[Long-term radiologic evolution of coral implanted in cancellous bone of the lower limb. Madreporic coral versus coral hydroxyapatite]. / Evolution radiologique à long terme du corail implanté en os spongieux au membre inférieur. Corail madréporique versus hydroxyapatite de corail.

PURPOSE: The study aimed to compare two successive series of procedure using first pieces of natural madreporic coral, then coralline hydroxyapatite in traumatology. The goal of this work was to evaluate long term radiological features of absorption and influencing factors. MATERIAL: Within six years, 65 pieces were used; only impaction articular fracture on the lateral tibial plateau (31) and calcaneum fractures (23) were included 21 cases completed inclusion criteria. As there were 3 secondary infections, 18 had therefore, an aseptic evolution and were included in this series. METHODS: Absorption was evaluated in five degrees according to the volume of the remaining piece on X-rays. We also took into consideration the position of the pieces and the amount of surfaces in contact with cancellous bone. RESULTS: Among the 18 pieces followed up for more than 19 months (average 40), there were 4 sequestrations (1 tibia, 3 calcaneum), all were natural madreporic coral, the others presented osteointegration. Only natural madreporic coral pieces were absorbed, but never completely and the absorption was slow (3 to 7 years) and never complete. A radiolucent line was present in 7 out of 10 pieces of madreporic coral and none around coralline hydroxyapatite. Three developed favorably. Four were sequestred. Number of surfaces in contact with cancellous bone was correlated with osteointegration. DISCUSSION: The calcium carbonate which makes up the natural madreporic coral pieces is the site of destruction by osteoclasts. The radiolucent line is the specific witness of this biochemical activity. The radiolucent line proceeds sequestrations. No substitute of coralline hydroxyapatite has presented the same sign of peripheral absorption or sequestration. Coralline hydroxyapatite could be the correct coralline material to fill cancellous defect after elevation of an articular depression. In every case, the piece of coral must be fitted in contact with the cancellous bone in the depth of the bone. Conditions of osteoconduction of madreporic coral must be reconsidered. The degree and speed of peripheral absorption must be controlled before weight bearing is allowed.

[Conditions for the consolidation of fractures of the femoral diaphysis after locked intramedullary flexible osteosynthesis]. / Conditions de consolidation des fractures de la diaphyse fémorale après ostéosynthèse centromédullaire flexible verrouillée.

PURPOSE OF THE STUDY: We present here the clinical results achieved with locked intramedullary flexible osteosynthesis (LIFO) system in femoral fractures. The two major advantages of the device are its flexibility an ease of use. MATERIAL AND METHODS: Over a period of 6 years, 42 patients were treated. Two died and 7 were lost for follow-up, leaving 33 cases available for evaluation. Fourteen cases were specifically analysed for callus morphological characteristics: volume, length and width, and compared to 18 cases from a previous series involving 27 intramedullary unmatched appaired nailings. RESULTS: Results in the 33 patients of this series are: no nonunion, 1 osteomyelitis in an open fracture, 4 malunions (2 primary iatrogenic malunions, 2 secondary cases due to a weak fixation). Fractures united at a mean delay of 10 weeks, that is, 4 weeks less than with a nail. Morphological callus study also showed that the LIFO system generates about 25 per cent longer of peripheral new bone formation than a nail. CONCLUSION: A LIFO construct with 3 or even 4 pins yields a shorter healing time than a nail. Furthermore, it generates a callus of 25 per cent longer around the fracture. The four pins construct was found to be as dependable as a locked nail, and minimizes inventory. This technique is indicated in the treatment of all common femoral shaft fractures in adults.

[Long-term osseous changes in the posterior arch after laminectomy for lumbar stenosis]. / Les remaniements à long-terme de l'arc postérieur restant après laminectomie pour sténose lombaire.

PURPOSE OF THE STUDY: Many studies have indicated favorable results of decompressive surgery for symptomatic lumbar spinal stenosis. However, little is known about the osseous changes that occur at the operative sites. Postacchini in 1992 and Chen in 1994 have studied, only from plain radiographs, osseous changes at the operative sites, and have suggested that bone regrowth possibly affects the neurologic result. The aims of this study were: to assess bone regrowth at the operative site, to compare the bone regrowth rate calculated from plain radiographs and CT-Scan examinations, to determine the effects of bone regrowth on clinical outcome, to investigate the factors promoting the bone regrowth. MATERIAL AND METHOD: 28 patients who underwent decompressive surgery for lumbar spinal stenosis were retrospectively studied with an average follow-up of 8.4 years. In order to evaluate the degree of bone regrowth at the posterior arch, early postoperative radiographs and CT images of the operative sites were compared with those obtained at final follow-up. Bone regrowth at the sites operated upon was evaluated as a percentage of regrowth of the original laminectomy site based upon plain radiographs and CT images. RESULTS: Decompressive lumbar spinal stenosis is responsible for bone regrowth at the operative site in most patients. However, this regrowth was mild, the mean bone regrowth rate evaluated from plain radiographs was 12 per cent in average and the obtained from CT images was 8.2 per cent in average. Changes were found to be predominant at the facet joint level compared to the pedicle level. The evaluation of regrowth obtained from plain films and CT image examinations were compared. Radiographs seem to overestimate bone regrowth. Postoperative spinal instability was statistically significantly associated with new bone development. This variable was the only factor that affected the degree of bone regrowth. No relationship between bone regrowth and clinical outcome was found. DISCUSSION AND CONCLUSION: Natural course of laminectomy defect includes probably new bone formation in most patients. New bone results from gradual regrowth of the laminae and articular processes partially resected at surgery and from coalescence of islets of bone tissue within the tissue filling the laminectomy defect. In the present study bone regrowth rate was moderate but in other ones it was marked. If some factors (like postoperative destabilization) promoting bone regrowth were identified many remain unknown. Factors influencing rapidity of regrowth progression remain also unknown. Patient's intrinsic features such as spinal stenosis characteristics are probably closely related to quantitative and kinetic characteristics of regrowth. Consequences of bone regrowth are also variable: in some cases regrowth may reproduce pathological conditions identical previous ones, in other ones new bone spreads around the dura a mater without any nerves roots compression. Study of bone regrowth requires further research including prospective studies and using a more precise method for the regrowth evaluation.

