Anatomical basis of digital rectal examination.

PURPOSE: Rectal examination is difficult to carry out by students because of their lack of knowledge and fear. It is therefore necessary to search for methods in order to facilitate its practice. This work mainly focuses on the palpation of the posterior lateral area of the rectum. METHODS: This work bases itself on the study of the average length of indexes and on the anatomical study of the dissection and prints of two pelvises. In the lithotomy position, we can identify three successive levels of exploration of the posterior and lateral area of the rectum. These three levels are defined by the extremity of the index, and the distal and proximal interphalangeal articulations placed successively on the tip of the coccyx. A 180° rotation of the hand enables at each level to identify the parietal structures that the pad of the index comes across, but excludes the palpation of genital organs and rectum. RESULTS: The first level corresponds to the higher part of the anal canal, the ischioanal fossa and the ischium. The second level corresponds to the levator ani muscle, the ischioanal fossa and the pudendal canal. The third level corresponds to the sacrospinous ligament, the ischiatic spine and the internal obturator muscle. CONCLUSIONS: In spite of the significant differences between the lengths of the indexes, the use of these landmarks will facilitate the identification of parietal anatomical structures. The internal organs' palpation will depend on the patient's position, his efforts in pushing, the length of the index, and the way the examiner presses on the perineum.

[Symptomatic approach to musculoskeletal dysfunction and chronic pelvic and perineal pain]. / Approche symptomatique des dysfonctions musculosquelettiques et douleurs pelvipérinéales chroniques.

INTRODUCTION: Clinical examination of a patient with chronic pelvic and perineal pain often demonstrates muscle hypertonia or muscle contracture sometimes associated with local tenderness or real muscle trigger points. It is sometimes very difficult to determine whether this muscle pain detected on clinical examination is the cause or a consequence of the pain. The purpose of this article is to review musculoskeletal dysfunction in the context of chronic pelvic and perineal pain. MATERIAL AND METHODS: Review of the literature devoted to musculoskeletal aspects of pelvic and perineal pain. RESULTS: Definitions of pelvic floor dysfunction, hyperactive pelvic floor, myofascial pain and muscle trigger points, and the concept of fibromyalgia. CONCLUSION: Musculoskeletal pain is certainly underestimated in the management of chronic pelvic and perineal pain. The pathophysiology of musculoskeletal pain involves disorders of the lumbar, pelvic and femoral equilibrium, myofascial pain characterized by the presence of trigger points for which the pathophysiology remains controversial: a purely muscle disease, reaction to adjacent inflammatory reactions causing hypersensitization, or simply a sign of central hypersensitization in a context of chronic pain syndrome.

[Treatment of the musculoskeletal component of chronic pelvic and perineal pain]. / Traitement de la composante musculosquelettique des douleurs pelvipérinéales chroniques.

OBJECTIVE: To describe muscle examination in patients with chronic pelvic and perineal pain and to determine the results that can be expected from specific treatments (physiotherapy and botulinum toxin). MATERIAL AND METHODS: Review of the literature, especially the Medline indexed literature. Description of the physical rehabilitation techniques that can be used in this context. RESULTS: The management of patients with chronic pelvic and perineal pain requires preliminary clinical analysis designed to identify trigger points responsible for myofascial pain, pelvic floor muscle tension, and lumbar-pelvic-hip instability. Physiotherapy must be initiated early in the course of the disease by therapists trained in these recent techniques. Botulinum toxin injections have been shown to be effective in piriformis syndrome, but a review of the literature indicates more controversial results in the other chronic pelvic and perineal pain syndromes.

[Obturator neuralgia: treatment and preliminary results of laparoscopic neurolysis]. / Névralgies obturatrices : prise en charge et résultats préliminaires de la neurolyse laparoscopique.

