Morphological and molecular characterization of larval trematodes infecting the assassin snail genus Anentome in Thailand.

The assassin snail genus Anentome is widespread in Southeast Asia, and is distributed all over the world via the aquarium trade. One species of genus Anentome, Anentome helena, is known to act as intermediate host of parasitic trematodes. This study investigates the taxonomic diversity of larval trematodes infecting A. helena and Anentome wykoffi in Thailand. Larval trematodes were identified by combining morphological and DNA sequence data (cytochrome c oxidase I and internal transcribed spacer 2). Species delimitation methods were used to explore larval trematode species boundaries. A total of 1107 specimens of Anentome sp. were collected from 25 localities in Thailand. Sixty-two specimens of A. helena (n = 33) and A. wykoffi (n = 29) were infected by zoogonid cercariae, heterophyid metacercariae and echinostome metacercariae, with an overall prevalence of 5.6% (62/1107) and population-level prevalences in the range of 0.0-22.3%. DNA sequence data confirmed that the larval trematodes belong to the families Echinostomatidae, Heterophyidae and Zoogonidae. As such, this is the first report of zoogonid cercariae and heterophyid metacercariae in A. helena, and echinostome metacercariae in A. wykoffi. Moreover, this study provides evidence of tentative species-level differentiation between Thai Echinostoma sp. and Cambodian Echinostoma mekongi, as well as within Echinostoma caproni, Echinostoma trivolvis and Echinostoma revolutum.

Preliminary results from a prospective study of laparoscopic aortobifemoral bypass using a clampless and sutureless aortic anastomotic technique.

OBJECTIVE: This prospective study describes the feasibility and safety of a new clampless and sutureless aortic anastomotic technique used during retroperitoneal laparoscopic aortobifemoral bypass in extensive aortoiliac occlusive lesions. This is a case series of a previously published technique, demonstrating wider applicability of the technique. MATERIALS AND METHODS: Twelve patients underwent a clampless and sutureless laparoscopic bypass for TASC D aortoiliac occlusive lesions using the EndoVascular REtroperitoneoScopic Technique (EVREST). Dissection of the retroperitoneal space and the infrarenal aorta was performed laparoscopically. A bifurcated graft was inserted into the retroperitoneal space. The main body of the graft was connected on the left side of the aorta by an intra- and extra-aortic covered stent-graft. An aortic clamp was used temporarily on four patients because of excessive bleeding when the connector was deployed. The femoral anastomoses were performed by classic open surgery. Initial technical success, complications, and bypass patency were assessed. RESULTS: Median follow-up was 9.3 months. Median operative time was 265 minutes. Median duration of aorto-prosthetic connection was 60 seconds. Thirty-day postoperative mortality was 0%. No major postoperative complications were observed. All grafts were patent at the end of follow-up and there was no early or late disruption of the proximal assembly. CONCLUSIONS: EVREST greatly facilitates laparoscopic aortic surgery in occlusive disease with no need for suture or clamping of the aorta. This technique performed in a single center on 12 patients, seems to be feasible and safe. It offers the advantages of laparoscopy and those of endovascular surgery, especially in the challenging conditions encountered during aortic laparoscopic surgery. Early experience supports procedural and initial postprocedural safety and demonstrates proof-of-concept for EVREST.

Ruptured aneurysm of the popliteal artery. Is the diagnosis still difficult?

The aneurysm of the popliteal artery is the most commonly treated non-aortoiliac aneurysm, accounting for more than 70% of all peripheral aneurysms. The rupture of a popliteal aneurysm is rare and it is often misdiagnosed. In the case of a 46-year old female patient here reported, the patient was referred to our department with the diagnosis of ruptured aneurysm of the right popliteal artery with formation of a large pseudo-aneurysm. We operated the patient on a semi-urgent basis and performed a replacement of the popliteal artery by a saphenous vein graft. Three months after the operation, the patient was free of symptoms. This article's aim was to emphasize on how the pitfalls during clinical examination, as well as the problems of imaging interpretation, can make the diagnosis of ruptured popliteal aneurysm still difficult.

Totally retroperitoneal laparoscopic aortobifemoral bypass.