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions.

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.

Purification and mechanism of action of a nucleosome assembly factor from Xenopus oocytes.

Chromatin with nucleosomes spaced at 180-base pair intervals can be formed in vitro on circular DNA molecules using a Xenopus oocyte S-150 extract, but the ability to form a periodic chromatin structure is lost upon fractionation of this extract. To identify factors other than the known ones involved in chromatin assembly, we have first depleted the extract by incubating it in batch with charged resins, and we have subsequently reconstituted it with purified fractions. Studies performed with the fractionated components indicate that formation of periodically spaced nucleosomes on the relaxed, closed circular DNA proceeds in two steps and does not require DNA topoisomerases. In a first step, histones H3/H4 are transferred from the endogenous H3/H4-N1 complex to the DNA, forming a nascent chromatin structure. This structure can then be rapidly complemented in a subsequent and independent step with a stoichiometric amount of histone H2A/H2B dimers. Under these experimental conditions, excess histone H2A/H2B dimers inhibit DNA supercoiling and nucleosome formation. We describe the purification of a factor from the Xenopus oocyte S-150 which permits DNA supercoiling and nucleosome formation under conditions of excess histone H2A/H2B. The activity purifies as a complex of five nonacidic polypeptides with apparent molecular masses ranging between 56 and 62 kDa. This factor prevents the binding of excess histone H2A/H2B to the DNA, and it can also remove excess histone H2A/H2B already bound to the DNA, thus ensuring that stoichiometric amounts of all four nucleosomal histones associate with the DNA.

The transcription complex of the 5 S RNA gene, but not transcription factor IIIA alone, prevents nucleosomal repression of transcription.

Assembly of nucleosomes on a 5 S DNA plasmid with histone H3.H4-N1 complex and histone H2A-H2B dimers causes a marked, 30-300-fold repression of 5 S RNA transcription. This repression is a time-dependent process that parallels the process of nucleosome formation. At physiological histone levels, DNA plasmids carrying nucleosomes with only histones H3 and H4 are transcriptionally permissive. The histone H3-H4 chromatin becomes transcriptionally nonpermissive when histone H2A-H2B dimers complement the nucleosome assembly reaction. H3-H4-H2A-H2B nucleosomes, but not H3-H4 nucleosomes, displace a DNA-bound transcription factor IIIA. In contrast, a preassembled 5 S RNA transcription complex is refractory to inactivation by nucleosomes.

Histone H1 represses transcription from minichromosomes assembled in vitro.

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.

Assembly and properties of chromatin containing histone H1.

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.

The assembly of regularly spaced nucleosomes in the Xenopus oocyte S-150 extract is accompanied by deacetylation of histone H4.

Histone proteins, which were assembled into chromatin using the Xenopus oocyte S-150 extract, were analyzed on acid-urea gels and Triton-acid-urea gels to determine their state of modification. We find that histone H4, which is present in a diacetylated form in the oocyte S-150, gradually loses its acetate groups as the DNA is packaged into chromatin. Thus, this process parallels the one observed in vivo during chromatin formation in growing eucaryotic cells. Histone H4 deacetylation in the oocyte S-150 is a DNA-dependent reaction. This reaction is blocked when butyrate (an inhibitor of histone deacetylase) is added at the onset of the chromatin assembly process. When butyrate is added at the end of the assembly process, no de novo acetylation of the nucleosomal histone H4 is observed. Chromatin with regularly spaced nucleosomes, displaying periodicities ranging from 160 to 220 base pairs, can be assembled in vitro with the oocyte S-150 (Rodríguez-Campos, A., Shimamura, A., and Worcel, A. (1989) J. Mol. Biol., in press). This chromatin may contain either deacetylated histone H4 when assembled under standard conditions or diacetylated H4 when assembled in the presence of butyrate. Both types of chromatin display identical structures upon digestion with nucleases. The potential applications of this system toward the study of the naturally occurring diacetylated histone H4 are discussed.

