Human Intestinal Enteroids as a Model System of Shigella Pathogenesis.

The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease; thus, cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells, grown ex vivo, and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigella pathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneri invades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE proinflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneri invasion via the apical surface.

New developments in rabies vaccination.

Current rabies vaccines are safe and, when administered properly, they are highly effective. In addition, they elicit long-lasting immunity, with virus-neutralising antibody titres persisting for years after vaccination. However, current regimens require multiple doses to achieve high neutralising titres and they are costly, which means that it is difficult for developing countries, where rabies deaths are highest, to implement widespread vaccination. New innovations are the only way to reduce rabies disease to acceptable rates. Numerous preclinical and clinical studies are under way, testing novel vaccines, adjuvants and injection methods. Research into the use of live vaccines and alternative vaccine vectors is ongoing, while attempts to develop DNA vaccines have so far failed to match the immunogenicity and neutralising capability of traditional vaccines. The development of molecular adjuvants that induce faster, stronger immune responses with less antigen has yielded exciting preclinical results and appears to edge us closer to a better rabies vaccine. However, steep challenges remain: molecular adjuvants require administration with live vaccines, and differences in species specificity of immune molecules complicate development. Over all, the array of research undertaken over the past decade is impressive and encouraging, but most new vaccines have yet to be tested in clinical trials, and the viability of such experimental vaccines in the global market remains to be seen. Only a vaccine that outperforms currently available vaccines in every area will have a chance at widespread adoption. Nevertheless, the authors are confident that some vaccine candidates will meet these criteria.

Les vaccins actuels contre la rage sont sûrs et très efficaces lorsqu'ils sont administrés correctement. En outre, ils confèrent une immunité durable, avec le maintien de titres neutralisants d'anticorps plusieurs années après la vaccination. Néanmoins, les régimes actuels nécessitent l'administration de plusieurs doses pour obtenir des titres élevés d'anticorps neutralisants et ils sont onéreux, de sorte que la vaccination à grande échelle est difficile à mettre en oeuvre dans les pays en développement, pourtant les plus touchés par la mortalité par rage. Seule l'adoption de solutions innovantes permettra de ramener l'incidence de la rage à un niveau acceptable. De nombreuses études précliniques et cliniques sont en cours, visant à tester les innovations en matière de vaccins, de modes d'injection et d'adjuvants. La recherche sur l'utilisation de vaccins à virus vivant et sur de nouveaux vecteurs vaccinaux se poursuit, alors que les tentatives de développement de vaccins à ADN n'ont pas réussi jusqu'à présent à obtenir un effet immunogène ou des capacités de neutralisation virale équivalents à ceux des vaccins traditionnels. Les résultats d'essais précliniques sur de nouveaux adjuvants moléculaires induisant une réponse immune plus rapide et plus puissante avec moins d'antigène sont extrêmement prometteurs et semblent annoncer l'imminence de meilleurs vaccins contre la rage. Il subsiste toutefois d'importantes difficultés : les adjuvants moléculaires ne peuvent être administrés qu'avec des vaccins vivants et les différences de spécificité d'espèce des molécules immunes rendent le développement plus complexe. Globalement, les efforts déployés depuis une décennie par la recherche sont impressionnants et encourageants mais la plupart des nouveaux vaccins doivent encore être soumis à des essais cliniques ; d'autre part la viabilité de ces vaccins expérimentaux dans le marché mondial reste à démontrer. Seul un vaccin capable de surpasser les performances des vaccins actuels dans chaque domaine aura une chance d'être largement adopté. Les auteurs estiment cependant que certains vaccins candidats pourront satisfaire à ces exigences.

