human specimens (82-100 years) were instrumented percutaneously at L2, L3 and L4 by non-augmented screws, vertebroplasty augmentation and fenestrated screws. Cement distribution (2 ml PMMA) was analyzed on CT. Vertebral endplates and the rod were oriented at 45° to the horizontal plane. The vertebral body was held by resin in a cylinder, linked to an unconstrained pivot, on which traction (10 N/s) was applied until rupture. Load-displacement curves were compared to simultaneous video recordings. RESULTS: Median pullout forces were 488.5 N (195-500) for non-augmented screws, 643.5 N (270-1050) for vertebroplasty augmentation and 943.5 N (750-1084) for fenestrated screws. Cement augmentation through fenestrated screws led to significantly higher rupture forces compared to non-augmented screws (P=0.0039). The pullout force after vertebroplasty was variable and linked to cement distribution. A cement bolus around the distal screw tip led to pullout forces similar to non-augmented screws. A proximal cement bolus, as it was observed in fenestrated screws, led to higher pullout resistance. This cement distribution led to vertebral body fractures prior to screw pullout. CONCLUSION: The experimental setup tended to reproduce a pullout mechanism observed on radiographs, combining axial pullout and a bending moment. Cement augmentation with fenestrated screws increased pullout resistance significantly, whereas the fixation strength with the vertebroplasty prefilling method was linked to the cement distribution.

Crystallization of fragment D from human fibrinogen.

Fragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide-linked chains (alpha' beta' gamma' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 A; the space group is P2(1), unit cell dimensions a = 108 A, b = 48 A, c = 167 A, beta = 106 degrees. Fragment D was also co-crystallized with the ligand GPRP-amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 A, b = 82 A, c = 432 A.

Effects of cyclosporin A on progressive and regressive tumors induced by two cancer lines derived from a single colon carcinoma chemically induced in the rat.

The effects of cyclosporin-induced immunosuppression were assessed in a rat model of progressive and regressive colonic tumors. Two cloned cell variants, obtained from the same chemically induced colonic carcinoma, differ in their capacity to grow when injected into the syngeneic rat. PROb cells yield progressive tumors and often metastases; in contrast, REGb cells produce tumors which regress in 3 to 6 weeks. Cyclosporin A (CsA) administered daily, 20 mg/kg subcutaneously (s.c.) for 30 days after tumor cell inoculation, drastically enhanced the local growth of PROb tumors and increased the number of metastases. It increased the local growth and prevented the regression of REGb tumors which persisted even as long as 8 weeks after the termination of CsA administration and occasionally yielded metastases. CsA prevented the accumulation of inflammatory cells with the T lymphocyte phenotype at the periphery of both PROb and REGb tumors but did not alter the tumor infiltration by macrophages and NK cells. CsA did not modify the natural cytotoxicity of peripheral blood mononuclear cells against PROb and REGb target cells. These results suggest that CsA-induced suppression of T lymphocyte activity may enhance tumor progression and suppress tumor regression in this model.

Cytotoxic activity of lymphocytes infiltrating progressive and regressive tumor variants from a rat colonic cancer.

Two tumor cell variants (PROb and REGb) isolated from a single chemically-induced rat colon adenocarcinoma were previously shown to differ in their tumorigenicity. When injected into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells give rise to tumors which always regress. PROb and REGb variants also differ in their capacity to induce an immune response in the syngeneic host. Regression of REGb tumors could have been mediated by cytotoxic effector cells acting at the tumor site. To test this hypothesis, the cytolytic activity of non-adherent lymphoid cells isolated from PROb and REGb tumors and from the spleen of the same animals were compared. The same number of infiltrating lymphocytes was recovered per gram of PROb or REGb tumor. The cytolytic activity of tumor infiltrating lymphocytes, as that of spleen lymphocytes, was predominantly non specific, as demonstrated by their ability to kill YAC-1 cells, an NK-sensitive cell line. PROb cells were relatively resistant to the cytotoxic activity of spleen or tumor infiltrating lymphocytes. In the regressing REGb tumors, the cytotoxic activity of tumor infiltrating lymphocytes to homologous cells or to YAC-1 cells was low and significantly inferior to that of the corresponding spleen lymphocytes. These results suggest that the cytotoxic activity of lymphocytes was impaired at the local, intratumoral level, even in spontaneously regressing tumors.

In vitro natural killer activity against progressive and regressive variants of a rat colon adenocarcinoma. Effect of treatments with anti-asialo GM1 plus complement.

