Pharmacokinetics of danofloxacin and N-desmethyldanofloxacin in adult horses and their concentration in synovial fluid.

The objectives of this study were to investigate the pharmacokinetics of danofloxacin and its metabolite N-desmethyldanofloxacin and to determine their concentrations in synovial fluid after administration by the intravenous, intramuscular or intragastric routes. Six adult mares received danofloxacin mesylate administered intravenously (i.v.) or intramuscularly (i.m.) at a dose of 5 mg/kg, or intragastrically (IG) at a dose of 7.5 mg/kg using a randomized Latin square design. Concentrations of danofloxacin and N-desmethyldanofloxacin were measured by UPLC-MS/MS. After i.v. administration, danofloxacin had an apparent volume of distribution (mean ± SD) of 3.57 ± 0.26 L/kg, a systemic clearance of 357.6 ± 61.0 mL/h/kg, and an elimination half-life of 8.00 ± 0.48 h. Maximum plasma concentration (Cmax ) of N-desmethyldanofloxacin (0.151 ± 0.038 µg/mL) was achieved within 5 min of i.v. administration. Peak danofloxacin concentrations were significantly higher after i.m. (1.37 ± 0.13 µg/mL) than after IG administration (0.99 ± 0.1 µg/mL). Bioavailability was significantly higher after i.m. (100.0 ± 12.5%) than after IG (35.8 ± 8.5%) administration. Concentrations of danofloxacin in synovial fluid samples collected 1.5 h after administration were significantly higher after i.v. (1.02 ± 0.50 µg/mL) and i.m. (0.70 ± 0.35 µg/mL) than after IG (0.20 ± 0.12 µg/mL) administration. Monte Carlo simulations indicated that danofloxacin would be predicted to be effective against bacteria with a minimum inhibitory concentration (MIC) ≤0.25 µg/mL for i.v. and i.m. administration and 0.12 µg/mL for oral administration to maintain an area under the curve:MIC ratio ≥50.

Complications during catheter-mediated patent ductus arteriosus closure and pulmonary balloon valvuloplasty.

OBJECTIVES: To evaluate and illustrate complications of cardiac catheterisation and the associated risk factors of the most common cardiac intervention procedures. MATERIALS AND METHODS: Retrospective study of clinical records of client-owned dogs presented to a cardiology referral centre between January 2006 and December 2017. RESULTS: Five hundred and twenty-four dogs were included, 62 of which had complications. Complications were divided into technical complications and those due to unexpected additional anatomical abnormalities. Seven procedures (1.33%) were interrupted; five dogs (0.95%) subsequently underwent surgery, and four dogs died during the interventional procedure, indicating a mortality rate of 0.76% CLINICAL SIGNIFICANCE: There is a low risk of complications following closure of patent ductus arteriosus or pulmonary balloon valvuloplasty when carried out by a trained team using standardised procedures in a referral centre.

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells.

Non-homologous end joining (NHEJ) and homologous recombination (HR) are two pathways that can compete or cooperate for DNA double-strand break (DSB) repair. NHEJ was previously shown to act throughout the cell cycle whereas HR is restricted to late S/G2. Paradoxically, we show here that defect in XRCC4 (NHEJ) leads to over-stimulation of HR when cells were irradiated in G1, not in G2. However, XRCC4 defect did not modify the strict cell cycle regulation for HR (i.e. in S/G2) as attested by (i) the formation of Rad51 foci in late S/G2 whatever the XRCC4 status, and (ii) the fact that neither Rad51 foci nor HR (gene conversion plus single-strand annealing) events induced by ionizing radiation were detected when cells were maintained blocked in G1. Finally, both gamma-H2AX analysis and pulse field gel electrophoresis showed that following irradiation in G1, some DSBs reached S/G2 in NHEJ-defective cells. Taken together, our results show that when cells are defective in G1/S arrest, DSB produced in G1 and left unrepaired by XRCC4 can be processed by HR but in late S/G2.

Bax and Bid, two proapoptotic Bcl-2 family members, inhibit homologous recombination, independently of apoptosis regulation.

