Prolonged bleeding from the bite of the Asian medicinal leech Hirudo nipponia

Selected antihaemostatic parameters associated with the bite of the Asian medicinal leechHirudo nipponia were investigated in human volunteers. This study confirms earlier work onHirudo medicinalis, in that the wound from a leech bite bleeds for hours even though blood coagulates normally after about 15 min. The whole blood clotting time forH. nipponia after 1 min was 12.88±3.15 min, comparable to that forH. medicinalis, but in both cases clotting time returned to control levels after about 15 min. However, the duration of bleeding from the bite ofH. nipponia (mean=210 min) was consistently shorter thanH. medicinalis, even when adjusted for size differences (mean=490 min). Similarly, the blood flow rate fromH. nipponia (mean-39 μl/min) is markedly slower thanH. medicinalis (mean =200 μl/min). The total blood lost from the host, therefore, was approximately ten times more withH. medicinalis thanH. nipponia. Collagen-induced platelet aggregation was significantly impaired in blood from theH. nipponia bite wound in 25 min. This may indicate that the prolongation of bleeding is caused by inhibition of platelet aggregation rather than by thrombin inhibition alone. In both species, the saliva of the leech contains a potent antithrombin whose inhibitory activity returns to normal levels approximately 15 min after cessation of feeding. The internal amino acid sequence of the thrombin inhibitor secreted byH. nipponia into the saliva is unexpectedly different (55%) from that of hirudin secreted byH. medicinalis. This difference in sequence is reflected in the lack of neutralisation by the polyclonal antibody to hurudin fromH. medicinalis. Blockage of the N-terminal in antithrombin fromH. nipponia appears to be a further real difference compared to hirudin fromH. medicinalis. The biological significance, if any, of these differences between species in bleeding time and antithrombin structure remains an open, but intriguing, question.     

Bleeding in human volunteers from the bite of the American medicinal leech Macrobdella decora compared with its European counterpart Hirudo medicinalis

In this paper the effectiveness of the so-called American medicinal leech Macrobdella decora in overcoming human haemostasis is compared to that of the European medicinal leach Hirudo medicinalis. Thrombin-clotting times indicated that M. decora prevents coagulation of human blood by means of anti-thrombin activity. Our findings suggest that its role is similar to that of hirudin. In human volunteers, the duration of bleeding from the bite of M. decora (mean = 73 min; n = 18) is significantly shorter than H. medicinalis (mean = 600 min; n = 15). The mean concentration of anti-thrombin units in each specimen of M. decora and H. medicinalis is 100 and 285 AT-U, respectively. These findings support the concept that H. medicinalis is more advanced in terms of haematophagous predation. Despite similar feeding durations by both species of leech (means = 68 min and 70.5 min by M. decora and H. medicinalis, respectively), the mean increase in body weight of M. decora was only 58% compared to 460% in H. medicinalis.     

Alpha-conotoxin ImI disrupts central control of swimming in the medicinal leech

Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal's midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, α-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals.     

Effect of mu and kappa opioids on injury-induced microglial accumulation in leech CNS: involvement of the nitric oxide pathway

Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. Opioids have been postulated to modulate various parameters of the immune response. Morphine and leech morphine-like substance are shown to release NO and suppress microglial activation. Regarding the known immuno-modulatory effects of selective mu and kappa ligands, we have assessed the effect of these agents on accumulation of microglia at the site of injury in leech CNS. Leech nerve cords were dissected, crushed with fine forceps and maintained in different concentrations of opiates in culture medium for 3 h and then fixed and double stained with Hoechst 33258 and monoclonal antibody to endothelial nitric oxide synthase (NOS). Morphine and naloxone (> or =10(-3) M) but not selective mu agonist, DAMGO [d-Ala2, N-Me-Phe-Gly5(ol)-enkephalin] and antagonist, CTAP [D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] inhibited the microglial accumulation. The effect of morphine was abrogated by pre-treatment with naloxone and also non-selective NOS inhibitor, l-NAME [N(omega)-nitro-l-arginine-methyl-ester; 10(-3) M] implying an NO-dependent and mu-mediated mechanism. These results are similar to properties of recently found mu-3 receptor in leech, which is sensitive to alkaloids but not peptides. Both selective kappa agonist, U50,488 [3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)-benzeneacetamide; > or =10(-3) M], and antagonist, nor-binaltorphimine (nor-BNI; > or =10(-3) M), inhibited the accumulation. The effect of nor-BNI was reversed by l-NAME. Immunohistochemistry showed decreased endothelial NOS expression in naloxone and U50,488-treated cords. Since, NO production at the injury site is hypothesized to act as a stop signal for microglias, opioid agents may exert their effect via changing of NO gradient along the cord resulting in disruption of accumulation. These results suggest an immuno-modulatory role for mu and kappa opioid receptors on injury-induced microglial accumulation which may be mediated via NO.     