In a previous work, a cell line (DHD/K12) was established from a colon adenocarcinoma induced in a BDIX rat by 1,2-dimethylhydrazine. From this line, two cloned sublines, PROb and REGb, were then isolated. When subcutaneously inoculated into syngeneic rats, PROb cells yield progressive tumors, whereas REGb cells yield tumors which regress. In this study, in a 16-h 51Cr release assay, natural cytotoxicity mediated by BDIX splenic nonadherent lymphoid cells (NK cells) was shown to be much higher against REGb cells than against PROb cells. Whatever the target cells, NK cytotoxicity was always higher when the effector cells were obtained from males rather than from females. Treatment of BDIX splenic lymphocytes by anti-asGM1 serum plus complement revealed that both anti-asGM1 sensitive and non-sensitive NK cells exist. The activity of anti-asGM1 non-sensitive NK cells appeared to be minor and to be detected only when the level of cytotoxicity before treatment was sufficiently high. The difference between PROb and REGb tumor growth appears to be linked, at least in part, to a higher sensitivity of REGb cells to NK cells and especially to anti-asGM1-sensitive NK cells.

In vivo and in vitro effects of fish-containing diets on colon tumour cells in rats.

Polyunsaturated n-3 fatty acids, abundant in sea fish, can inhibit the growth of chemoinduced or transplanted mammary tumours in the rat. Since mammary and colonic cancers have both been linked to a high fat consumption, we studied the effect of 2 diets moderately (7% fish meal) or strongly (9% fish oil) enriched in fish fatty acids on the growth of colon cancer cells subcutaneously inoculated into syngeneic rats. The diets had no effect on the in vivo tumor growth and on the in vitro tumouricidal activity of peritoneal macrophages or splenic lymphocytes.

Involvement of effector cells in the treatment with endotoxins of peritoneal carcinomatosis induced by colon tumour cells.

Peritoneal carcinomatoses, an ordinary way for human colon carcinoma to spread, are incurable. When rat peritoneal carcinomatoses of colon origin were treated with endotoxins i.p. administered 3 days after the tumour cell injection, 70% of the BDIX rats were alive 30 weeks after the PROb tumour cell injection whereas all the untreated rats died of their tumour within 14 weeks. The study of the effector cells involved in the antitumour effect of endotoxins suggests that T lymphocytes are required for this effect. The endotoxin effect is local and is not reflected by the cytolytic activity of peritoneal cells.

Serum concentrations of amiodarone required for an in vivo modulation of anthracycline resistance.

We demonstrated previously that amiodarone is able to circumvent in vitro the inherent resistance to anthracyclines of the DHD/K12 rat colon cancer cell line. We have now determined in the rat the amiodarone seric concentrations required to enhance the in vitro cytotoxicity of 4'-deoxydoxorubicin (deoDX) against DHD/K12 cells. A maximal deoDX potentiation was obtained in vitro when anthracycline was diluted in the serum of rats receiving at least 75 mg/kg of intravenous amiodarone resulting in seric concentrations of more than 40 micrograms/ml. In patients treated with amiodarone, the mean serum concentrations were 0.9 +/- 0.1 microgram/ml after an one month's oral administration of 200 mg/day, 2.2 +/- 1.0 micrograms/ml after a 24 hr continuous infusion of 300 to 900 mg/day and 5.4 +/- 1.1 micrograms/ml after a brief 3 hrs infusion of 450 mg amiodarone. Such amiodarone concentrations in human serum are much lower than those necessary to produce a significant anthracycline potentiation. In rats receiving amiodarone at a maximal tolerated dose (100 mg/kg) minutes before the injection of 10 mg/kg doxorubicin (DX), we observed an increased accumulation of the anthracycline in the liver and kidney compared to rats receiving DX alone. The DX content was not modified by amiodarone in the other organs studied (heart, lung, spleen and pancreas). An amiodarone pretreatment accelerated the death of rats receiving 5 or 10 mg/kg DX did not provoke lethality for a lower dose of 2.5 mg/kg DX. The very high doses required and the risk of increased toxicity seem to preclude the use of amiodarone for the modulation of anthracycline resistance in cancer patients.

Use of a specific monoclonal antibody for studying the liver metastatic invasion of a rat colon cancer.