In order to analyse the relationships between regulation of apoptosis and homologous recombination (HR), we overexpressed proapoptotic Bax or only-BH3 Bid proteins or antiapoptotic Bcl-2 or Bcl-XL, in hamster CHO cells or in SV40-transformed human fibroblasts. We measured HR induced by gamma-rays, UVC or a specific double-strand cleavage targeted in the recombination substrate by the meganuclease I-SceI. We show here that the induction of both recombinant cells and recombinant colonies was impaired when expressing Bcl-2 family members, in hamster as well as in human cells. Moreover, the pro- as well as antiapoptotic Bcl-2 family members inhibited HR, independently of degradation of the RAD51 recombination protein and of their impact on apoptosis. These data reveal a mechanism of HR downregulation by potentially proapoptotic proteins, distinct from and parallel to degradation of recombination proteins, a situation that should also optimize the efficiency of programmed cell death.

Human 100-kDa homologous DNA-pairing protein is the splicing factor PSF and promotes DNA strand invasion.

Proteins promoting homologous pairing could be involved in various fundamental biological processes. Previously we detected two mammalian nuclear proteins of 100 and 75 kDa able to promote homologous DNA pairing. Here we report isolation and characterisation of the human (h) 100-kDa DNA-pairing protein, hPOMp100, from HeLa nuclei. The peptide sequences of hPOMp100 revealed identity to the human splicing factor PSF and a DNA-binding subunit of p100/p52 heterodimer of unknown function. Bacterially expressed PSF promotes DNA pairing identical to that of hPOMp100. hPOMp100/PSF binds not only RNA but also both single-stranded (ss) and double-stranded (ds) DNA and facilitates the renaturation of complementary ssDNAs. More important, the protein promotes the incorporation of a ss oligonucleotide into a homologous superhelical dsDNA, D-loop formation. A D-loop is the first heteroduplex DNA intermediate generated between recombining DNA molecules. Moreover, this reaction could be implicated in re-establishing stalled replication forks. Consistent with this hypothesis, DNA-pairing activity of hPOMp100/PSF is associated with cellular proliferation. Significantly, phosphorylation of hPOMp100/PSF by protein kinase C inhibits its binding to RNA but stimulates its binding to DNA and D-loop formation and may represent a regulatory mechanism to direct this multifunctional protein to DNA metabolic pathways.

Role of RAD51 in sister-chromatid exchanges in mammalian cells.

To measure the impact of the RAD51 pathway on Sister-Chromatid Exchanges (SCE), we used hamster cells expressing either the wild-type MmRAD51, which stimulates, or the dominant negative SMRAD51, which inhibits, gene conversion without affecting cell viability of untreated as well as gamma-rays irradiated cells. We show that MmRAD51 did not affect the rate of spontaneous SCE while it strongly stimulated spontaneous recombination between tandem repeats. No spontaneous recombinant was detected when expressing SMRAD51 while spontaneous SCE were only slightly diminished. After treatment by an alkylating agent (MNU), MmRAD51 stimulated MNU-induced recombination whereas no recombinant was detected when expressing SMRAD51. MNU induced SCE in all cell lines, even in the SMRAD51 expressing lines, but the induction of SCE was slightly more efficient in lines expressing MmRAD51 and less efficient in lines expressing SMRAD51. Thus, in mammalian cells, the RAD51-dependent gene conversion pathway drastically affects recombination between intrachromosomal tandem repeats, whereas it only partially participates in SCE formation, measured at a chromosomal level. These results show that RAD51-gene conversion can participate in induced SCE but that alternative pathways should exist.

Human POMp75 is identified as the pro-oncoprotein TLS/FUS: both POMp75 and POMp100 DNA homologous pairing activities are associated to cell proliferation.