Las actuales vacunas antirrábicas son seguras y, si se administran debidamente, muy eficaces. Además, inducen inmunidad duradera, con títulos de anticuerpos neutralizantes que subsisten años después de la vacunación. Sin embargo, los regímenes actuales resultan costosos y exigen dosis múltiples para lograr títulos de neutralización elevados, lo que dificulta a los países en desarrollo, que son los más golpeados por la rabia, la implantación generalizada de la vacunación. El único camino para reducir la rabia a niveles aceptables pasa por la innovación. Están en marcha numerosos estudios preclínicos y clínicos en los que se ensayan vacunas, adyuvantes y métodos de inyección novedosos. También sigue adelante la investigación sobre el uso de vacunas vivas y vectores vacunales alternativos, mientras que ninguna de las tentativas realizadas hasta la fecha con vacunas de ADN ha deparado niveles de inmunogenicidad y capacidad de neutralización equiparables a los de las vacunas tradicionales. La obtención de adyuvantes moleculares que inducen una respuesta inmunitaria más rápida y vigorosa en presencia de menos cantidad de antígeno ha dado resultados preclínicos muy interesantes y poco a poco parece acercarnos al logro de una mejor vacuna antirrábica. Subsisten, empero, arduas dificultades: los adyuvantes moleculares solo funcionan si se administran con vacunas vivas, y las diferencias existentes entre las especies en cuanto a la especificidad de las moléculas inmunitarias complican las labores de desarrollo. Globalmente, el conjunto de investigaciones emprendidas en el último decenio es impresionante y alentador, pero la mayoría de las nuevas vacunas aún deben pasar por la fase de ensayo clínico, y está por ver qué viabilidad tienen estas vacunas experimentales en el mercado mundial. Solo una vacuna que supere a las actuales en todos los aspectos tiene posibilidades de ser adoptada a gran escala. Pese a todo, los autores expresan su confianza en que algunas de las vacunas candidatas cumplan estos criterios.

Role of intracellular carbon metabolism pathways in Shigella flexneri virulence.

Shigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellular S. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkAB and pykAF mutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway gene eda resulted in small plaques, but the double eda edd mutant formed normal-size plaques. This suggested that the plaque defect of the eda mutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellular S. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-type S. flexneri also formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate of S. flexneri in vitro, suggesting that it may be a preferred carbon source inside host cells.

Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth.

Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.

Systematic review: Helicobacter pylori and the risk of upper gastrointestinal bleeding risk in patients taking aspirin.

BACKGROUND: Aspirin is widely used to modify the risk of recurrent vascular events. It is, however, associated with increased upper gastrointestinal bleeding risk. The influence of Helicobacter pylori on this risk is uncertain. AIM: To determine the influence of H. pylori on upper gastrointestinal bleeding risk in patients taking aspirin. METHODS: MEDLINE and EMBASE databases were searched. All studies providing data regarding H. pylori infection in adults taking aspirin and presenting with upper gastrointestinal bleeding were included. RESULTS: A total of 13 studies that included 1 case-control, 10 cohort studies and 2 randomized-controlled trials (RCTs) were analysed. The case-control study (n = 245) determined H. pylori to be a significant independent risk factor for upper gastrointestinal bleeding. The cohort studies were heterogeneous, varying in inclusion criteria, doses and duration of aspirin used, mode of H. pylori testing and causative GI pathology considered. Comprising 5465 patients, H. pylori infection was tested for in 163 (0.03%) aspirin users with upper gastrointestinal bleeding. The RCTs yielded no significant results. CONCLUSIONS: The current data are not sufficient to allow meta-analyses. The widely held belief that H. pylori is a risk factor for upper gastrointestinal bleeding in regular aspirin users is not supported by the very limited evidence available.

A paradox resolved: sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy.

Vestimentiferan tubeworms, symbiotic with sulfur-oxidizing chemoautotrophic bacteria, dominate many cold-seep sites in the Gulf of Mexico. The most abundant vestimentiferan species at these sites, Lamellibrachia cf. luymesi, grows quite slowly to lengths exceeding 2 meters and lives in excess of 170-250 years. L. cf. luymesi can grow a posterior extension of its tube and tissue, termed a "root," down into sulfidic sediments below its point of original attachment. This extension can be longer than the anterior portion of the animal. Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 microM in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and rarely detectable only around the bases of mature aggregations. Respiration experiments, which exposed the root portions of L. cf. luymesi to sulfide concentrations between 51-561 microM, demonstrate that L. cf. luymesi use their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 micromol x g(-1) x h(-1). Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior (root) portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L. cf. luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.

Hemoglobin from a deep-sea hydrothermal-vent copepod.

