Tyrannobdella rex N. Gen. N. Sp. and the Evolutionary Origins of Mucosal Leech Infestations

Background

Leeches have gained a fearsome reputation by feeding externally on blood, often from human hosts. Orificial hirudiniasis is a condition in which a leech enters a body orifice, most often the nasopharyngeal region, but there are many cases of leeches infesting the eyes, urethra, vagina, or rectum. Several leech species particularly in Africa and Asia are well-known for their propensity to afflict humans. Because there has not previously been any data suggesting a close relationship for such geographically disparate species, this unnerving tendency to be invasive has been regarded only as a loathsome oddity and not a unifying character for a group of related organisms.

Principal Findings

A new genus and species of leech from Perú was found feeding from the nasopharynx of humans. Unlike any other leech previously described, this new taxon has but a single jaw with very large teeth. Phylogenetic analyses of nuclear and mitochondrial genes using parsimony and Bayesian inference demonstrate that the new species belongs among a larger, global clade of leeches, all of which feed from the mucosal surfaces of mammals.

Conclusions

This new species, found feeding from the upper respiratory tract of humans in Perú, clarifies an expansion of the family Praobdellidae to include the new species Tyrannobdella rex n. gen. n.sp., along with others in the genera Dinobdella, Myxobdella, Praobdella and Pintobdella. Moreover, the results clarify a single evolutionary origin of a group of leeches that specializes on mucous membranes, thus, posing a distinct threat to human health.     

Highly Active AntiRetroviral Therapy and cryptosporidiosis

In HIV infected persons, Cryptosporidium parvum causes chronic diarrhoea, which can be life-threatening in persons with AIDS and with a low CD4+ T cell count. However, a specific and effective therapy for this opportunistic infection does not yet exist. Since the use of a combination therapy with a highly active antiretroviral therapy (HAART), the prevalence of C. parvum infection in persons with AIDS has been strongly reduced. This favorable outcome was usually attributed to the recovery of the host immunity, however improvements from this opportunistic infection have been demonstrated even in the absence of immunological recovery. The aim of the present study was to determine whether HIV protease inhibitors (PIs) exert an anti-C. parvum activity. We selected the indinavir (an aspartyl protease inhibitor included in HAART) for our experiments, since a resolution of cryptosporidial enteritis in a person with AIDS after treatment with this drug has been reported. Human ileocecal adenocarcinoma tumor cells (HCT-8) were used as in vitro model. To determine whether or not indinavir had an effect on the parasite attachment to, or invasion of the HCT-8 cells, indinavir was added to the cultures at the same time as the infective dose (3 oocysts/cell) at one of the following concentrations: 0.1, 0.5, 5, 10, 20, and 50 microM (maximum DMSO content 0.5% vol/vol). To determine whether or not indinavir had an effect on established C. parvum infection, HCT-8 cells were infected with excysted oocysts at a ratio of 3 oocysts/cell at day 0, and then indinavir at a concentration of 50 microM was added to the cultures every 24 h for 4 days. The infection level was evaluated at 2, 3, 4 and 5 days p.i. using a flowcytometric assay. Three-day-old Balb/c mice were used as animal model, animals were infected per os with 50 microl of PBS containing 10(5) oocysts. The infected mice were divided into two groups (Group A and Group B), both of which received per os indinavir diluted in PBS with 0.1% DMSO at a concentration of 10 microM (24 mg/kg). For Group A, which consisted of 15 mice (3 litters), indinavir was administered at the same time that experimental infection was performed and then every day until the mice were sacrificed (i.e., 5 days p.i.), to determine the effect of indinavir on the attachment/invasion of the enterocytes. For Group B, which also consisted of 15 mice (3 litters), indinavir was administered after the infection was established (i.e., 72 h p.i.) and every day until being sacrificed, to determine the effect of indinavir on established infection. The mice of Group B were sacrificed 7, 10, 11 and 13 days p.i., corresponding to 4, 7, 8, and 10 days of treatment with indinavir. In vitro, the treatment of the excystated oocysts with different concentrations of indinavir reduced the percentage of HCT-8 infected cells in a dose-dependent manner. For established infection, the treatment with 50 microM of indinavir decreased the percentage of infected cells in a time-dependent manner. Treatment for 48 h resulted in a 40.1% reduction in infected cells (from 90% to 53%). After 72 h of treatment, the percentage of infected cells did not substantially differ from that observed after 48 h. Treatment for 96 h resulted in a 57.8% reduction (from 90 to 38%). In vivo, mice treated with indinavir at the same time they were infected with the oocysts showed a 93% reduction in the number of oocysts present in the entire intestinal contents and a 91% reduction in the number of intracellular parasites in the ileum. For established infection, indinavir treatment reduced the number of oocysts in the entire intestinal content by about 50% and the number of intracellular parasites in the ileum by about 70%. These data demonstrate that PIs directly exert an inhibitory effect on C. parvum and the extent of this effect depended on the specific dose and the duration of treatment. Although there are no reports of aspartyl proteases in C. parvum, the inhibitory effect of PIs on C. parvum growth in vitro suggests that aspartyl proteases could have some important functions for this parasite. In fact, proteolytic activities have been demonstrated during peak periods of excystation in C. parvum oocysts and cysteine and serine protease classes have been functionally associated with this process. Moreover, we identified several different C. parvum sequences that showed homology with a protein family related to aspartyl proteases. In prospect, PIs could be valuable for the chemotherapy of cryptosporidiosis.     