We have previously developed an assay to measure DNA homologous pairing activities in crude extracts: The POM blot. In mammalian nuclear extracts, we detected two major DNA homologous pairing activities: POMp100 and POMp75. Here, we present the purification and identification of POMp75 as the pro-oncoprotein TLS/FUS. Because of the pro-oncogene status of TLS/FUS, we studied in addition, the relationships between cell proliferation and POM activities. We show that transformation of human fibroblasts by SV40 large T antigen results in a strong increase of both POMpl00 and TLS/POMp75 activities. Although detectable levels of both POMp100 and TLS/POMp75 are observed in non-immortalized fibroblasts or lymphocytes, fibroblasts at mid confluence or lymphocytes stimulated by phytohaemaglutinin, show higher levels of POM activities. Moreover, induction of differentiation of mouse F9 line by retinoic acid leads to the inhibition of both POMp100 and TLS/POMp75 activities. Comparison of POM activity of TLS/FUS with the amount of TLS protein detected by Western blot, suggests that the POM activity could be regulated by post-translation modification. Taken together, these results indicate that POMp100 and TLS/POMp75 activities are present in normal cells but are connected to cell proliferation. Possible relationship between cell proliferation, response to DNA damage and DNA homologous pairing activity of the pro-oncoprotein TLS/FUS are discussed.

Mutant p53 proteins stimulate spontaneous and radiation-induced intrachromosomal homologous recombination independently of the alteration of the transactivation activity and of the G1 checkpoint.

We report here a systematic analysis of the effects of different p53 mutations on both spontaneous and radiation-stimulated homologous recombination in mouse L cells. In order to monitor different recombination pathways, we used both direct and inverted repeat recombination substrates. In each line bearing one of these substrates, we expressed p53 proteins mutated at positions: 175, 248 or 273. p53 mutations leading to an increased spontaneous recombination rate also stimulate radiation-induced recombination. The effect on recombination may be partially related to the conformation of the p53 protein. Moreover, p53 mutations act on recombination between direct repeats as well as between inverted repeats indicating that strand invasion mechanisms are stimulated. Although all of the p53 mutations affect the p53 transactivation activity measured on the WAF1 and MDM2 gene promoters, no correlation between the transactivation activity and the extent of homologous recombination can be drawn. Finally, some p53 mutations do not affect the G1 arrest after radiation but stimulate radiation-induced recombination. These results show that the role of p53 on transactivation and G1 cell cycle checkpoint is separable from its involvement in homologous recombination. A direct participation of p53 in the recombination mechanism itself is discussed.

Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein.

Homologous recombination plays an essential role in processes involved in genome stability/instability, such as molecular evolution, gene diversification, meiotic chromosome segregation, DNA repair and chromosomal rearrangements. p53 devoid cells exhibit predisposition to neoplasia, defects in G1 checkpoint and high genetic instability but a normal rate of point mutations. We investigated the effect of a p53 mutation, on spontaneous homologous recombination between intrachromosomal direct repeat sequences, in mouse L cells. In these cells, wild type for the p53 gene, we have overexpressed the mutant p53(175(Arg>His)) protein leading to a p53 mutant phenotype, as verified by the absence of a G1 arrest after gamma-irradiation. We show that the rate of spontaneous recombination is increased from five- to 20-fold in the mutant p53 lines. Moreover, this increase is observed in gene conversion as well as in deletion events. Our results provide new insights into the molecular mechanisms of genetic instability due to a defect of p53.

Characterization of Reca Mediated homologous pairing on nitrocellulose membrane.

Reactions between a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) provide an efficient model to study RecA promoted homologous recombination. We have devised an assay in which the ssDNA is first bound to a nitrocellulose membrane. RecA protein is loaded on this membrane (loading step) which is then incubated with a labelled homologous dsDNA (incubation step). Since this assay can be used for study of mutant RecA proteins or RecA-like activities in crude extracts from other organisms, we have characterized the reaction promoted on the membrane. Under these new conditions, the reaction keeps the main characteristics observed with classical assays performed in solution: increasing NaCl concentration destabilized the RecA-DNA complex, ATP gamma S was required for formation of stable RecA-DNA complex, initiation of the reaction exhibits the same polarity as in classical assays, a complete strand exchange with a 44 bp long duplex oligonucleotide has been recorded under our conditions. Moreover, our results indicate that the binding of RecA protein itself to the nitrocellulose membrane did not impair its ability to promote homologous pairing. Pairing reactions involving long dsDNA (6407 bp) were more efficient with hydrolysable ATP than with ATP gamma S only when the ssDNA was bound to the membrane. Furthermore, ATP hydrolysis was not required when using short dsDNA (44 bp). These results constitute experimental support for a new role for the ATPase activity of RecA protein: the energy produced could favor the initiation of RecA mediated recombination involving long stretches of DNA which have restricted freedom to rotation.

Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences.

In all the organisms, homologous recombination (HR) is involved in fundamental processes such as genome diversification and DNA repair. Several strategies can be devised to measure homologous recombination in mammalian cells. We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination. The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells. The possibility to use two different orientations of tandem repeats (direct or inverted repeats) in parallel constitutes also an advantage. While inverted repeats measure only events arising by strand exchange (gene conversion and crossing over), direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage. In yeast, these processes depend on different pathways, most of them also existing in mammalian cells. These data permit to devise substrates adapted to specific questions about HR in mammalian cells. The effect of substrate structures (heterologies, insertions/deletions, GT repeats, transcription) and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease (especially the rare-cutting endonuclease ISce-I) on HR are discussed. Finally, transgenic mouse models using tandem repeats are briefly presented.

[Double strand break repair, one mechanism can hide another: alternative non-homologous end joining]. / Réparation des cassures double-brin de l'ADN, un mécanisme peut en cacher un autre : la ligature d'extrémités non homologues alternative.

DNA double strand breaks are major cytotoxic lesions encountered by the cells. They can be induced by ionizing radiation or endogenous stress and can lead to genetic instability. Two mechanisms compete for the repair of DNA double strand breaks: homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires DNA sequences homology and is initiated by single strand resection. Recently, advances have been made concerning the major steps and proteins involved in resection. NHEJ, in contrast, does not require sequence homology. The existence of a DNA double strand break repair mechanism, independent of KU and ligase IV, the key proteins of the canonical non homologous end joining pathway, has been revealed lately and named alternative non homologous end joining. The hallmarks of this highly mutagenic pathway are deletions at repair junctions and frequent use of distal microhomologies. This mechanism is also initiated by a single strand resection of the break. The aim of this review is firstly to present recent data on single strand resection, and secondly the alternative NHEJ pathway, including a discussion on the fidelity of NHEJ. Based on current knowledge, canonical NHEJ does not appear as an intrinsically mutagenic mechanism, but in contrast, as a conservative one. The structure of broken DNA ends actually dictates the quality repair of the alternative NHEJ and seems the actual responsible for the mutagenesis attributed beforehand to the canonical NHEJ. The existence of this novel DNA double strand breaks repair mechanism needs to be taken into account in the development of radiosensitizing strategies in order to optimise the efficiency of radiotherapy.

S-phase progression stimulates both the mutagenic KU-independent pathway and mutagenic processing of KU-dependent intermediates, for nonhomologous end joining.

We used intrachromosomal substrates to directly monitor the effect of the cell cycle on the efficiency and the accuracy of nonhomologous end joining (NHEJ) in mammalian cells. We show that both KU and KU-independent (KU-alt) pathways are efficient when maintaining cells in G1/S, in G2/M or during dynamic progression through S phase. In addition, the accuracy of NHEJ is barely altered when the cells are blocked in G1/S or in G2/M. However, progression through S phase increases the frequency of deletions, which is a hallmark of the KU-alt pathway. Moreover, we show that the intermediates that are generated by the KU-dependent end joining of non-fully complementary ends, and which contain mismatches, nicks or gap intermediates, are less accurately processed when the cells progress through S phase. In conclusion, both KU and KU-alt processes are active throughout the cell cycle, but the repair is more error prone during S phase, both by increasing the mutagenic KU-alt pathway and decreasing the accuracy of the repair of the intermediates generated by the KU-dependent pathway.

Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms.

