Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems.

Biogenesis of the toxin-coregulated pilus (TCP) of Vibrio cholerae 01 is essential for successful bacterial colonization of the small intestine. Pilus assembly requires the products of at least seven genes located on the chromosome adjacent to the pilin-encoding gene, tcpA. Previously reported TnphoA insertions in the TCP-assembly-deficient V. cholerae strains, KP2.21 and KP4.2, were isolated from the chromosome for further analysis. Nucleotide sequencing of the tcpE::phoA and tcpF::phoA fusions and corresponding clones of the region containing the intact genes revealed the presence of two open reading frames (ORFs) of 340 and 338 amino acids, designated TcpE and TcpF, respectively. The partial sequence of an ORF downstream from the TcpF coding sequence was determined to correspond to the global virulence regulator, ToxT. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Computer-generated alignment algorithms predict that a homology exists between TcpE and the Klebsiella pneumoniae pullulanase secretion proteins PulD and PulF, the Xanthomonas campestris extracellular enzyme secretion factor XpsF, the Bacillus subtilis DNA competence protein ComG-ORF2, and the Yersinia enterocolitica Yop secretion determinant YscC. These observations provide a model to investigate further the relationship between the secretion mechanisms utilized by these seemingly diverse virulence determinants. Additionally, an extreme C-terminal segment of TcpE shows striking homology to the transmembrane segment of the eukaryotic integrin beta-1 chain, which could imply a role for TcpE in not only TCP secretion, but also host cell interaction.

Identification of bacterial cell-surface virulence determinants with TnphoA.

Insertion mutagenesis using TnphoA has proved to be a potent device for the creation of easily screened knockout mutations in genes encoding virulence determinants in a variety of pathogenic bacteria. Initial identification of genes with TnphoA directly initiates more sophisticated genetic and biochemical studies on these factors essential to our understanding of bacterial pathogenesis.

Thymopharyngeal duct cyst: an unusual variant of cervical thymic anomalies.

BACKGROUND: The thymus develops from the third pharyngeal pouch and descends from the neck into the anterior-superior mediastinum. Thus, it is possible to have thymic remnants in the neck, which most often present as a cervical mass during childhood. One type of cystic thymic remnant is the thymopharyngeal duct cyst, a remnant of one of the paired tracts of embryological thymic descent. Thymopharyngeal duct cysts are rare lesions that can have a similar presentation to more commonly encountered childhood neck masses. OBJECTIVES: To review the embryological development of cervical thymic remnants and to report our experience with the thymopharyngeal duct cyst. DESIGN: Case series. SETTING: Tertiary care center. PATIENTS: Two children who presented with asymptomatic neck masses that were caused by cystic remnants of the thymopharyngeal duct. RESULTS: Both patients underwent preoperative computed tomography, which revealed a multiloculated mass coursing adjacent to the carotid sheath. Surgical treatment was the definitive therapy for both patients, although neither patient had a definitive preoperative diagnosis. In both cases, the mass was approached through an incision anterior to the sternocleidomastoid muscle, and dissection proceeded along the length of the carotid sheath. A fibrous cord extending into the mediastinum was found in both patients. There were no postoperative complications. Histopathologic evaluation revealed the presence of mature thymic elements within the wall of a multiloculated cyst. CONCLUSIONS: Thymopharyngeal duct cysts must be considered in the differential diagnosis of pediatric neck masses. Computed tomography is helpful to delineate the relationship to the carotid sheath. Complete surgical excision is the appropriate therapy in a majority of cases, with minimal morbidity when careful attention is paid to vital structures.

Age-related changes in muscle fiber regeneration in the human thyroarytenoid muscle.

BACKGROUND: Muscle fiber regeneration is essential to maintain normal muscle fiber populations and muscle mass by continuous replacement of fibers lost to acute muscle injury or overuse. However, the extent of ongoing muscle fiber regeneration in the laryngeal muscles is unknown. OBJECTIVE: The present study provides statistically unbiased, quantitative estimates of the content of regenerating fibers in the human thyroarytenoid muscle over the adult lifespan. DESIGN: In the adult, only regenerating muscle fibers express the developmental myosin isoform. Therefore, regenerating fibers were identified using immunohistochemical techniques. The content of regenerating muscle fibers in the entire muscle volume was then estimated using stereological techniques. Through the use of a computer-automated sampling protocol, stereological data were collected from sets of isotropic uniform random cryostat sections. Overprojection error was minimized by using a confocal laser-scanning microscope to image thin optical sections for use as sample fields. SUBJECTS: Eight autopsy cases, subjects ranging in age from 19 to 81 years. RESULTS: The summed length of fibers expressing developmental myosin increased significantly (P=.02) with age when compared with the overall muscle fiber length. CONCLUSIONS: This finding indicates that muscle fibers maintain the capability for spontaneous regeneration, and that the proportion of regenerating fibers increases as the thyroarytenoid muscle ages. This increase is possibly a compensatory response to an age-related increase in muscle fiber injury or death.

