Primary carnitine deficiency and sudden death: in vivo evidence of myocardial lipid peroxidation and sulfonylation of sarcoendoplasmic reticulum calcium ATPase 2.

OBJECTIVES: Primary carnitine deficiency is an autosomal recessive disorder caused by mutations in the SLC22A5 gene which results in impaired carnitine transport, cytosolic fatty acid accumulation and impaired beta oxidation. The disease is associated with cardiomyopathy and arrhythmias, but the mechanism is unknown. We hypothesized that carnitine deficiency results in increased myocardial oxidative stress. METHODS: We evaluated a 22-year-old woman with primary carnitine deficiency and ventricular fibrillation, as well as her first-degree relatives. RESULTS: Sequencing of SLC22A5 identified two deleterious mutations (A142S and R488H) and a novel mutation predicted to be a splice variant. Histology demonstrated increased myocardial lipid deposition and swollen mitochondria. Immunohistochemistry demonstrated accumulation of the reactive aldehyde 4-hydroxy-2-nonenal, indicative of increased lipid peroxidation, and sulfonylation of sarcoendoplasmic reticulum calcium ATPase 2 at cysteine 674. CONCLUSIONS: These findings suggest that increased oxidant stress may contribute to myocardial dysfunction and arrhythmogenesis in this disorder.

Differential tissue expression of a calpastatin isoform in Xenopus embryos.

This study is aimed at demonstrating the role played by a calpastatin isoform (Xcalp3) in Xenopus embryos. A specific monoclonal antibody (mAb) was raised against a glutathione S-transferase (GST)-Xcalp3 fusion protein and characterized by immunoblotting and confocal fluorescence microscopy on stage 20-36 embryos. Under these conditions, calpastatin reactivity is associated with a major 110kDa protein fraction and preferentially expressed by notochord and somitic cells. In notochord cells, anti-calpastatin reactive sites were initially restricted to the luminal space of the vacuoles and later became diffused throughout the cytoplasm. In contrast, anti-calpastatin reactive sites in somitic cells were initially diffused throughout the cytoplasm and became restricted to a few intracellular granules in the later developmental stages. At the ultrastructural level, notochord cells appeared as flattened discs containing several vacuoles and numerous electron-dense granules. During transition from stages 26 to 32, electron-dense granules were gradually reduced in number as vacuoles enlarged in size and losed their calpastatin reactivity. Electron-dense granules were also present in myoblast cells and their number gradually reduced during development. To determine whether these observations bear any causal relationship to the calpain/calpastatin system, a number of Xenopus embryos were examined both ultrastructurally and histochemically following exposure to a specific calpain inhibitor (CI3). Under these conditions, Xenopus embryos exhibited an altered right-left symmetry and an abnormal axial shortening. In CI3-treated stage 32 embryos, notochord cells had a reduced vacuolar extension and exhibited at the same time an increase in granular content. The overall morphology of the somites was also distorted and myoblasts were altered both in shape and granular content. Based on these findings, it is concluded that the calpain/calpastatin may play an important role in the control of notochord elongation and somite differentiation during Xenopus embryogenesis.

Viral particles of the endogenous retrovirus ZAM from Drosophila melanogaster use a pre-existing endosome/exosome pathway for transfer to the oocyte.

BACKGROUND: Retroviruses have evolved various mechanisms to optimize their transfer to new target cells via late endosomes. Here, we analyzed the transfer of ZAM, a retroelement from Drosophila melanogaster, from ovarian follicle cells to the oocyte at stage 9-10 of oogenesis, when an active yolk transfer is occurring between these two cell types. RESULTS: Combining genetic and microscopic approaches, we show that a functional secretory apparatus is required to tether ZAM to endosomal vesicles and to direct its transport to the apical side of follicle cells. There, ZAM egress requires an intact follicular epithelium communicating with the oocyte. When gap junctions are inhibited or yolk receptors mutated, ZAM particles fail to sort out the follicle cells. CONCLUSION: Overall, our results indicate that retrotransposons do not exclusively perform intracellular replication cycles but may usurp exosomal/endosomal traffic to be routed from one cell to another.

Biological activity of cytokines: an evolutionary perspective.

It appears evident that teleost fish are at present the vertebrate group in which, excluding mammals, most information on the immune system is available. However, despite the great impetus on the discovery of genes homologous to mammalian immunomodulatory molecules, the knowledge on biological activities exerted by cytokines is meager. This review reports the present knowledge on the biological activities of cytokine-like and cytokines in invertebrates and cold-blooded vertebrates.