In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SCE:I cleavage site. We characterized conservative recombination after I-SCE:I cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two dominant-negative RAD51 forms that specifically inhibit conservative recombination: the yeast ScRAD51 or the yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates conservative recombination. None of these RAD51 forms affects non-conservative recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD51 prevents radiation-induced recombination. This suggests that mRAD51 does not significantly affect the global DSB repair efficiency but controls the classes of recombination events. Finally, both ScRAD51 and SMRAD51 drastically inhibit spontaneous recombination but not cell proliferation, showing that RAD51-dependent spontaneous and DSB-induced conservative recombination can be impaired significantly without affecting cell viability.

A novel role for the Bcl-2 protein family: specific suppression of the RAD51 recombination pathway.

The oncogenic role of Bcl-2 is generally attributed to its protective effect against apoptosis. Here, we show a novel role for Bcl-2: the specific inhibition of the conservative RAD51 recombination pathway. Bcl-2 or Bcl-X(L) overexpression inhibits UV-C-, gamma-ray- or mutant p53-induced homologous recombination (HR). Moreover, Bcl-2 recombination inhibition is independent of the role of p53 in G1 arrest. At an acute double-strand break in the recombination substrate, Bcl-2 specifically inhibits RAD51-dependent gene conversion without affecting non-conservative recombination. Bcl-2 consistently thwarts recombination stimulated by RAD51 overexpression and alters Rad51 protein by post-translation modification. Moreover, a mutant (G145A)Bcl-2, which is defective in Bax interaction and in apoptosis repression, also inhibits recombination, showing that the death and recombination repression functions of Bcl-2 are separable. Inhibition of error-free repair pathways by Bcl-2 results in elevated frequencies of mutagenesis. The Bcl-2 gene therefore combines two separable cancer-prone phenotypes: apoptosis repression and a genetic instability/mutator phenotype. This dual phenotype could represent a mammalian version of the bacterial SOS repair system.

Homologous pairing between single-stranded DNA immobilized on a nitrocellulose membrane and duplex DNA is specific for RecA activity in bacterial crude extract.

Reaction between a circular single stranded and a linear double stranded DNA molecule (ssDNA and dsDNA) provides an efficient system to study recombination mediated by RecA protein. However, classical assays using reaction in solution require highly purified enzymes. This limits biochemical studies of mutant RecA proteins from Escherichia coli or of RecA proteins from other organisms. We describe here an assay that is specific for RecA activity even in bacterial crude extracts. In this assay, the ssDNA is bound to a nitrocellulose membrane, proteins are loaded on this membrane and it is then incubated with a labeled homologous dsDNA. Joint molecules are visualized by autoradiography. We have shown that, despite the reduced mobility of the DNA due to its binding to the membrane, RecA protein is able to promote formation of stable plectonemic joints, in a homology dependent manner. Fourteen other proteins involved in DNA metabolism were checked and did not produce a signal in our assay. Moreover, in Dot blot analysis as well as after native electrophoresis and electrotransfer on a ssDNA coated membrane, production of a signal was strictly dependent on the presence of active RecA protein in the bacterial crude extracts used. We named this assay Pairing On Membrane blot (POM blot).

Directional recombination is initiated at a double strand break in human nuclear extracts.

The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast.

Characterization of two nuclear mammalian homologous DNA-pairing activities that do not require associated exonuclease activity.

We have developed an assay to study homologous DNA-pairing activities in mammalian nuclear extracts. This assay is derived from the POM blot assay, described earlier, which was specific for RecA activity in bacterial crude extracts. In the present work, proteins from mammalian nuclear extracts were resolved by electrophoresis on SDS/polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane coated with circular single-stranded DNA (ssDNA). The blot obtained was incubated with a labeled homologous double-stranded DNA (dsDNA). Homologous pairing between the ssDNA and the labeled dsDNA was detected by autoradiography as a radioactive spot on the membrane. In nuclear extracts from mammalian cells, we found two major polypeptides of 100 and 75 kDa, able to promote the formation of stable plectonemic joints. Joint molecule formation required at least one homologous end on the dsDNA, but either end of the dsDNA could be recruited to initiate the reaction. For each polypeptide, the reaction required divalent cations such as Mg2+, Ca2+, or Mn2+. Although ATP was not necessary, ADP was inhibitory in each case. Unlike most of the known eukaryotic DNA-pairing proteins, both activities identified here were able to promote the formation of joint molecules without requiring an associated exonuclease activity. In addition, these two proteins were detected in cell lines from different tissues and from different mammalian species (human, mouse, and hamster).