A simple model for liver metastasis from colon cancer resulted from the intraportal injection of 2 x 10(7) highly tumorigenic DHD/K12/PROb cells into syngeneic BDIX rats. Early detection and development of cancer invasion were studied by conventional microscopy and immunoenzymatic staining using a specific monoclonal antibody. Metastases developed either from isolated cancer cells early disseminated in sinusoid network or from intraportal microthrombi. An intense immune reaction developed until day 15 after injection but decreased and disappeared at the latest stages of evolution.

Cariogenic feeding habits and fluoride supplementation during infancy and early childhood.

Parents of 240 children between nine months and three years of age were interviewed using a questionnaire in order to determine cariogenic feeding habits and fluoride supplementation. Mean age of weaning from the bottle was 14.6 months. After 18 months of age, children from minority ethnic groups were more frequently bottle-fed than French-Canadian children (p < .005). Giving a bottle in bed (34.6% of cases) was more often practised by less educated mothers (p = .007) or by minority ethnic groups (p = .002), and was seen as a cariogenic factor by 31% of parents. Fluoride was given in half of cases, mainly by highly educated mothers (p = .001) and was mentioned as a preventive measure by 27% of parents. Physicians should be aware of poor parental knowledge and practices of preventive dentistry, and must discuss cariogenic feeding habits and fluoride supplementation during well-baby visits.

Surface roughness and morphology of three nanocomposites after two different polishing treatments by a multitechnique approach.

OBJECTIVES: The purpose of this study was to assess the surface roughness and morphology of three nanocomposites polished with two different polishing systems. METHODS: Specimens made of hybrid composite (Tetric Ceram [TC] as control) and nanocomposites: nanofilled (Filtek Supreme [FS]), nanofilled hybrid (Grandio [Gr]), complex nanofilled hybrid (Synergy D6 [Syn]) were polished with CompoSystem [CS] or Sof-Lex [SL] polishing discs. The average surface roughness (Ra) before and after polishing was measured using optical profilometry. Both AFM and SEM techniques were additionally used to analyze the surface morphology after polishing with the aim of relating the surface morphology and the surface roughness. Statistical analysis was done by ANOVA using a general linear model (alpha=0.05) with an adjustment for multiple comparisons. RESULTS: Within the same polishing system, FS exhibited the smoothest surface, followed by Syn, TC and Gr (p<0.0001). Sof-Lex polishing discs produced the smoothest surface compared to CompoSystem (p<0.0001). AFM and SEM observations confirmed that the surface roughness was related to the surface morphology and to the average filler size. SIGNIFICANCE: Positive correlation between the average filler size and the surface roughness suggest that using nanoparticles in the formulation does not necessary improve the surface texture. The nanofilled composite FS, which contains only nanofillers, showed the best results when associated to Sof-Lex polishing discs.

Specificity of the immune response leading to protection or enhancement by regressive and progressive variants of a rat colon carcinoma.

Two cell clones, K12/TRb (PROb) and K12/TSb (REGb), have been isolated from the same serially transplantable tumor, DHD, established from a colon carcinoma chemically induced in the rat. Inoculation of REGb cells gives a tumor which regresses within 4 to 8 weeks and generates immune protection against subsequent injection of the progressive tumor cells, PROb. Inoculation of PROb cells gives a progressive tumor and generates tolerance allowing progressive growth of contralaterally injected REGb cells. Inoculation of REGb cells fully protects the host against growth of a DHD tumor graft, the tumor from which REGb and PROb cells were originally obtained. On the other hand, inoculation of REGb cells does not confer any protection against growth of 4 other syngeneic tumor grafts, DHA, DHB, DHC and DHE. These tumors were obtained from other colonic tumors induced as DHD by 1.2 dimethylhydrazine (DMH). Progressive growth of the tumor induced by inoculation of REGb cells is observed in animals bearing a contralateral DHD tumor, but not in animals bearing tumor from other transplantable lines, DHA, DHB, DHC and DHE. Our results show that immune enhancement of a regressive tumor and the immune protection that it confers constitute specific responses to a tumor-specific transplantation antigen present on a single transplantable colon tumor.

Characterization of the metal ion binding helix-hairpin-helix motifs in human DNA polymerase beta by X-ray structural analysis.