Phase transitions and critical phenomena in the two-dimensional Ising model with dipole interactions: A short-time dynamics study.

The ferromagnetic Ising model with antiferromagnetic dipole interactions is investigated by means of Monte Carlo simulations, focusing on the characterization of the phase transitions between the tetragonal liquid and stripe of width h phases. The dynamic evolution of the physical observables is analyzed within the short-time regime for 0.5≤δ≤1.3, where δ is the ratio between the short-range exchange and the long-range dipole interaction constants. The obtained results for the interval 0.5≤δ≤1.2 indicate that the phase transition line between the h=1 stripe and tetragonal liquid phases is continuous. This finding contributes to clarifying the controversy about the order of this transition. This controversy arises from the difficulties introduced in the simulations due to the presence of long-range dipole interactions, such as an important increase in the simulation times that limits the system size used, strong finite size effects, as well as to the existence of multiple metastable states at low temperatures. The study of the short-time dynamics of the model allows us to avoid these hindrances. Moreover, due to the fact that the finite-size effects do not significantly affect the power-law behavior exhibited in the observables within the short-time regime, the results could be attributed to those corresponding to the thermodynamic limit. As a consequence of this, a careful characterization of the critical behavior for the whole transition line is performed by giving the complete set of critical exponents.

Association of melasma with thyroid autoimmunity and other thyroidal abnormalities and their relationship to the origin of the melasma.

Melasma is localized hyperpigmentation over the forehead, upper lips, cheeks, and chin. In this study, evidence suggesting an association between autoimmune thyroid disorders and melasma and the relationship of thyroid disorders to the origin of melasma is presented. A total of 108 nonpregnant women, aged 20-56 yr, were divided into 2 groups for the purpose of this study: 1) melasma, 84 patients; 2) control group, 24 patients from the Dermatology Clinic matched for age and sex. Microsomal thyroid autoantibodies (MCHA) were sought in all subjects. TRH-TSH tests were performed in patients with melasma and in those women with goiter and/or positive MCHA tests from the control group. Studies were completed with serum T4, T3, and antithyroglobulin antibody (TGHA) measurements in all patients with thyroid abnormalities. In patients with melasma, the frequency of thyroid disorders (58.3%) was 4 times greater than in the control group. The MCHA-negative patients had 1) simple goiter (13.1%), 2) Plummer's disease (2.4%), and 3) TSH hyperresponse to TRH in nongoitrous patients (10.7%). Patients with positive MCHA tests (32.1%) were divided into 2 subgroups. One comprised those women with an apparently normal thyroid gland and thyroid function (n = 7), while the other included all patients with goiter and/or subclinical hypothyroidism (n = 20). Regarding the origin of the melasma, it was found that 70% of women who developed melasma during pregnancy or while using oral contraceptives had thyroid abnormalities compared to 39.4% of patients with idiopathic melasma. Subjects from the control group had a 12.5% incidence of thyroid abnormalities, and only 8.3% had positive MCHA. Estrogen, progesterone, or both could be the triggering factor in the development of melasma in women who have a particular predisposition toward both melasma and thyroid autoimmunity. Patients with idiopathic melasma had a lower frequency of thyroid abnormalities, suggesting that there may be different genetic patterns linked to autoimmune thyroid disease. We conclude that there is a true association between thyroid autoimmunity and melasma, mostly in women whose melasma develops during pregnancy or after ingestion of oral contraceptive drugs.

The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture.

The sea bass Dicentrarchus labrax is one the most important seawater fish species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scientific and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response.

Internet oncology: increased benefit and risk for patients and oncologists.

Whether the benefits will outweigh the risks of electronic health care remains to be seen, and to a large extent the question is moot; consumers, providers, and interested third parties have ensured its existence. One can surmise, or at least hope, that all interested parties will become increasingly sophisticated in both the delivery and consumption of e-health information, and that ultimately consumer, advocate, and physician demands will help shape the industry into a self-regulated entity that balances the need for high-grade information with a commitment to the principles of privacy and autonomy.

Carbohydrates of the surface of the normal human spermatozoon.

Autoproteolysis of human spermatozoa produces oligopeptides with oligosaccharide chains of the N-glycosidic-linked type that are released from the "surface exposed" parts of glycoproteins. The products eluted in the previous washing of the spermatozoa have the same composition and solubility characteristics as the oligopeptides from the digestion. This suggests that autoproteolysis is a constant process that normally occurs on the spermatozoa membrane. The cytochemical characterization and localization of the N-glycosidic-linked oligosaccharide receptors on the human spermatozoa membrane after digestion, in the presence or absence of seminal plasma, indicates that only part of the oligosaccharides are cleaved. Their distribution on the different zones of the spermatozoon changed as the probability of detecting these receptors in the intermediate segment increased after proteolysis; this indicates that in this zone the receptors are cryptic ones that become exposed by the action of the proteolytic enzymes. In the presence of seminal plasma most receptors on the acrosome are eliminated.