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts.

The effect of the structure of donor DNA molecules on the initiation of recombination for double strand break repair in human nuclear extracts, was investigated here. A unique double strand break was introduced into M13 duplex derivatives by digestion with restriction enzymes. After coincubation of the cleaved DNA in human nuclear extracts, with a plasmid containing M13 sequences spanning the break, double strand break repair was estimated by the plating efficiency in JM109 (RecA1) bacteria. We first confirm that a short heterologous insert (8bp) close to the break on the recipient cleaved M13 DNA inhibits recombination with circular as well as with linear donor molecules. The results indicate that, with these substrates, recombination is initiated at the level of the break, requires uninterrupted homology on both sides of the break, and is associated with a decreasing gradient of gene conversion. When the heterologous insertion is located on the plasmid donor DNA, similar results are obtained with a circular donor DNA. In contrast, with a linear donor molecule, bearing the insert, homology requirements, in the region of the break in M13 DNA, are abolished. This last result suggests that recombination could be initiated at the extremities of the linear donor DNA.

Determining optimal surface roughness of TiO(2) blasted titanium implant material for attachment, proliferation and differentiation of cells derived from human mandibular alveolar bone.

In the complex process of bone formation at the implant-tissue interface, implant surface roughness is an important factor modulating osteoblastic function. In this study, primary cultures of osteoblast-like cells, derived from human mandibular bone, were used. The aim was to examine the effect of varying surface roughness of titanium implant material on cellular attachment, proliferation and differentiation. A recognized method of increasing surface roughness and enlarging the surface area of titanium implants is by blasting with titanium dioxide particles: the four specimen types in the study comprised surfaces which were machine-turned only, or blasted after turning, with 63-90 microm, 106-180 microm, or 180-300 microm TiO(2) particles, respectively. The specimens were analyzed by scanning electron microscopy and confocal laser scanning. The turned samples had the smoothest surfaces: average height deviation (S(a)) of 0.20 microm. The roughest were those blasted with 180-300 microm particles, S(a) value 1.38 microm. Blasting with intermediate particle sizes yielded S(a) values of 0.72 microm and 1.30 microm, respectively. Cell profile areas were measured using a semiautomatic interactive image analyzer. Figures were expressed as percentage of attachment. DNA synthesis was estimated by measuring the amount of [(3)H]-thymidine incorporation into trichloroacetic acid (TCA) insoluble cell precipitates. The specific activity of alkaline phosphatase was assayed using p-nitrophenylphosphate as a substrate. The ability of the cells to synthesize osteocalcin was investigated in serum-free culture medium using the ELSA-OST-NAT immunoradiometric kit. After 3 h of culture, the percentage of cellular attachment did not differ significantly between specimens blasted with 180-300 micromparticles and the turned specimens. All blasted surfaces showed significantly higher [(3)H]-thymidine incorporation than the turned surfaces (P<0.05), with the highest on the surfaces blasted with 180-300 microm particles. Osteocalcin synthesis by the cells in response to stimulation by 1,25(OH)2D3, was also significantly greater (P<0.05) on the surfaces blasted with TiO(2) particles. However, analysis of alkaline phosphatase activity disclosed no significant differences among the four surface modifications. It is concluded that in this cellular model, the proliferation and differentiation of cells derived from human mandibular bone is enhanced by surface roughness of the titanium implant. However, increasing the size of the blasting particles to 300 microm does not further increase the initial attachment of the cells compared to turned surfaces and those blasted with 63-90 microm particles.