Deep-sea hydrothermal-vent fauna live in a highly variable environment where oxygen levels can be very low, and carbon dioxide and sulfide can reach high concentrations (1). These conditions are harsh for most aerobic metazoans, yet copepods can be abundant at hydrothermal vents. Here we report the structure and functional properties of hemoglobin extracted from the copepod Benthoxynus spiculifer, which was found in large numbers in a paralvinellid/gastropod community collection made during a cruise to the Juan de Fuca Ridge in 1998. Although hemoglobin has been reported in some littoral copepods (2), this is the first study of the structure and functional properties of copepod hemoglobin. Hemoglobin represents about 60% of the total soluble proteins extracted from B. spiculifer, and although it imparts a red color to the copepod, it does not provide a significant storage pool of oxygen. It is a 208-kDa protein, composed of 14 globin chains--7 of 14.3 kDa and 7 of 15.2 kDa. The hemoglobin has a very high and temperature-sensitive oxygen affinity, with no cooperativity or Bohr effect. These properties are adaptive for an animal living in a low-oxygen environment in which the primary function of the hemoglobin is most likely oxygen acquisition to support aerobic respiration.

Methane ice worms: Hesiocaeca methanicola colonizing fossil fuel reserves.

During a research cruise in July 1997 in the Gulf of Mexico we discovered a gas hydrate approximately 1 m thick and over 2 m in diameter which had recently breached the sea floor at a depth of 540 m. The hydrate surface visible from the submarine was considerably greater than that of any other reported hydrate. Two distinct color bands of hydrate were present in the same mound, and the entire exposed surface of the hydrate was infested (2500 individuals/m2) with 2 to 4 cm-long worms, since described as a new species, Hesiocaeca methanicola, in the polychaete family Hesionidae (Desbruyères and Toulmond 1998). H. methanicola tissue stable isotope values are consistent with a chemo-autotrophic food source. No evidence of chemo-autotrophic symbionts was detected, but geochemical data support the presence of abundant free living bacteria on the hydrate. The activities of the polychaetes, grazing on the hydrate bacteria and supplying oxygen to their habitats, appears to contribute to the dissolution of hydrates in surface sediments.

Herbal preparations: a primer for the aeromedical physician.

PURPOSE: This study was an effort to identify the botanical preparations of potential risk to the aviator and aviation safety, and to ascertain whether aviators are using dietary supplements despite extensive educational efforts discouraging over-the-counter medication use. Herbal preparations may be used by nearly 20% of the adult population. Although the aviator population may be presumed to use them as well, the actual degree of use among aviators is unknown. Use of such substances as health promotion or therapeutic agents may provide health benefits, but may also carry risk. Military and civilian aviators are not currently required to disclose such use, nor are examiners obligated to inquire or counsel aviators about them. This paper examines the trends in post-mortem toxicological samples suggesting botanical preparation use, and develops a rational method for determining suitability for use by the aviator. METHOD: The toxicological test results from 3177 mishap pilots performed at the Civil Aeromedical Institute from 1989-1997 were examined for the presence of substances suggesting botanical preparation use. The prevalence of positive test results for ephedrine among mishap pilots was compared with the prevalence of tests positive for chemically and biologically similar non-botanical substances among mishap pilots. A review of existing literature was also performed to identify substances posing possible risk to the aviator health or aviation safety. RESULTS: Ephedrine was found to be the only substance routinely screened on toxicological specimens that was suitable for association with botanical substance utilization. The percent of specimens positive for ephedrine increased three- to four-fold while the percent of specimens positive for similar non-botanical substances decreased overall. The literature revealed sufficient evidence that a number of open market botanical agents are capable of causing incapacitation by cardiovascular or neuropsychiatric mechanisms, yet are legally permitted for use by aviators. CONCLUSION: Aviators are using botanical products with increasing frequency, and many of those substances may pose significant risk of incapacitation, altered sensorium, or adverse health effects. The flight surgeon must be diligent in eliciting a history of use and assisting aviators to minimize personal risk and risks to flight safety. A rational approach to assessing risk is presented.

Tubeworm succession at hydrothermal vents: use of biogenic cues to reduce habitat selection error?