Recombinant Semliki forest virus infects and kills human prostate cancer cell lines and prostatic duct epithelial cells ex vivo.

Semliki Forest Virus (SFV) is a broad host range RNA virus capable of high-level recombinant protein expression and apoptosis induction in many cell types. We have successfully used a recombinant, replication deficient SFV vector to express the LacZ marker gene product in seven human prostate cell lines, as well as in human prostate tissue explants. Flow cytometry revealed that 40-60% of PPC-1 prostate cancer cells died 24-72 h after infection with SFV-LacZ virus. Most human prostate cancer cell lines expressed high levels of recombinant protein. Infection of human prostate tissue ex vivo led to similarly high expression levels but the recombinant beta-galactosidase was confined to duct epithelial cells. Infection of cell and tissue cultures resulted in detachment of adherent cells from the culture surface and detachment of epithelial cells from the basement membrane of tissue. Our results indicate that SFV may be useful in targeting recombinant protein expression and apoptosis to prostatic duct epithelial cells.

Formation of the egg envelope of a teleost, Dicentrarchus labrax (L.): immunochemical and cytochemical detection of multiple components.

The formation of the egg envelope in a teleost, Dicentrarchus labrax (L.), was analysed at histological and ultrastructural level. The sequential deposition of three main layers (Z1, Z2 and Z3) constitutes the extracellular matrix throughout oocyte development. Various findings indicate that these subunits are biochemically distinct: (1) periodate- and phosphotungstic acid-reactive carbohydrates are obviously detected only in the Z1, that constitutes the initial deposit of the egg envelope in early lipidic oocytes; (2) a monoclonal antibody (DLE7) against egg envelope polypeptides did not immunostain the Z1 and the underlying Z2; (3) the antigenic determinants recognised by DLE7, thought to be exogenous in origin (synthesised in the liver), are incorporated in the inner layer (Z3). In addition, DLE7 immunostained a thin layer, assembled together with Z3. This line has not yet been described in teleost eggs and was named Z1a. This study first describes at fine cytological level the contribution of exogenous proteins to formation of the different egg envelope layers. Results obtained with conventional, immunochemical and cytochemical techniques suggest multiple synthetic sources (exogenous and follicular) of egg envelope proteins.

Ultrastructure of the spermatozoon ofElenchus japonicus and its bearing on the phylogeny of strepsiptera.

The fine structure of the mature spermatozoon of the strepsipteranElenchus japonicus Esaki and Hashimoto (Elenchidae) is described using transmission electron microscopy. The spermatozoon was seen to have an elongated head, a tail containing a 9 + 9 + 2 axoneme, two mitochondrial derivatives and two accessory sheaths. The monolayered acrosome is conical in shape while the nucleus exhibits an internal channel of uncondensed chromatin. The tail is long, and in its final portion, the axoneme, loses its elements progressively. These results are compared with the sperm ultrastructure ofXenos moutoni De Buysson (Stylopidae) and with those of other insect orders, particularly the Coleoptera.

Fine structure of the spermatozoon of the strepsipteran Xenos moutoni.

Spermatozoa of Xenos moutoni De Buysson belonging to the order Strepsiptera (Insecta) were examined by electron microscopy. The spermatozoon was seen to have an elongated head and a tail containing a 9+9+2 axoneme and two mitochondrial derivatives of equal size. The pear-shaped acrosome is characterised by a mono-layered structure and terminates anteriorly forming two pyramidal evaginations. The nucleus exhibits an external portion of dense chromatin and an internal one of uncondensed material. The latter occupies a central position at the base and becomes progressively peripheral at the apex. The tail is long and in its final portion the axoneme loses its elements progressively. These results have been compared with the ultrastructure of the spermatozoa of Coleoptera which have been considered as a sister group of Strepsiptera.

The secretory pathway of vitellogenin in the fat body of the stick insect bacillus rossius: An ultrastructural and immunocytochemical study.