AIMS: Obturator neuralgia is a pain which is ill-defined and particularly less well-known to practitioners. Here we report on the etiologies, the treatment and the results of conservative laparoscopic treatment by neurolysis of the obturator nerve in cases of obturator neuralgia. PATIENTS AND METHOD: Thirteen patients (15 nerves) who had obturator neuralgia have been treated in our service since 2005. The etiologies were idiopathic (four cases), following surgery for an inguinal hernia (two cases), trauma of the pelvis (one case), a TVT strip (three cases) and a TOT strip (three cases). The diagnosis was based on the pain, which was neuropathic, of the antero-internal side of the thigh. It was confirmed under block anesthetic by tomodensitometry using a posterior approach. The treatment consisted of laparoscopic neurolysis. RESULTS: The patients suffered pain measured at a rate of 8/10 on the visual analogical scale before the operation. In each case, neurolysis was carried out by transperitoneal laparoscopy by dissecting the nerve and sectionning the scarring fibrosis where the prothesis was in contact. In the idiopathic cases, the liberation of the nerve was carried out by a section of the internal obturator muscle and of the obtured membrane, allowing for the blocked canal to be widened. Seventeen months later, a rate of improvement of at least 50% of pain was found in 77% of cases (10/13), of whom pain had totally disappeared in 54% of cases (7/13). There was no improvement at all for 23% of cases (3/13). CONCLUSION: The mini-invasive conservative treatment of obturator neuralgia by laparascopic neurolysis of the obturator nerve, after confirmed diagnosis by selective infiltration allowed for a rate of 75% of improvement to be obtained after a period of 17 months.

Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes.

A Streptomyces pristinaespiralis strain, which produces a streptogramin antibiotic pristinamycin II (PII) as a mixture of two biologically active molecules PIIB and PIIA, was genetically engineered to produce exclusively PIIA. The snaA,B genes, which encode a PIIA synthase that performs oxidation of the precursor (PIIB) to the final product (PIIA), were integrated in the chromosome of S. pristinaespiralis using an integrative derivative of the pSAM2 genetic element from Streptomyces ambofaciens. Integration was due to site-specific recombination at the attB site of S. pristinaespiralis, and no homologous recombination at the snaA,B locus was observed. The attB site of S. pristinaespiralis was sequenced and found to overlap the 3' end of a pro-tRNA gene. The integrants were stable in industrial conditions of pristinamycin production and showed no decrease in PII biosynthesis. Western blot analysis showed a constant production of the PIIA synthase in the overall fermentation process due to expression of the cloned snaA,B genes from the constitutive ermE promoter. This allows the complete conversion of the PIIB form into PIIA.

Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2.

In Saccharomyces cerevisiae, the alcohol dehydrogenase genes ADH1 and ADH5 are part of a duplicated block of genome, thought to originate from a genome-wide duplication posterior to the divergence from the Kluyveromyces lineage. We report here the characterization of Kluyveromyces marxianus ADH2 and the five genes found in its immediate downstream region, MRPS9, YOL087C, RPB5, RIB7 and SPP381. The order of these six genes reflects the structure of the ancestral S. cerevisiae genome before the duplication that formed the blocks including ADH1 on chromosome XV and ADH5 on chromosome II, indicating these ADH genes share a direct ancestor. On the one hand, the two genes found immediately downstream of KmADH2 are located, for the first, downstream ADH5 and, for the second, downstream ADH1 in S. cerevisiae. On the other hand, the order of the paralogs included in the blocks of ADH1 and ADH5 in S. cerevisiae suggests that two of them have been inverted within one block after its formation, and that inversion is confirmed by the gene order observed in K. marxianus.

Construction of a series of pSAM2-based integrative vectors for use in actinomycetes.

We have developed vectors which allowed integration of cloned DNA at a single site in the chromosome of Streptomyces lividans 66. These vectors made use of (1) an Escherichia coli replicon, (2) a thiostrepton (Th)- and a streptomycin/spectinomycin-resistance gene for selection in Streptomyces, (3) a 3.5-kb fragment of the Streptomyces integrative plasmid pSAM2 containing its xis and int genes as well as its attachment site, attP, to direct the integration of the vectors at the chromosomal pSAM2 attachment site attB, (4) the origin of transfer of the IncP broad-host-range plasmid RK2 which allowed the mobilization of the vectors from E. coli to S. lividans, and (5) the Th-inducible tipA promoter to permit regulated transcription of cloned genes. We demonstrated that pPM927, a plasmid which contained all of these elements, was able to transfer cloned fragments from E. coli to S. lividans by conjugation, stably integrate into the chromosome, and express cloned genes from the tipA promoter. Furthermore, since pPM927 contained the pBR322 replicon, cloned fragments could be conveniently recovered from the S. lividans chromosome for analysis in E. coli by cleavage of genomic DNA isolated from transformed strains, intramolecular ligation and transformation. Since we have shown that the pSAM2 attB site forms part of a conserved prokaryotic tRNA gene, these integrative vectors are potentially useful tools for analysis and expression of genes in diverse bacteria.

Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens.

The Streptomyces ambofaciens genome contains four rRNA gene clusters. These copies are called rrnA, B, C and D. The complete nucleotide (nt) sequence of rrnD has been determined. These genes possess striking similarity with other eubacterial rRNA genes. Comparison with other rRNA sequences allowed the putative localization of the sequences encoding mature rRNAs. The structural genes are arranged in the order 16S-23S-5S and are tightly linked. The mature rRNAs are predicted to contain 1528, 3120 and 120 nt, for the 16S, 23S and 5S rRNAs, respectively. The 23S rRNA is, to our knowledge, the longest of all sequenced prokaryotic 23S rRNAs. When compared to other large rRNAs it shows insertions at positions where they are also present in archaebacterial and in eukaryotic large rRNAs. Secondary structure models of S. ambofaciens rRNAs are proposed, based upon those existing for other bacterial rRNAs. Positions of putative transcription start points and of a termination signal are suggested. The corresponding putative primary transcript, containing the 16S, 23S and 5S rRNAs plus flanking regions, was folded into a secondary structure, and sequences possibly involved in rRNA maturation are described. The G + C content of the rRNA gene cluster is low (57%) compared with the overall G + C content of Streptomyces DNA (73%).

Excision and integration of a self-transmissible replicon of Streptomyces ambofaciens.

When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.

Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24.

Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.

amlC, another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24.

The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.

Rearrangement of mitochondrial DNA molecules during the differentiation of mitochondria in yeast. II. - Labelling studies of the precursor product relationship.

The length distribution in sucrose sedimentation gradient of the newly-synthesized pulse-labelled mitochondrial DNA has been established at an early stage of depression in wild type yeast (Saccharomyces cerevisiae). This stage corresponded to the beginning of mitochondrial differentiation. The radioactive DNA was longer (mean lengths 5, 10 and 22-25 mu) than the preexisting cold DNA (mean length 6.5 mu with two shoulders at 4 mum and 10 mum and one minor peak at 2-2.5 mum). These date confirm that the mean size of the different length populations of linear yeast mitochondrial DNA are under physiological control. Chase experiments were undertaken as follows. The yeast cells were uniformly prelabelled under anaerobiosis. Therefore the mitochondrial DNA molecules were short. Respiratory adaptation was performed in a cold medium and the lengthening process was induced. The specific activities of the long molecules made up during the respiratory adaptation did mot markedly differ from that of prelabelled DNA (decrease of specific activity less than 18 per cent). Molecules as long as 40 mum were also recorded. This lengthening seems to proceed through a non reciprocal exchange of polynucleotide stretches between preexisting molecules. We call it rearrangement. It occurs during the differentiation of mitochondria. Much of the mitochondrial DNA is maintained whereas a small amount of DNA is synthesized. This hypothesis is favoured by recent genetical and physical studies on mitochondrial recombination in yeast.

Rearrangement of mitochondrial DNA molecules during the differentiation of mitochondria in yeast. I.-Electron microscopic studies of size and shape.

Size and shape of purified mitochondrial DNA was analyzed by electron microscopy as a function of mitochondrial differentiation. The mitochondrial DNA was extracted at fourth growth stages corresponding to different steps of mitochondria repression and depression. It was heterogeneous both in form and length. The size of linear molecules ranged from 1 mu to 25 mu but most of the molecules could be assigned into four Gaussian subpopulations with mean lengths of 2.2 mu to 4.0 mu, 6.0 mu and 10.0 mu. The circular molecules were all open and sized varied from 0.5 mu to 10 mu. Their length repartition was congruent with a logarithmic Gaussian distribution. The relative proportion of the different classes of molecules changed according to the stage of the growth cycle: during the repression most of the mitochondrial DNA molecules were short: the population of 2.2 mu was predominant. The longest linear molecules were observed during derepression where the populations of 4.0 mu and 10.0 mu were only found as well as the highest proportion of circular molecules. At the stationary phase the mitochondrial DNA became short again and the circles disappeared completely. The mitochondrial DNA extracted from a cytoplasmic "petite" was composed of linear and circular molecules. The linear molecules ranged from 0.1 mu to 32 mu and most of them could be assigned to two subpopulations of 1.3 mu and 4.2 mu. The circular molecules which accounted for 11 percent had contour lengths of 0.7 mu and 1.5 mu. The physiological meaning of the change in the relative proportion of different classes of mitochondrial DNA is discussed.