A 3' exonuclease activity degrades the pseudogene 5S RNA transcript and processes the major oocyte 5S RNA transcript in Xenopus oocytes.

Transcription of the major oocyte 5S RNA gene (o) and pseudogene (psi) of Xenopus laevis yields different RNAs with three different homologous systems: oocyte microinjection, whole oocyte extract, and fractionated TFIIIA + TFIIIB + TFIIIC components. Those peculiar results are caused by a 3' RNA exonuclease activity, which is inhibited in the oocyte extract, that rapidly degrades the pseudogene 5S RNA but does not degrade as readily the chimeric RNA transcripts generated by HindIII-truncated 5S RNA pseudogenes. The same, or a similar, RNase activity processes the 130- and the 142-base-long transcripts of the major oocyte 5S RNA gene into mature 120-base-long 5S RNA. We performed site-specific mutagenesis on the somatic 5S RNA gene and changed specific nucleotides on the somatic 5S RNA. These studies indicated that the structure that confers stability to the 5S RNA in vivo and in vitro is the 9-bp helix formed in 5S RNA, but not in psi 5S RNA, by the complementary 5' and 3' ends of the molecule.

The C-terminal domain of transcription factor IIIA interacts differently with different 5S RNA genes.

DNase I footprints and affinity measurements showed that the C-terminal arm of Xenopus transcription factor IIIA interacts differently with different Xenopus 5S DNAs, forming three distinct types of transcription factor IIIA-5S DNA complexes: a somatic type, a major-oocyte (and pseudogene) type, and a trace-oocyte type. Site-directed mutagenesis on the major-oocyte 5S gene revealed that somatic-type changes at positions 53, 55, and 56 changed the structure of the transcription factor IIIA-5S DNA complex from major-oocyte to somatic, and a single trace-oocyte change at position 56 caused the change from major-oocyte to trace-oocyte complex. We further show that the somatic-type changes are accompanied by a marked enhancement in the rate of 5S RNA transcription, and we discuss the possible biological relevance of these findings.

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

The positive transcription factor of the 5S RNA gene proteolyses during direct exchange between 5S DNA sites.

We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor transfers promptly from the complex to the free 5S DNA site. Unexpectedly, the direct exchange of factor between 5S DNA sites leads to proteolysis at the C-terminal arm of TFIIIA.

Mechanism of chromatin assembly in Xenopus oocytes.

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.

The role of DNA-mediated transfer of TFIIIA in the concerted gyration and differential activation of the Xenopus 5S RNA genes.

The transcription factor of the 5S RNA gene, TFIIIA, induces gyration of oocyte- and somatic-type 5S DNA plasmids in Xenopus oocyte extracts, but oocyte 5S gyration requires a 5-fold higher TFIIIA concentration than somatic 5S gyration. Concomitant with the differential gyration at intermediate TFIIIA concentrations, the oocyte genes are repressed and the somatic genes become activated, a situation that mimics the one seen in Xenopus somatic cells. Data obtained with plasmids immobilized in agarose indicate that TFIIIA finds its site via a DNA-mediated transfer mechanism, and that all-or-none gyration is a consequence of TFIIIA transfer between 5S DNA sites. Based on these results, we present a model that explains the differential all-or-none activation of the 5S RNA genes.

Gyration is required for 5S RNA transcription from a chromatin template.

We have assembled transcriptionally active chromatin on 5S DNA plasmids by using a Xenopus oocyte supernatant and the 5S-specific transcription factor IIIA (TFIIIA). In this system, the 5S RNA gene is accurately transcribed at a rapid rate of 50 transcripts per gene per hr. By following the time course of RNA synthesis during chromatin assembly, the dose response to TFIIIA addition, and the effect of novobiocin on the assembled nucleoprotein, we show that there is a strict correlation between transcriptional activity and the generation of torsionally strained DNA supercoils in "dynamic chromatin." Transcription cannot be the cause of the dynamic structure, because the assembly of this chromatin is unaffected by alpha-amanitin levels that completely block RNA polymerase III. Surprisingly, the dynamic chromatin remains transcriptionally active after relaxation with DNA topoisomerase I, which implies that the essential parameter for chromatin transcription is gyration per se, and not its effect on DNA topology.

The positive transcription factor of the 5S RNA gene induces a 5S DNA-specific gyration in Xenopus oocyte extracts.

We report that TFIIIA, the positive transcription factor of the 5S RNA gene, induces DNA gyration in Xenopus oocyte extracts. The reaction uses one molecule of TFIIIA per molecule of DNA and is highly specific for 5S DNA plasmids. DNA gyration also requires the oocyte supernatant, ATP, and Mg2+, and is inhibited by novobiocin, suggesting that it is catalyzed by a type II DNA topoisomerase. The chromatin assembled with TFIIIA is dynamic and rapidly relaxed by novobiocin; the chromatin assembled without TFIIIA is static and unaffected by novobiocin. The torsionally strained DNA is produced in a novel concerted reaction: all of the 5S DNA molecules gyrate at TFIIIA-5S DNA ratios equal to or above 1, and none of them gyrate at TFIIIA-5S DNA ratios below 1. We discuss the biological implications of this eukaryotic DNA gyration.