Helicobacter pylori and cagA and vacA gene status in children from Brazil with chronic gastritis

Abstract. Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1–16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Antigenic similarities in lipopolysaccharides of Haemophilus and Neisseria and expression of a digalactoside structure also present on human cells

Monoclonal antibodies raised against Haemophilus influenzae and Neisseria gonorrhoeae were used to investigate similarities or differences in the lipopolysaccharide antigens of pathogenic and commensal strains of several Gram-negative bacteria indigenous to mucosal surfaces of humans. In immunoblotting experiments, 20 of 36 monoclonal antibodies showed cross-reactions between species of Neisseria and Haemophilus. The common epitopes were present on N. gonorrhoeae, N. meningitidis, N. lactamica, H. influenzae including biogroup aegyptius, and occasionally H. parainfluenzae. No other commensal Neisseria or Gram-negative organisms tested reacted with the monoclonal antibodies with one exception; a single strain of pathogenic Escherichia coli was recognised by a N. gonorrhoeae-specific monoclonal antibody. One monoclonal antibody, raised against N. gonorrhoeae lipopolysaccharide, reacted with N. gonorrhoeae (32 of 59 strains), N. meningitidis (9 of 26 strains), H. influenzae (6 of 16 strains). An epitope expressed by H. influenzae and implicated in its virulence was also present on 14 of 59 strains of N. gonorrhoeae and was shown to comprise a digalactoside structure, α-galactosyl-1,4-β-galactose (Galα1, 4Galβ), also found on human cells.     

Phylogeny and evolution of the NS1 and VP1/VP2 gene sequences from porcine parvovirus

The objective of the present study was to gain new insights on the evolution and phylogeny of the PPV genome, specifically on the NS1 and VP1/VP2 genes. Moreover, two new complete sequences from PPV isolates from China (BQ and ZJ strains) were generated and included in the study. The data set studied contained available NS1 and VP1/VP2 sequences at the GenBank database, plus those corresponding to the mentioned Chinese isolates. PPV sequences were divided into two major groups, with one group separated into two branches. Both phylogenetic groups were homogeneous and several marker aminoacidic changes and synapomorphic positions were identified along both genes. Despite the two genes were satisfactory molecular markers, the absence of selection pressure on the VP1/VP2 fragment makes it a preferential option compared to the NS1 one. Furthermore, NS1 gene showed a biased mutation pattern compared with VP1/VP2 genes, which is compatible with the existence of selection in the first but not in the second gene (as indicated by the negative difference between non-synonymous and synonymous values). No correlation between NS1 and VP1/VP2 phylogenetic groups and/or branches and health status was observed. However, a relationship among virulence and the absence of the 127-bp repeat located downstream the part of ORF2 encoding the structural proteins VP1 and VP2 cannot be excluded.     

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials

cDNAs encoding IgM heavy chain constant region (Cμ) were isolated from two metatherians (marsupials) — the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cμ in both species. The domain size and structure of the marsupial Cμ sequences were compared with other Cμ sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.     

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio – Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n = 20), Typhimurium (n = 4), Zanzibar (n = 2), Manhattan (n = 1), Bovismorbificans (n = 1), Amsterdam (n = 1), Saint Paul (n = 1), Kentucky (n = 1) and Muenster (n = 1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1–5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.     