The classic procedure for aortobifemoral bypass is open surgery. Since the first totally laparoscopic aortobifemoral bypass reported in 1997 by Yves-Marie Dion, laparoscopy has been accepted by several authors as a possible minimally invasive alternative for aorto-iliac occlusive disease. The transperitoneal left retrocolic and retrorenal ways are generally used. The totally retroperitoneal laparoscopic procedure has been described as an alternative to the transperitoneal approach. We report here a totally laparoscopic retroperitoneal approach to performing aortobifemoral bypass. This approach was proposed to a 51-year-old man with aorto-iliac occlusive disease. There was no indication for endovascular revascularization. The patient suffered from 10 metres of bilateral intermittent claudication and lower limb ulcers. During the surgical procedure our patient was placed in a 30-degree right lateral decubitus position. The optical system was first placed in an intra-abdominal position to check the positioning of the trocars in the left retroperitoneal space. The dissection of the retroperitoneal space was performed by CO2 insufflation and by blunt dissection using laparoscopic forceps. The infrarenal aorta was exposed and clamped by laparoscopic clamps. A bifurcated graft was sutured on the left-hand side of the aorta by a running suture. Both prosthetic limbs were tunnelized retroperitoneally to the groin under optical control. The femoral anastomoses were performed by classic open surgery.

Retroperitoneal laparoscopic bilateral lumbar sympathectomy.

The first retroperitoneal lumbar sympathectomy was performed in 1924 by Julio Diez. The classic procedure for sympathectomy is open surgery. We report a unilateral laparoscopic retroperitoneal approach to perform bilateral lumbar sympathectomy. This approach was performed for a 43-year-old man with distal arterial occlusive disease and no indication for direct revascularization. His predominant symptoms were intermittent claudication at 100 metres and cold legs. The patient was placed in a left lateral decubitus position. The optical system was placed first in an intra-abdominal position to check that the trocars were well positioned in the retroperitoneal space. The dissection of retroperitoneum was performed by CO2 insufflation. The inferior vena cava was reclined and the right sympathetic chain was individualized. Two ganglia (L3-L4) were removed by bipolar electro-coagulation. The aorta was isolated on a vessel loop and careful anterior traction allowed a retro-aortic pre-vertebral approach between the lumbar vessels. The left sympathetic chain was dissected. Two ganglia (L3-L4) were removed by bipolar electro-coagulation.

Isolated supradiaphragmatic descending thoracic aorta stenosis in a Takayasu's disease: surgical cure.

A 21-year-old male patient presented with a typical middle aortic syndrome. Echography disclosed a severe narrowing of the lower thoracic aorta with parietal thickening. The isolated character of the lesion was confirmed by magnetic resonance imaging and aortography. The surgical cure was realized by a Dacron bypass between the upper thoracic descending aorta and the juxta-diaphragmatic thoracic aorta. Aortic biopsy confirmed Takayasu's disease. Postoperative course was uneventful with normalized blood pressure. The therapeutic options, surgery versus percutaneous dilatation and stent, are discussed.

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.

Refined localization of the alpha 1-subunit of the skeletal muscle L-type voltage-dependent calcium channel (CACNL1A3) to human chromosome 1q32 by in situ hybridization.

We isolated and partially sequenced a cosmid clone containing the human skeletal muscle L-type voltage-dependent calcium channel gene (CACNL1A3). The cosmid clone, which was also found to contain a novel dinucleotide repeat marker for the CACNL1A3 gene, was used for the chromosomal localization of CACNL1A3 by in situ hybridization. Our results refine the localization of CACNL1A3 on the long arm of human chromosome 1 to band q32.

Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart.

A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from the rabbit heart alpha 1 include a shortened N-terminus, a unique C-terminal insertion, and both forms of an alternatively spliced motif IV S3 region. The shortened N-terminus provides optimal access to consensus sequences thought to facilitate translation. Northern blot analysis revealed a single hybridizing mRNA species of 9.4 kb. The gene for the human heart alpha 1 subunit was localized specifically to the distal region of chromosome 12p13. The cloned alpha 1 subunit was expressed in Xenopus oocytes and single-channel analyses revealed native-like pharmacology and channel properties.

Genetic mapping of the beta 1- and gamma-subunits of the human skeletal muscle L-type voltage-dependent calcium channel on chromosome 17q and exclusion as candidate genes for malignant hyperthermia susceptibility.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant disorder of skeletal muscle which manifests as a life-threatening hypermetabolic crisis triggered by commonly-used inhalation anaesthetics and depolarizing muscle relaxants. Defects in the ryanodine receptor (RYR1) protein have been proposed to underly MHS, but significant genetic heterogeneity in MHS has recently been demonstrated. In order to investigate the potential roles played by other skeletal muscle calcium channels in MHS, we isolated cosmids containing the gene encoding the beta 1-subunit of skeletal muscle L-type voltage-dependent calcium channel (CACNLB1). We identified a new, highly polymorphic dinucleotide repeat motif close to this gene, and linkage analysis placed the marker proximal to the HOX2B locus, previously localized to chromosome segment 17q21-q22. We recently identified a novel marker within the gamma-subunit locus (CACNLG) at band 17q24, and since both markers are within the 17q11.2-q24 region reported to contain the MHS2 locus, we tested them for linkage in MHS families whose disease trait has been shown not to co-segregate with markers for the RYR1 region on chromosome 19q13.1. Our results exclude CACNLB1 and CACNLG as candidate genes for MHS2, and do not support the reported chromosome 17q localization for the MHS2 locus in our families.

Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene.

The mutation causing myotonic dystrophy (DM) has recently been identified as an unstable CTG trinucleotide repeat located in the 3' untranslated region of a gene encoding for a protein with putative serine-threonine protein kinase activity. In this report we present the genomic sequences of the human and murine DM kinase gene. A comparison of these sequences with each other and with known cDNA sequences from both species, led us to predict a translation initiation codon, as well as determine the organization of the DM kinase gene. Several polymorphisms within the human DM kinase gene have been identified, and PCR assays to detect two of these are described. The complete sequence and characterization of the structure of the DM kinase gene, as well as the identification of novel polymorphisms within the gene, represent an important step in a further understanding of the genetics of myotonic dystrophy and the molecular biology of the gene.

Localization of the gamma-subunit of the skeletal muscle L-type voltage-dependent calcium channel gene (CACNLG) to human chromosome band 17q24 by in situ hybridization and identification of a polymorphic repetitive DNA sequence at the gene locus.

The skeletal muscle dihydropyridine receptor consists of five subunits and fulfils an essential role in excitation-contraction coupling. A genomic clone for the human gamma subunit was used to map the gene (CACNLG) to chromosome band 17q24 by in situ hybridization. Contained within the gene is a 416-bp polymorphic repetitive DNA element that is potentially useful as a genetic marker.

High-resolution physical mapping of four microsatellite repeat markers near the RYR1 locus on chromosome 19q13.1 and apparent exclusion of the MHS locus from this region in two malignant hyperthermia susceptible families.

Malignant hyperthermia susceptibility (MHS) is a potentially lethal, hereditary disorder of skeletal muscle that may be triggered by inhalation anesthetics and depolarizing muscle relaxants. Defects in the gene encoding the ryanodine receptor (RYR1) localized on human chromosome 19q13.1 have been proposed to be responsible for MHS. Using a chromosome 19-specific human/hamster somatic cell hybrid mapping panel, we were able to determine that four closely linked microsatellite repeat markers bracket RYR1 with the order 19cen-D19S75-D19S191-RYR1-(D19S47, D19S190)-19ter. Application of the four markers to genetic studies of MHS showed recombination between the markers and MHS in two families, with linkage analysis apparently excluding the MHS locus from the RYR1 region of 19q13.1. These results therefore support the recent observations of genetic heterogeneity in MHS.

Characterization of the myotonic dystrophy region predicts multiple protein isoform-encoding mRNAs.

The mutation underlying myotonic dystrophy (DM) has been identified as an expansion of a polymorphic CTG-repeat in a gene encoding protein kinase activity. Brain and heart transcripts of the DM-kinase (DMR-B15) gene are subject to alternative RNA splicing in both human and mouse. The unstable [CTG]5-30 motif is found uniquely in humans, although the flanking nucleotides are also present in mouse. Characterization of the DM region of both species reveals another active gene (DMR-N9) in close proximity to the kinase gene. DMR-N9 transcripts, mainly expressed in brain and testis, possess a single, large open reading frame, but the function of its protein product is unknown. Clinical manifestation of DM may be caused by the expanded CTG-repeat compromising the (alternative) expression of DM-kinase or DMR-N9 proteins.