Tubed gastro-omental free flap for pharyngoesophageal reconstruction.

BACKGROUND: Malignant lesions of the pharyngoesophagus often require total laryngopharyngectomy and mediastinal dissection. As a result of the current treatment paradigms for advanced laryngopharyngeal cancers, it is common that the surgical field has been previously irradiated or exposed to systemic chemotherapy, resulting in fistula rates as high as 78% and mortality as high as 8%. The free vascularized tubed gastric antrum and the accompanying greater omentum offer a single-staged method of pharyngoesophageal reconstruction, with the added benefit of protection of the great vessels, the tracheal stump, and the mediastinal contents in a high-risk surgical field. OBJECTIVE: To assess the gastro-omental free flap as a method of pharyngoesophageal reconstruction in patients who have been previously treated with multimodality therapy. METHODS: Five consecutive cases of gastro-omental free flap reconstruction after total laryngopharyngectomy were retrospectively reviewed. Each case was assessed for intraoperative, perioperative, and postoperative complications at the primary site of reconstruction and the donor site. Patients were also evaluated for their ability to maintain an oral diet. Patients were followed up for a minimum of 6 months after surgery. RESULTS: Five patients aged 44 to 70 years (mean, 59 years) underwent gastro-omental free flap reconstruction after total laryngopharyngectomy. Five patients had received previous external beam irradiation, 2 had received systemic chemotherapy, and 4 had undergone previous surgery. There were no fistulae or flap complications. Three patients were successfully treated with esophageal dilation for strictures sustained 2 to 5 months after surgery, and a third patient was successfully treated with conservative management for a partial gastric outlet obstruction sustained 2 months after surgery. One patient died 3 months after surgery of distant metastatic disease. The remaining 4 patients currently tolerate an oral diet. CONCLUSION: The tubed gastro-omental free flap offers a safe method of reconstructing the pharyngoesophageal segment in a surgical field compromised by previous multimodality therapy.

Effect of guanosine nucleotides on the respiratory burst oxidase from human neutrophils.

The respiratory burst is a series of metabolic events which result in the production of microbicidal oxidants by phagocytes. The enzyme responsible for the respiratory burst is a membrane-bound oxidase which catalyzes the production of O-2 by the NADPH-dependent reduction of oxygen. Activity of this oxidase is diminished by a variety of guanosine-containing nucleotides. The decrease in activity caused by GTP, the most potent of these, occurs rapidly and is not solely dependent on hydrolysis of terminal phosphate groups. GTP appears to diminish oxidase activity through both inhibition and inactivation. GTP-mediated inhibition is kinetically mixed with respect to NADPH.

Polytetrafluoroethylene injection for urinary incontinence in children.

We have treated 11 children for urinary incontinence with one or more endoscopic injections of polytetrafluoroethylene (Teflon) into the external urethral sphincter region. The procedure is simple to perform and without significant complications. Long-term results have been gratifying, with cure or improvement in 8 of the 11 patients.

Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria.

Biogenesis of the Vibrio cholerae toxin-coregulated pilus (TCP) requires the activities of at least seven accessory proteins. We demonstrate that a portion of this pathway involves a novel processing step in which a hydrophilic leader peptide is proteolytically removed from TcpA by the gene product characterized in this report, TcpJ, to yield the mature, export-competent form of the pilin. Cleavage of the pilin leader peptide is independent of known signal peptidases as demonstrated by pilin-processing profiles in Escherichia coli strains conditionally defective for production of leader peptidase or grown in the presence of the antibiotic globomycin. Additionally, pilin cleavage did not rely on the SecA protein, as evidenced by TcpA processing in azide-treated cells. These results suggest that TcpJ is representative of a new class of proteins involved in SecA-independent proteolytic cleavage of a set of atypical leader peptides during extracellular export.

Genetic and cytological control of the asymmetric divisions that pattern the Volvox embryo.

