Spermatozoa of tapeworms (Platyhelminthes,Eucestoda): advances in ultrastructural and phylogenetic studies

New data on spermiogenesis and the ultrastructure of spermatozoa of ‘true’ tapeworms (Eucestoda) are summarized. Since 2001, more than 50 species belonging to most orders of the Eucestoda have been studied or reinvestigated, particularly members of the Caryophyllidea, Spathebothriidea, Diphyllobothriidea, Bothriocephalidea, Trypanorhyncha, Tetraphyllidea, Proteocephalidea, and Cyclophyllidea. A new classification of spermatozoa of eucestodes into seven basic types is proposed and a key to their identification is given. For the first time, a phylogenetic tree inferred from spermatological characters is provided. New information obtained in the last decade has made it possible to fill numerous gaps in the character data matrix, enabling us to carry out a more reliable analysis of the evolution of ultrastructural characters of sperm and spermiogenesis in eucestodes. The tree is broadly congruent with those based on morphological and molecular data, indicating that convergent evolution of sperm characters in cestodes may not be as common as in other invertebrate taxa. The main gaps in the current knowledge of spermatological characters are mapped and topics for future research are outlined, with special emphasis on those characters that might provide additional information about the evolution of tapeworms and their spermatozoa. Future studies should be focused on representatives of those major groups (families and orders) in which molecular data indicate paraphyly or polyphyly (e.g. ‘Tetraphyllidea’ and Trypanorhyncha) and on those that have a key phylogenetic position among eucestodes (e.g. Diphyllidea, ‘Tetraphyllidea’, Lecanicephalidea, Nippotaeniidea).     

Suitability of flow cytometry for estimating bacterial biovolume in natural plankton samples: comparison with microscopy data

The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4',6'-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4',6'-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R2 = 0.545, P < 0.001, n = 80) and the largest (R2 = 0.544, P < 0.001, n = 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods.     

Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations

Immunotherapy against beta-amyloid peptide (Abeta) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Abeta immunization reduces Abeta plaque pathology and improves cognitive function. However, the biological mechanisms by which Abeta antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Abeta antibodies decreases levels of intracellular Abeta. Antibody-mediated reduction in cellular Abeta appears to require that the antibody binds to the extracellular Abeta domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Abeta antibodies protects against synaptic alterations that occur in APP mutant neurons.     

N-glycosylation of the Xenopus laevis ClC-5 protein plays a role in cell surface expression, affecting transport activity at the plasma membrane

Mutations in the gene encoding ClC-5 lead to X-linked hypercalciuric nephrolithiasis (XLHN), characterized by proteinuria, hypercalciuria, and phosphaturia. In renal proximal tubule cells, ClC-5 was identified as an important player in endocytosis, which ensures reabsorption of filtered protein. However, the recent finding that ClC-5 is a Cl(-)/H(+) antiporter and not a Cl(-) channel as long thought points to the lack of understanding of its functional role. Also, little biochemical data are available about ClC-5 and its post-translational modifications have not been investigated. Here, we examined the role of N-glycosylation of xClC-5 in the Xenopus oocyte expression system by comparing wild-type (WT) xClC-5 and N-glycosylation site mutants. We found that xClC-5 is N-glycosylated on asparagines 169 and 470, which are the only N-glycosylated sites. xClC-5 mutants have an increased susceptibility to polyubiquitination and proteasomal degradation; however, without a notable impact on the expression level. Using a cross-linking reagent, we showed that xClC-5 assembles into protein complexes, independent of its N-glycosylation. Voltage-clamp measurements showed a reduced conductance in the presence of tunicamycin and with xClC-5 N-glycosylation site mutants. Using immunocytochemistry, we localized xClC-5 mainly in intracellular compartments, and found that its cell surface pool is reduced in the absence of N-glycans. We further examined the plasma membrane retrieval of WT and mutant xClC-5 in the presence of Brefeldin A (BFA), and found that the non-glycosylated mutant was retrieved more than five times faster than the WT protein. We conclude that N-glycosylation enhances cell surface expression of xClC-5, increasing its plasma membrane transport activity.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

Effect of testosterone on oxidative stress and cell damage induced by 3-nitropropionic acid in striatum of ovariectomized rats

This paper evaluates the effects of testosterone (0.5 mg/kg subcutaneously (s.c.) for 8 days) on oxidative stress and cell damage induced by 3-nitropropionic acid (20 mg/kg intraperitoneally (i.p.) for 4 days) in ovariectomized rats. Gonadectomy triggered oxidative damage and cell loss, evaluated by the detection of caspase-3, whereas 3-nitropropionic acid increased the levels of oxidative stress induced by ovariectomy and prompted cell damage characterized by enhanced levels of lactate dehydrogenase. These changes were blocked by testosterone administration. Our results support the following conclusions: i) ovariectomy triggers oxidative and cell damage via caspase-3 in the striatum; ii) 3-nitropropionic acid exacerbates oxidative stress induced by ovariectomy and leads to cell damage characterized by increased levels of lactate dehydrogenase; iii) testosterone administration decreases oxidative stress and cell damage. Additionally, these data support the hypothesis that testosterone might play an important role in the onset and development of neurodegenerative diseases.     

Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

Los mamíferos insulares del Mioceno medio y superior de Menorca (islas Baleares, Mediterráneo occidental)

The insular mammals from the karstic deposits of Punta Nati-2 and the marine beds of es Cul de sa Ferrada, placed in the northwest coast of Minorca (Balearic islands, Spain, western Mediterranean) are described in this work. One of the mammals (only present in Punta Nati-2) is a new glirid species, Margaritamys adroveri, closely related with Margaritamys llulli Mein and Adrover, 1982 and Pseudodryomys granatensis Agustí, 1993, from the middle Miocene of Santa Margalida and Sant Llorenç (Mallorca) and Murchas (Granada), respectively. M. adroveri shows more derived characters than P. granatensis and is more archaic than M. llulli, the size being similar to P. granatensis. The ochotonid present in Minorca is very similar to Gymnesicolagus gelaberti Mein and Adrover, 1982 from Mallorca. In the two minorcan deposits were recovered the first mandibles of this ochotonid, characterized by their big size. The medium weight (estimated from the length of the lower row) for G. aff. gelaberti is 5.4 kg, very similar to that some extant leporids like Lepus alleni Mearns, 1890 or Lepus arcticus Ross, 1819 and higher than any other living ochotonid. Doubtless, the big size of G. aff. gelaberti is a consequence of insular evolution, but not the short diastema, a primitive character shared with continental ochotonids and insular leporids. The discovery of a mandible in the marine beds of es Cul de sa Ferrada permits to place G. aff. gelaberti in the lower Tortonian, which represents the youngest record for this species with respect the faunal associations of Mallorca and Granada, Langhian-Serravalian in age (middle-upper Miocene). The fauna from Punta Nati-2 may represent an endemic association or a faunal group closely related to the fauna of Mallorca and Granada, but in an older context. The age of Gymnesicolagus and the presence of a similar fauna in Mallorca and Minorca suggest a connection between both islands during the Serravalian and the existence of an emerged area in Minorca after the Tortonian transgression.     

S-Nitrosation of proteins during D-galactosamine-induced cell death in human hepatocytes

Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocytes.