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.

[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.

The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer.

pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer. A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer. Initiation (i.e. excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain. pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome. Transfer generally involves single-stranded DNA. In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer. Using the differential sensitivity to the SalI restriction-modification system of transfers involving single- and double-stranded DNA, we found that pSAM2 was probably transferred to the recipient as double-stranded DNA. This provides the first experimental evidence for the transfer of double-stranded DNA during bacterial conjugation. Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double-stranded DNA actively, either from donor to recipient or from mother cell to prespore.

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.

KorSA from the Streptomyces integrative element pSAM2 is a central transcriptional repressor: target genes and binding sites.

pSAM2, a 10.9-kb mobile integrative genetic element from Streptomyces ambofaciens, possesses, as do a majority of Streptomyces conjugative plasmids, a kil-kor system associated with its transfer. The kor function of pSAM2 was attributed to the korSA gene, but its direct role remained unclear. The present study was focused on the determination of the KorSA targets. It was shown that KorSA acts as a transcriptional repressor by binding to a conserved 17-nucleotide sequence found upstream of only two genes: its own gene, korSA, and pra, a gene positively controlling pSAM2 replication, integration, and excision. A unique feature of KorSA, compared to Kor proteins from other Streptomyces conjugative plasmids, is that it does not directly regulate pSAM2 transfer. KorSA does not bind to the pSAM2 genes coding for transfer and intramycelial spreading. Through the repression of pra, KorSA is able to negatively regulate pSAM2 functions activated by Pra and, consequently, to maintain pSAM2 integrated in the chromosome.

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.

Construction of efficient centromeric, multicopy and expression vectors for the yeast Kluyveromyces marxianus using homologous elements and the promoter of a purine-cytosine-like permease.

Efficient centromeric and multicopy vectors have been constructed for the yeast Kluyveromyces marxianus using homologous ARS and centromere sequences. A homologous promoter of a purine-cytosine permease gene called PCPL3 has been cloned, using an expression system based on GUS. Its strength has been estimated in K. marxianus by putting the homologous beta-glucosidase gene under its control. This promoter is very efficient as activities higher than the ones obtained with the Saccharomyces cerevisiae PGK promoter were obtained. This promoter appears to be constitutive in various conditions tested. Its five transcription start sites have been mapped, and a derivative expression vector for K. marxianus has been constructed.

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.

Replicase, excisionase, and integrase genes of the Streptomyces element pSAM2 constitute an operon positively regulated by the pra gene.

pSAM2 is a site-specific integrative element from Streptomyces ambofaciens. The pra gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which the pra gene was placed under the control of the inducible tipAp promoter. Transformation of Streptomyces lividans by these derivatives was efficient only when pra expression was induced, indicating its involvement in pSAM2 integration activation. Once established, these constructions remained integrated in the chromosome under noninduced conditions. Activation of the pra expression provoked strong activation of their excision, leading to the appearance of free forms. The results of functional, transcriptional, and sequence analyses allowed to conclude that the three genes repSA, xis, and int coding for the pSAM2 replicase, excisionase, and integrase, respectively, constitute an operon directly or indirectly activated by pra.

Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli.

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

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.

Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene.

A regulatory gene, reg1, was identified in Streptomyces lividans. It encodes a 345-amino-acid protein (Reg1) which contains a helix-turn-helix DNA-binding motif in the N-terminal region. Reg1 exhibits similarity with the LacI/GalR family members over the entire sequence. It displays 95% identity with MalR (the repressor of malE in S. coelicolor), 65% identity with ORF-Sl (a putative regulatory gene of alpha-amylase of S. limosus), and 31% identity with CcpA (the carbon catabolite repressor in Bacillus subtilis). In S. lividans, the chromosomal disruption of reg1 affected the expression of several genes. The production of alpha-amylases of S. lividans and that of the alpha-amylase of S. limosus in S. lividans were enhanced in the reg1 mutant strains and relieved of carbon catabolite repression. As a result, the transcription level of the alpha-amylase of S. limosus was noticeably increased in the reg1 mutant strain. Moreover, the induction of chitinase production in S. lividans was relieved of carbon catabolite repression by glucose in the reg1 mutant strain, while the induction by chitin was lost. Therefore, reg1 can be regarded as a pleiotropic regulatory gene in S. lividans.

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.

The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats flanking the extremities of the linear chromosomal DNA.

Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Asel fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dral map was obtained by positioning the Dral sites relative to the Asel map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Asel and Dral fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The two terminal Asel fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.

Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens.

pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2, which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.

Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2.

pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism. Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin). We report here the study of the second region. Its nucleotide sequence was determined and analysed for open reading frames (ORFs). Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa). orf183 is not necessary for replication. The function of orf50 is unknown. repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism. A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2. repSA might be co-transcribed with the genes involved in integration and excision of pSAM2.