Correction of image instability in confocal microscopy using image realignment. Effects on the analysis of intracellular calcium

Using confocal microscopy, we have examined the increases in [Ca(2+)](i) evoked by sodium channel toxins in cells labelled with the fluorescent dye INDO-1. We describe a new image analysis method that improves the detection of region-specific, toxin-induced patterns of change of intracellular calcium. This method is based on correction of global image motion followed by calculation of the strength of correlation between calcium changes in "seed" or reference pixels chosen to represent different regions of cells and those in other regions of the image. When the selected "seed" pixel was chosen to be in either varicosities or neurites, correlations were detected in the same regions of other cells as well as in the soma, indicating specific but spatially distinct patterns of behaviour. Control images (without changes in [Ca(2+)](i)) did not reveal significant interpixel correlations. The ability to recognize correlated patterns of calcium change in different regions of cells was greatly improved by correction for global motion.     

Plasma determination of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide and preliminary pharmacokinetic studies in the rat

A sensitive gas chromatographic method with flame ionization detection was developed for the analysis in plasma of the novel anticonvulsant d,l-3-hydroxy-3-ethyl-3-phenylpropionamide (HEPP), using d,l-2-hydroxy-2-ethyl-2-phenylacetamide as the internal standard. HEPP was extracted from alkalinized plasma into dichloromethane and quantified after derivatization with bis(trimethylsilyl)-trifluoroacetamide. Standard curves were linear from 0.5 to 50 and from 2 to 100 μg/ml of plasma, using 1.5 and 5 μg of the internal standard, respectively. The lower limit of detection at a signal-to-noise ratio of 3 standard deviations was 0.33 μg/ml of sample. The sensitivity, accuracy and reproducibility of the method were shown to be satisfactory for pharmacokinetic studies of HEPP. After intraperitoneal administration of 50 mg/kg to Wistar rats, the principal kinetic parameters were: absorption half-life = 0.04 h; volume of distribution = 1.32 l/kg; clearance = 4.40 ml/min; peak concentration = 50 μg/ml; peak time = 0.25 h; mean residence time = 4.55 h.     

Sodium chloride absorption across the ileal epithelium of the lizardGallotia galloti

Summary Ion transport processes in the ileum of the lizard,Gallotia (=Lacerta) galloti was examined in vitro by measuring Na²² and Cl³⁶ fluxes across short-circuited preparations.In Ringer-bicarbonate solution there was both a net sodium flux ( ) and a net chloride flux ( ) from mucosa to serosa. The inequality between and short-circuit current (I _sc) suggests that part of the net sodium transport is the result of an electrically neutral transport mechanism or that another electrogenic mechanism opposite in sign is contributing to the short-circuit current.In the absence of sodium, the short-circuit current and net chloride flux were abolished. In the absence of chloride, the net sodium was reduced but not abolished and the short-circuit current was unchanged.From an analysis of the effects of the inhibitors furosemide, amiloride, disulfonic stilbene (DIDS) and acetazolamide, a plausible model was developed to explain the characteristics of these transports. It is proposed that the entry of sodium into the cell across the luminal membrane occurs by two pathways. Part occurs by the antiport Na⁺H⁺ and part by an electrogenic pathway. The entry of chloride is by the antiport Cl^–HCO ₃ ^– .Symbols and abbreviations DIDS 4,4 diisothiocyanatostilbene-2,2-disulfonic acid - G _t tissue conductance - I _sc short circuit current - m mucosal - PD potential difference - s serosal     