Sarcocystis arctosi sp. nov. (Apicomplexa, Sarcocystidae) from the brown bear ( Ursus arctos ), and its genetic similarity to schizonts of Sarcocystis canis -like parasite associated with fatal hepatitis in polar bears ( Ursus maritimus )

The tissues of herbivores are commonly infected with cysts of parasites belonging to the apicomplexan genus Sarcocystis, but such sarcocysts are rare in bears. Here, we describe a new species, Sarcocystis arctosi, based on the mature sarcocysts identified in two brown bears (Ursus arctos) from Alaska, USA. Microscopic sarcocysts (37–75 × 20–42 μm) had thin walls (<1 μm). The outer layer of the sarcocyst, the parasitophorous vacuolar membrane (pvm), was wavy in outline and had minute undulations that did not invaginate towards the sarcocyst interior; these undulations occurred at irregular intervals and measured up to 100 nm in length and up to 60 nm width. The ground substance layer beneath the pvm was smooth and lacked microtubules. Longitudinally cut bradyzoites measured 5.6–6.8 × 0.7–1.8 μm. A major portion of nuclear small subunit rDNA sequence obtained from these sarcocysts was similar to that previously obtained from the hepatic schizonts of a S. canis-like parasite from polar bears (Ursus maritimus).     

Investigation of Batten Disease with the YeastSaccharomyces cerevisiae

TheCLN3gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned theSaccharomyces cerevisiaehomolog to the humanCLN3gene, designatedBTN1,whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p,btn1-Δ deletion yeast strains, are more resistant to -(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the humanCLN3gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced intoBTN1,altered the degree of ANP resistance. Severity of Batten disease due to mutations inCLN3and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.     

A comparison of the leech Theromyzon tessulatum angiotensin I-like molecule with forms of vertebrate angiotensinogens: a hormonal system conserved in the course of evolution

After five steps of purification including gel permeation, anti-angiotensin I affinity column chromatography followed by reverse-phase HPLC, a peptide immunoreactive to two different antisera (anti-angiotensin I) was purified to homogeneity from extracts of the leech Theromyzon tessulatum. The first 14 amino acid residues of the purified peptide (DRVYIHPFLLXWG) established by automated Edman degradation, reveal the existence in leeches of an angiotensin I-like molecule close to human angiotensin I. The sequence of the purified peptide presents 78.5% of homology with the N-terminal part of human angiotensin. Moreover, in its sequence, this peptide presents the cleavage sites of vertebrate angiotensin metabolic enzymes, i.e. the renin and the angiotensin-converting enzyme. This finding constitutes the first biochemical characterization of an angiotensin I in Invertebrates. It also reflects the high conservation of angiotensins in the course of evolution, suggesting a fundamental role of this family in fluid homeostasis.     

RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi

In this study, we used real-time PCR to simultaneously monitor the responses of 12 key genes of the shrimp innate immune system in Litopenaeus vannamei after challenge with Vibrio harveyi. In the proPO activating system, we found that proPO was up-regulated (3.3× control at 36 hpi). The hemolymph clotting genes transglutaminase (TGase) and clotting protein were also up-regulated, as were 5 genes in the antimicrobial peptide system (ALF, Crustin, Lyz, PEN2 and PEN4), with only PEN3 showing no significant changes. In the antioxidant defense system, SOD was slightly elevated while GPx was substantially down-regulated. In the pattern recognition receptor system, at 24 hpi, the Toll gene (LvToll) showed the highest relative increase in expression level of all the investigated genes (15× greater than the sterile seawater control). In the second part of this study, when LvToll was knocked down by RNAi silencing, there was no effect on either survival rates or bacterial number in unchallenged shrimp. There was also no difference in mortality rates between control shrimp and LvToll-silenced shrimp when these two groups were challenged with a viral pathogen (white spot syndrome virus; WSSV). However, when LvToll-silenced shrimp were challenged by V. harveyi, there was a significant increase in mortality and bacterial CFU counts. We note that the increase in bacterial CFU count occurred even though treatment with EGFP dsRNA had the opposite effect of reducing the CFU counts. We conclude that LvToll is an important factor in the shrimp innate immune response to acute V. harveyi infection, but not to WSSV.     

Promoter Methylations of p16 and p14 Genes in Early and Advanced Gastric Cancer. Correlations of the Modes of Their Occurrence with Histologic Type

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16^INK4a and p14^ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16^INK4a and p14^ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16^INK4a and p14^ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16^INK4a and p14^ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14^ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16^INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16^INK4a and p14^ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16^INK4a methylation was found irrespective of tumor stages and histology. Whereas p14^ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14^ARF methylation in cancers of the intestinal type. Thus, the significance of p14^ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16^INK4a methylation.     