Species colonizing new deep-sea hydrothermal vents along the East Pacific Rise show a distinct successional sequence: pioneer assemblages dominated by the vestimentiferan tubeworm Tevnia jerichonana being subsequently invaded by another vestimentiferan Riftia pachyptila, and eventually the mussel Bathymodiolus thermophilus. Using a manipulative approach modified from shallow-water ecological studies, we test three alternative hypotheses to explain the initial colonization by T. jerichonana and its subsequent replacement by R. pachyptila. We show that R. pachyptila and another vestimentiferan, Oasisia alvinae, colonized new surfaces only if the surfaces also were colonized by T. jerichonana. This pattern does not appear to be due to restricted habitat tolerances or inferior dispersal capabilities of R. pachyptila and O. alvinae, and we argue the alternative explanation that T. jerichonana facilitates the settlement of the other two species and is eventually outcompeted by R. pachyptila. Unlike the classic model of community succession, in which facilitating species promote their own demise by modifying the environment to make it more hospitable for competitors, we suggest that T. jerichonana may produce a chemical substance that induces settlement of these competitors. This process of selecting habitat based on biogenic cues may be especially adaptive and widespread among later-successional species that occupy a physically variable and unpredictable environment. In these cases, the presence of weedy species implies some integrated period of environmental suitability, whereas an instantaneous assessment of physical habitat conditions, such as water temperature for vent tubeworms, provides a poorer predictor of long-term habitat suitability.

Metazoans in extreme environments: adaptations of hydrothermal vent and hydrocarbon seep fauna.

Some of the most extreme environments where animals survive are associated with active vents and seeps in the deep sea. In addition to the extreme pressure, low temperatures, and lack of light that characterize the deep sea in general, a variety of other factors that are hostile to most animals prevail in these environments. Hydrothermal vent regions show extremes in temperature, areas of very low oxygen, and the presence of toxic hydrogen sulfide and heavy metals. Hydrocarbon seeps, though much cooler than vents, also have regions of very low oxygen and high hydrogen sulfide, as well as other potentially harmful substances such as crude oil and supersaturated brine. Specially adapted animals not only tolerate these conditions, they often thrive under them. In most cases this tolerance is due to a combination of physiological and behavioral adaptations that allow animals to avoid the extremes of their habitats and yet benefit from the chemoautotrophic production characteristic of these environments.

Roots as a site of hydrogen sulfide uptake in the hydrocarbon seep vestimentiferan Lamellibrachia sp.

Vestimentiferan tubeworms have no mouth or gut, and the majority of their nutritional requirements are provided by endosymbiotic bacteria that utilize hydrogen sulfide oxidation to fix CO(2) into organic molecules. It has been assumed that all vestimentiferans obtain the sulfide, O(2) and CO(2) needed by the bacteria across the plume (gill) surface, but some live in locations where very little sulfide is available in the sea water surrounding the plume. We propose that at least some of these vestimentiferans can grow a posterior extension of their body and tube down into the sea-floor sediment, and that they can use this extension, which we call the 'root', to take up sulfide directly from the interstitial water. In this study of the vestimentiferan Lamellibrachia sp., found at hydrocarbon seeps in the Gulf of Mexico at depths of approximately 700 m, we measured seawater and interstitial sulfide concentrations in the hydrocarbon seep habitat, determined the structural characteristics of the root tube using transmission electron microscopy, characterized the biochemical composition of the tube wall, and measured the sulfide permeability of the root tube. We found that, while the sulfide concentration is less than 1 (micro)mol l(-)(1) in the sea water surrounding the gills, it can be over 1.5 mmol l(-)(1) at a depth of 10-25 cm in sediment beneath tubeworm bushes. The root tube is composed primarily of giant (&bgr;)-chitin crystallites (12-30 % of total mass) embedded in a protein matrix (50 % of total mass). Root tubes have a mean diameter of 1.4 mm, a mean wall thickness of 70 (micro)m and can be over 20 cm long. The tubeworm itself typically extends its body to the distal tip of the root tube. The root tube wall was quite permeable to sulfide, having a permeability coefficient at 20 degrees C of 0. 41x10(-)(3 )cm s(-)(1), with root tube being 2.5 times more permeable to sulfide than trunk tube of the same diameter. The characteristics of the root suggest that it reaches down to the higher sulfide levels present in the deeper sediment and that it functions to increase the surface area available for sulfide uptake in a manner analogous to a respiratory organ.

Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene.

It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor alpha. These mice became infertile primarily because of an interruption of fluid reabsorption by the efferent ductules of the epididymis, thus leading to a disruption of the seminiferous epithelium [Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) Nature (London) 390, 509-512]. Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of the cyp19 gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia/hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.

Assisted bibliotherapy: effective, efficient treatment for moderate anxiety problems.

Specific psychological treatments of proven effectiveness for moderate anxiety disorders are not often easily accessible in general practice. In this study, selected patients were supported in learning skills to manage their symptoms. This approach was efficient, acceptable, and led to clinically significant symptom reduction for a high proportion of patients. This improvement was well sustained at three-month follow-up.

Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene.

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor alpha is deleted by targeted disruption.

Sulfide binding in the body fluids of hydrothermal vent alvinellid polychaetes.

Dissolved H2S is a major environmental factor in hydrothermal vent ecosystems. In a study of adaptations to sulfide by alvinellid polychaetes, the sulfide-binding capacity of body fluids was examined in Paralvinella palmiformis from northeast Pacific ridges and Alvinella species from the East Pacific Rise. Sulfide concentrations in vascular blood and coelomic fluid of freshly collected animals were notably variable. Separation of P. palmiformis body-fluid components revealed that most sulfide (ca. 77%) was accumulated in the dissolved fraction. In P. palmiformis, both vascular blood and coelomic fluid could reversibly bind sulfide in vitro with a low affinity, saturating only at high dialysate concentrations (ca. 2 mmol L-1). No sulfide-binding activity was observed in the vascular blood from Alvinella species. A dissolved protein component of greater than 90 kDa appears to be involved in sulfide binding in Paralvinella, probably a vascular extracellular high-molecular-weight hemoglobin. Some sulfide may also adsorb onto a 15.38-kDa intracellular hemoglobin present in the coelomic erythrocyte fraction. In the absence of epibiotic bacteria, Paralvinella body fluids may function as a sulfide buffer to protect tissues from deleterious effects of sulfide exposure.

Aromatase expression in health and disease.

Family 19 of the P450 superfamily is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization, since it involves conversion of the delta 4-3-one A-ring of the androgens to the corresponding phenolic A-ring characteristic of estrogens. Its members occur throughout the entire vertebrate phylum. The reaction mechanism of aromatase is very interesting from a chemical point of view and has been studied extensively; however, a detailed examination of structure-function relationships has not been possible due to lack of a crystal structure. Recent attempts to model the three-dimensional structure of aromatase have permitted a model that accounts for the reaction mechanism and predicts the location of aromatase inhibitors. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, since there are a number of untranslated first exons that occur in aromatase transcripts in a tissue-specific fashion, due to differential splicing as a consequence of the use of tissue-specific promoters. Thus, expression in ovary utilizes a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in placenta utilizes a distal promoter that is located at least 40 kb upstream of the start of transcription and that is regulated by retinoids. Other promoters are employed in brain and adipose tissue. In the latter case, class I cytokines such as IL-6 and IL-11 as well as TNF alpha are important regulatory factors. PGE2 is also an important regulator of aromatase expression in adipose mesenchymal cells via cAMP and PGE2 appears to be a major factor produced by breast tumors that stimulates estrogen biosynthesis in local mesenchymal sites. In all of the splicing events involved in the use of these various promoters, a common 3'-splice junction is employed that is located upstream of the start of translation; thus, the coding regions of the transcripts- and hence the protein-are identical regardless of the tissue site of expression; what differ in a tissue-specific fashion are the 5'-ends of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint as well as for the physiology and pathophysiology of estrogen formation. Recently, a number of mutations of the aromatase gene have been described, which give rise to complete estrogen deficiency. In females this results in virilization in utero and primary amenorrhea with hypergonadotropic hypogonadism at the time of puberty. In men the most striking feature is continued linear bone growth beyond the time of puberty, delayed bone age, and failure of epiphyseal closure, thus indicating an important role of estrogens in bone metabolism in men. In both sexes the symptoms can be alleviated by estrogen administration.