X-ray crystallographic studies have shown that DNA binding by human polymerase beta (pol beta) occurs primarily through two structurally and sequentially homologous helix-hairpin-helix (HhH) motifs, one in the fingers subdomain and the other in the 8-kDa domain [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996a) Biochemistry 35, 12742-12761]. In that DNA binding by each HhH motif is facilitated by a metal ion, we set out to determine the identity of the metal ion that most likely binds to the HhH motif in vivo. Crystal soaking experiments were performed on human pol beta-DNA cocrystals with Mg2+, Ca2+, Na+, and K+, the four most prevalent metal ions in the cell, and in each case a data set was collected and the resulting structure was refined. Under the conditions tested, the HhH motifs of pol beta have an affinity for these biologically prevalent metal ions in the order Mg2+ < Ca2+ < Na+ < K+, with K+ displaying the strongest binding. Crystals soaked in the presence of Tl+, a commonly used spectroscopic probe for K+, were too X-ray-sensitive to establish the binding behavior of Tl+, but soaking experiments with Ba2+ and Cs+ resulted in relatively stable crystals that gave evidence of metal ion binding in both HhH motifs, confirming that larger monovalent and divalent metal ions are capable of binding to the HhH metal sites. Although Mn2+, which has been categorized as a potent polymerase mutagen, binds to the HhH motifs with a greater affinity than Mg2+, Mn2+ does not bind to the HhH motifs in the presence of equimolar concentrations of Na+. These results suggest that in vivo, where Mn2+ is present only in trace amounts, Mn2+ probably does not have a large effect on DNA binding and may instead manifest a mutagenic effect on pol beta primarily by distorting nucleotide binding or by directly affecting the catalytic step [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996b) Biochemistry 35, 12762-12777]. Crystal soaking experiments with 31-kDa apoenzyme crystals show that, in the absence of DNA, the HhH motif in the fingers subdomain binds metal ions with either much lower occupancy or not at all, indicating that metal ion binding is dependent on the presence of the DNA substrate.

Tooth thickness at the furcation entrance of lower molars.

Better understanding of the furcation anatomy may serve to decrease the risk of pulpal injury during rotary odontoplasty, a procedure often used in conjunction with guided tissue regeneration. The purpose of this study was to determine (i) the tooth thickness about the furcation entrance of lower molars, and (ii) whether there is a relationship between tooth thickness and patient age. 40 mandibular 1st molars (M1) (mean age = 36.2; range 10-65 years) and 40 mandibular 2nd molars (M2) (mean age = 37.9; range 14-70 years) were collected. Age, gender and furcation involvement (if any) were noted for each tooth at the time of extraction. Teeth were sectioned in half, buccal-lingual, at the furcation entrance with a rotary diamond blade. A standardized linear reference scale was placed on each experimental section and an 8 x 10 in. photograph generated. The distance from the floor of the pulp chamber to 5 predetermined sites on the root surface was calculated. The data were expressed as (a) the mean of each site and (b) the mean of each tooth (the average of the 5 points of each tooth). Analysis of covariance failed to show a relationship between thickness measurements and gender or furcation involvement. Thus, the data was subjected to simple regression analysis to determine the relationship of age with tooth and cementum thickness. This study revealed that by site, the mean measurements ranged from 2.7-3.0 mm for both M1 and M2. The single least/greatest measurements of the 5 sites were for M1: 1.6/4.7 mm and for M2: 1.8/4.2 mm. By tooth, the average distance from the pulp to the root surface was 2.83 mm (+/- 0.49) for M1 and 2.88 mm (+/- 0.44) for M2. Regression analysis of tooth thickness with age was significant for M1 only. The maximum slope of the 5 sites was approximately 0.3 mm/10 years. No relationship was found between cementum thickness and age for either tooth group. The results of this study indicate that the majority of times the pulp is 1.6-4.2 mm from the root surface in the vicinity of the furcation entrance of lower 1st and 2nd molars. Although tooth thickness in this area may increase with age, the amount is not enough to forego judicious odontoplasty on older patients.

Potential usefulness of quinine to circumvent the anthracycline resistance in clinical practice.

Quinine, the widely used antimalaria agent, was found to increase the cytotoxicity of epideoxorubicin (epiDXR) in resistant DHD/K12 rat colon cancer cells in vitro. Quinine appeared as slightly less effective than quinidine or verapamil for anthracycline potentiation but its weaker cardiotoxicity could counterbalance this disadvantage in vivo. Serum from six patients treated by conventional doses of quinine (25-30 mg kg-1 day-1) was demonstrated to enhance the accumulation of epiDXR in DHD/K12 cells as judged by fluorescence microscopy and HPLC assay (1.6 to 6-fold compared with control serum). In this patients quinine concentrations in serum ranged from 4.4 to 10.1 micrograms ml-1. Our results suggest that quinine could be safely used as anthracycline resistance modifier in clinical practice.