Mitochondrial and nuclear mutagenicity of ellipticine and derivatives in the yeast Saccharomyces cerevisiae.

Haploid strains of the yeast Saccharomyces cerevisiae were tested for their sensitivity to ellipticine and nine derivatives; some of them exhibiting antitumor properties. Different mutagenic properties of ellipticines are described. Charged ellipticines, which were ineffective in Ames' bacterial assay, were found to be potent inducers of the mitochondrial p- mutation: induction proceeded even in the absence of growth. Uncharged ellipticines increased mitochondrial antibiotic resistance mutations, whereas charged derivatives decreased them. Ellipticine derivatives enhanced, reduced, or did not change the reversion frequencies of nuclear auxotrophic markers. The result depended on the strain being tested: no structure-effect relationship existed. As some ellipticine derivatives were mutagenic in Saccharomyces cerevisiae despite being ineffective in Ames' assay, multiple tests should be used to check that chemotherapeutic drugs are devoid of mutagenic properties.

Regulation of the expression of amy TO1 encoding a thermostable alpha-amylase from Streptomyces sp. TO1, in its original host and in Streptomyces lividans TK24.

In its original host, the thermophilic Streptomyces strain sp. TO1, the amy TO1 gene was expressed during growth but only in the presence of starch in the growth medium. When cloned in Streptomyces lividans, on a low copy number replicative plasmid, amy TO1 expression was detectable in fructose-, mannitol- and galactose-grown cultures but not in glucose- or glycerol-grown cultures. This basal expression could be further induced by maltotriose. In a mutant strain of S. lividans disrupted for the LacI-like negative transcriptional regulator (NTR) Reg1, and when the symmetry of the dyadic symmetry element located in the promoter region of amy TO1 was altered, the basal levels of amy TO1 expression were significantly higher than those of the wild-type strain, and the maltotriose inducibility was abolished. These results suggest that, in S. lividans, amy TO1 expression is under the control of the NTR Reg1 due to its interaction with the dyadic symmetry element.

Ethidium bromide mutagenesis in yeast: protection by anaerobiosis.

The mutagenesis by ethidium bromide, an intercalating dye, which induces the mutation from wild type (rho+) to the cytoplasmic respiratory deficient petite (rho-) in Saccharomyces cerevisiae, was studied under aerobic and anaerobic conditions. During growth of anaerobic cells at pH 6.5, ethidium bromide at a concentration of 2 mug/ml is unable to induce rho- mutants whereas under aerobic conditions the entire population is converted into rho- cells within 1 generation at the same drug concentration. With ethidium bromide 10 mug/ml 98% of the anaerobic cells are transformed into rho- in 5.5 h (more than 2 generations). In non-growing conditions, ethidium bromide 10 mug/ml has no effect in anaerobic cells. 3 h adapted cells used as control, are converted into rho- in 8 h. Increasing the ethidium bromide concentration to 20 mug/ml resulted in the appearance of some rho- mutants in the anaerobic population but marked at the same time the onset of a detectable toxic effect of the drug.

[Perineal pain]. / Le neurochirurgien face aux algies périnéales. Guide pratique.

The investigation of patients suffering from perineal pain when sitting led us to perform an anatomical study of the pudendal nerve. We dissected 50 cadavers and found areas of conflict for the nerve fibers. The nerve trunk can become entrapped at the level of the ischiatic spine, in the Alcock's canal and when it crosses the falciform process. Considering the clinical and neurophysiological data, this type of chronic pain may arise from compression of the nerve between the sacro-tuberal and the sacro-spinal ligaments, and/or in the fascia of the internal obturator muscle. Much like treatment of entrapment of the median nerve in the wrist, we decided to treat chronic perineal pain by nerve blocks, and later by surgery. We describe here the clinical symptoms, the neurophysiological data, and the technique of the nerve blocks. For patients with persistent pain, we propose a posterior surgical approach which has provided successful pain relief in two third of patients.

2 µm plasmid copy number in different yeast strains and repartition of endogenous and 2 µm chimeric plasmids in transformed strains.

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 µm plasmid in yeast cells. For this purpose we determined the 2 µm plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 µm chimeric plasmids.According to the strain used the proportion of 2µm DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 µm molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 µm copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 µm DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 µm copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 µm DNA copies per haploid genome is transformed with 2 µm chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 µm chimeric plasmids were introduced in our 2 µm-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.