Alterations in endothelial cell proteinase and inhibitor polarized secretion following treatment with interleukin-1, phorbol ester,and human melanoma cell conditioned medium

Polarized secretion of matrix metalloproteinases and plasminogen activators by monkey aortic endothelial cells was studied in vitro, using transwell inserts. The endothelial cells constitutively expressed matrix metalloproteinase-2, tissue inhibitors of metalloproteinases 1 and 2, urokinase, and tissue plasminogen activator, all with basal preference. Matrix metalloproteinase-9 activity was induced by phorbol 12-myristate 13-acetate (apical), interleukin-1α (basal), and by conditioned medium from DX3 human melanoma cells (basal). The DX3 melanoma conditioned medium also stimulated basal secretion of matrix metalloproteinase-2, urokinase, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. The rise in proteolytic activity in the basal direction was reflected by increased capacity to degrade subendothelial basement membrane type IV collagen, shown immunohistologically, using monkey kidney tissue sections and basement membrane deposited by endothelial cells into the transwell membrane. Thus, IL-1α and DX3 melanoma conditioned medium can stimulate endothelial cells in vitro to concentrate secretion of proteinases spatially onto the underlying basement membrane. We suggest that the stimulation of endothelial cell proteinase activity by tumor cells may facilitate tumor cell extravasation. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Synteny between a major heading-date QTL in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) and the<Emphasis Type="Italic"> Hd3</Emphasis> heading-date locus in rice

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.Communicated by Q. Zhang     

Patterns of variation in desiccation resistance in a set of recombinant inbred lines in Drosophila melanogaster

Desiccation, resulting from extremely dry environmental conditions, is a serious obstacle to the survival of organisms. Water is vital for the maintenance of intracellular structure and prevents the irreversible formation of aggregates, an occurrence leading to loss of cellular function. To characterize genetic variation in desiccation stress resistance (DSR) in Drosophila melanogaster Meigen, an intercontinental set of recombinant inbred lines (RIL) is used. Flies are exposed to a low humidity environment (<10% relative humidity) at a constant temperature of 25 °C. Desiccation stress resistance is higher in RIL derived from a backcross to the parental stock sensitive to heat stress (from Denmark) than in RIL derived from the reciprocal backcross to the heat‐stress resistant stock (from Australia). Composite interval mapping reveals significant quantitative trail loci (QTL) for DSR in the set of RIL. Both major and minor effects QTL are detected, suggesting a complex genetic architecture. When compared with a previous investigation performed on the same set of RIL, the present study indicates that not all traits of resistance to environmental stressors are affected in the same direction by segregating co‐localized QTL.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.     

Inter-male aggression with regard to polygynous mating system in Pampean grassland mouse, Akodon azarae (Cricetidae: Sigmodontinae)

Based on the hypothesis that, in Akodon azarae, polygyny operates through female defence, we studied inter-male aggression in order to test the following predictions: during the breeding period (1) resident males are more aggressive than intruder males in the presence of females (FP), and (2) aggressive behaviour is independent of male condition (resident or intruder) in the absence of females (FA). To test our predictions, we used the resident male behavioural response towards an intruder male in relation to FP or FA. We conducted 30 encounters in FP and 27 in FA in 0.79-m² round enclosures placed in the Espinal Reservation. Our results support the prediction that, in FP, the intensity of aggressive behaviour exhibited by males varied in relation to resident or intruder condition. Resident males showed high levels of aggression towards intruders, and intruders exhibited the greatest values of submissive behaviours with residents. In FA, the intensity of aggressive behaviour did not vary in relation to resident or intruder condition. Both resident and intruder males exhibited low aggressive behaviour and inter-male encounters resulted mainly in non interactive behaviours. Our results support the hypothesis that, in A. azarae, the polygynous mating system operates through female defence.