Some gonad-infecting species of Philometra (Nematoda, Philometridae) from offshore fishes of South Carolina and Georgia, USA, including Philometra charlestonensis sp. nov. from the scamp Mycteroperca phenax

Three gonad-infecting species of Philometra Costa, 1845 were, for the first time, recorded from perciform fishes from estuarine and marine waters in South Carolina and Georgia, USA: Philometra charlestonensis sp. nov. from the scamp Mycteroperca phenax (Jordan et Swain) (Serranidae), P. saltatrix Ramachandran, 1973 from the bluefish Pomatomus saltatrix (Linnaeus) (Pomatomidae), and Philometra sp. from the Atlantic croaker Micropogonias undulatus (Linnaeus) (Sciaenidae). The new species is characterized mainly by males (body length 2.65–3.14 mm) with equally long, needle-like spicules (length 132–141 μm) and the gubernaculum (81–93 μm) bearing dorsal transverse lamella-like structures on its distal portion, the body length of gravid females (168–247 mm), the presence of a well-developed anterior bulbous inflation on the female oesophagus, and by the length of the first-stage larvae (544–597 μm). A key to gonad-infecting species of Philometra parasitizing marine and brackish-water fishes is provided.     

Opecoelids (Platyhelminthes, Digenea) from the fork-tailed threadfin bream Nemipterus furcosus (Valenciennes, 1830) (Perciformes, Nemipteridae), with preliminary keys to the problematic genera Macvicaria Gibson et Bray, 1982 and Neolebouria Gibson, 1976

The opecoelid species Macvicaria jagannathi (Gupta et Singh, 1985) Bijukumar, 1997 (new syn. Plagioporus deeghaensis Gupta et Gupta, 1988) and Neolebouria lineatus Aken’Ova et Cribb, 2001 are redescribed from Nemipterus furcosus, from the waters off New Caledonia. Provisional keys to the genera Macvicaria Gibson et Bray, 1982 and Neolebouria Gibson, 1976 are presented. The following new combinations are made: Macvicaria yamagutii (Gupta et Ahmad, 1977), M. puriensis (Gupta et Govind, 1984) and M. chilkai (Gupta et Govind, 1984).     

Morphology of the accessory (pharyngeal) adenohypophysis and its relationship with the pharyngeal tonsil in the human fetus

The pharyngeal pituitary was examined in human fetuses during weeks 16 to 32 of development by light microscopy with routine and histochemical treatment of slices. All the fetuses examined possessed a pharyngeal pituitary, which develops from the epithelium of the upper pharyngeal wall, just like the main pituitary. Rathke's pouch becomes transformed into a cord and grows towards the midbrain forming the hypophysis cerebri, while some cells at the base of the cord remain in the integumentary epithelium of the pharynx and give rise to the pharyngeal pituitary. This organ represents a group of long epithelial cords under the integumentary epithelium within the connective tissue of the pharyngeal mucosa. The cords contain light and dark cells with signs of a secretory cycle. The pituitary cords grow into the lymphoid tissue of the pharyngeal tonsil. The integumentary epithelium does not contain protective structures at the site of origin of the pharyngeal pituitary. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 8, pp. 211–214, August, 1995 Presented by N. K. Permyakov, Member of the Russian Academy of Medical Sciences     

The first record of Macrogyrodactylus species (Monogenea, Gyrodactylidae) on freshwater fishes in Senegal with the description of Macrogyrodactylus simentiensis sp. nov., a parasite of Polypterus senegalus Cuvier

The first record of monogenean parasites of the genus Macrogyrodactylus Malmberg, 1957 on freshwater fish in Senegal is presented. Macrogyrodactylus congolensis Prudhoe, 1957 from the skin and Macrogyrodactylus heterobranchii N’Douba et Lambert, 1999 from the gills of Clarias anguillaris L. were found, representing new host records for these parasites. On Polypterus senegalus Cuvier, three Macrogyrodactylus species were identified, Macrogyrodactylus polypteri Malmberg, 1957, Macrogyrodactylus simentiensis sp. nov. and Macrogyrodactylus sp. M. simentiensis sp. nov. can be readily distinguished from the other Macrogyrodactylus species by the size of its hamuli and the shape of its marginal hook sickles. The marginal hooks on the anterolateral lobes of M. simentiensis differ in size and shape from those on the posterior margin of the haptor. Measurements and drawings of the haptoral sclerites of all five identified species are provided.