The highly regular pattern in which approximately 2000 small somatic cells and 16 large reproductive cells (or 'gonidia') are arranged in a typical asexual adult of Volvox carteri can be traced back to a stereotyped program of embryonic cleavage divisions. After five symmetrical divisions have produced 32 cells of equal size, the anterior 16 cells cleave asymmetrically, to produce one small somatic cell initial and one larger gonidial initial each. The gonidial initials then cease dividing before the somatic cell initials do. The significance of the visibly asymmetric divisions is underscored by genetic and experimental evidence that differences in size--rather than differences in cytoplasmic quality--are causally important in activating the programs that cause small cells to become mortal somatic cells and large cells to differentiate as reproductive cells. A number of loci, including at least five mul ('multiple gonidia') loci, appear to be responsible for determining where and when asymmetric divisions will occur, since mutations at these loci result in modified temporal and/or spatial patterns of asymmetric division in one or more portions of the life cycle. But the capacity to divide asymmetrically at all appears to require a function encoded by the gls (gonidialess) locus, since gls mutants fail to execute any asymmetric divisions. Second-site suppressors of gls that have been identified may encode other functions required for asymmetric division. Cytological and immunocytochemical studies of dividing embryos are being undertaken in an attempt to elucidate the mechanisms by which cell-division planes are established--and shifted--under the influence of such pattern-specifying genes. Studies to date clearly indicate a central role for the basal body apparatus, and particularly its microtubular rootlets, in establishing the orientation of both the mitotic spindle and the cleavage furrow; but it remains to be determined how behavior of the division apparatus becomes modified during asymmetric division.

Antihypertensive drugs and the risk of idiopathic aplastic anaemia.

AIMS: A recent report has raised concern that nifedipine may be associated with an increased risk of aplastic anaemia. This large population-based study evaluated the risk of idiopathic aplastic anaemia in users of calcium channel blockers compared with that of other antihypertensive drugs. METHODS: The study was based on information derived from the General Practice Research Database. We conducted a follow-up study with a nested case-control analysis of 322 448 subjects who received antihypertensive drugs. Cases were people who had a first-time diagnosis of aplastic anaemia during January 1, 1988 through September 30, 1997. The risk estimate of aplastic anaemia was calculated for all antihypertensive drugs. For the nested case-control analysis, six controls were matched to each case on age, sex and general practice attended. Odds ratios compared the risk of idiopathic aplastic anaemia for all antihypertensive drugs relative to nonusers. RESULTS: There were 13 cases of newly diagnosed idiopathic aplastic anaemia. The estimated risk of aplastic anaemia per 100 000 users was 0.8 (95% CI 0.1, 4.7) for calcium channel blockers, 1.4 (95% CI 0.5, 4.1) for beta-adrenoceptor blockers, 2.3 (95% CI 0.6, 8.6) for angiotension-converting enzyme (ACE) inhibitors and 5.9 (95% CI 1.6, 21.5) for users of other antihypertensive drugs. In the case-control analysis of 13 cases and 77 controls, the odds ratio was 0.3 (95% CI 0.02, 3.3) for calcium channel blockers, 0.5 (95% CI 0.1, 2.5) for beta-adrenoceptor blockers, 0.7 (95% CI 0.1, 5.6) for ACE inhibitors, 1.2 (95% CI 0.1, 11.8) for users of other antihypertensive drugs and 0.7 (95% CI 0.1, 7.2) for users of multiple drugs with a calcium channel blocker compared with nonusers. CONCLUSIONS: The present study suggests that the use of calcium channel blockers is not associated with an increased risk of aplastic anaemia.

Bacterial Symbionts Colonize the Accessory Nidamental Gland of the Squid Loligo opalescens via Horizontal Transmission.

The accessory nidamental gland (AN gland), a reproductive organ of the mature female squid Loligo opalescens, harbors a dense culture of bacteria of unknown function. A multilayered sheath surrounding the L. opalescens egg case is similarly colonized by bacteria that presumably originate in the AN gland, as evidenced by their presence in the egg case at oviposition. This study investigates how these bacteria are transmitted to juvenile squid and examines some morphological consequences of bacterial colonization of AN gland tissues. By observing the structure of the AN gland in adults and the development and bacterial colonization of the gland in juveniles raised in captivity, we determined that the AN gland was absent in newly hatched squid and did not appear until 87 days post-hatching. At 129 days posthatching, the organ displayed tubules composed of a single layer of epithelial cells and expressing numerous cilia and microvilli. These tubules were not yet fully formed and thus were open to the mantle cavity and external seawater, possibly to aid in the acquisition of microorganisms. Since the AN gland developed a considerable time after hatching, it most likely acquires its symbionts horizontally from environmental seawater and not vertically from the egg case sheath. The switch from expression of cilia to production of microvilli on the epithelial cell surface may dictate the competence of the tissue for bacterial colonization. Electron microscopic examination of juvenile and adult AN glands revealed that an analogous process occurs during the development of the related light organ of other cephalopod species that harbor symbiotic bacteria.