The fat body of the adult female stick insect Bacillus rossius was examined ultrastructurally with a view to clarifying the secretory pathway. The absence of lipid storage in the tissue allowed visualization of a polarized distribution of all organelles in the cell cytoplasm. Composite granules were distributed along the baso-apical axis of the cell according to progressive stages of maturation. At their final stage of maturation, these granules possess two distinct compartments, an electron-translucent compartment and a more electron-dense one. The origin of each of the two compartments was traced back to other organelles in the basal cytoplasm of the fat body cell. The differential origin of the two compartments contributing to the composite granules was further investigated by cytochemical analyses. Vitellogenin was detected both in the electrondense compartment of the composite granules and in the Golgi apparatus. The electron-translucent compartment of the composite granules appeared to consist mainly of urate crystals. Such enzyme activities as acid phosphatase, peroxidase and catalase were also detected in this latter compartment. The observations support the interpretation that secretion in the fat body of B. rossius entails fusion of Golgi-derived vesicles with a specialized kind of multivesicular body. While Golgiderived vesicles convey their load of newly synthesized vitellogenin to the electron-dense compartment, the multivesicular body develops the urate crystals of the electron-translucent compartment.

Morphological organization of the male brood pouch epithelium of Syngnathus abaster Risso (Teleostea, Syngnathidae) before, during, and after egg incubation.

The morphological organization of the male brood pouch epithelium of Syngnathus abaster, before, during, and after the breeding period, was observed by light and electron microscopy. Before gestation, the epithelium of the pouch wall was compact and consisted of three kinds of cells: typical epithelial cells (pavement cells), mitochondria-rich cells (MR cells), and, presumably, differentiating MR cells. In this stage, very few capillaries were observed beneath the epithelium. During egg incubation, the capillaries increased in number and size, large intercellular spaces formed among epithelial cells at their basal sides, MR cells were abundant, and differentiating MR cells were only occasionally observed. After incubation, MR cells degenerated by necrosis and apoptosis. The intercellular spaces between the epithelial cells disappeared and the number and size of the capillaries beneath the epithelium decreased. The presence of MR cells during gestation and their degeneration after incubation suggest that these cells play a pivotal role in the physiological functions of the brood pouch. The similar cytological characteristics of syngnathid pouch MR cells and chloride cells of the teleostean gills suggests that the Syngnathidae brood pouch is involved in osmoregulation of the fluid surrounding the developing embryos.

Cytology of lymphomyeloid head kidney of Antarctic fishes Trematomus bernacchii (Nototheniidae) and Chionodraco hamatus (Channicthyidae).

Species that live in extreme conditions have specially adapted physiology and tissue/organ organisation. The adaptation of lymphoid organs to low temperatures in polar species could be an original field of study, indicating how the immune system works under extreme conditions. In fishes, the head kidney is a key organ for immunity and here the cytology of this organ is studied in two common Antarctic species: Trematomus bernacchii and Chionodraco hamatus. Ultrastructural analysis revealed heterogeneity of epithelial cells, with reticular cells, subcapsular- and perivascular-limiting cells. Differences in the size and morphology of epithelial cells were observed between the polar species and warm water species of fish. Intermingled with epithelial cell leucocytes, such as lymphocytes, thrombocytes and macrophages, had comparable morphology in both species, contrary to sharp differences observed in the morphology of erythrocytes and granulocytes. The functional adaptation of the head kidney to the low temperatures of polar water is discussed.

The yolk sac in late embryonic development of the stick insect Carausius morosus (Br.).

Differentiation of the yolk sac was examined ultrastructurally and cytochemically in late embryonic development of the stick insect Carausius morosus. During migration along the yolk sac, endodermal cells form a discontinuous cell epithelium, leaving wide intercellular channels between neighbouring cell clusters. Within the same cell cluster, cells are all joined by septate junctions. In the proximity of the proctodeum region, intercellular channels are filled with numerous cell debris which are shown to derive from vitellophages undergoing cell lysis. Yolk sacs resolved by gel electrophoresis are shown to release a number of vitellin polypeptides into the culture medium. These are equivalent in molecular weight to those present in the vitellophage yolk granules This observation is consistent with the evidence that the basement lamina may act as a course physical filter, retaining particles larger than colloidal thorium dioxide and allowing free percolation of peroxidase. Differentiating endodermal cells form a microvillar striated border along the apical plasma membrane. A number of vesicular criptae were frequently seen in these differentiating endodermal cells. Electron dense granules released by endodermal cells are suggested to play a role in vitellophage lysis and vitellin release from the enclosed yolk granules.

An autoradiographic analysis of vitellogenin synthesis and secretion in the fat body of the stick insect Bacillus rossius.

The fat body of the stick insect Bacillus rossius was studied with a view to clarifying the metabolic pathway leading to secretion of vitellogenin (VG). Electrophoretic analysis of ovarian follicles and hemolymph from egg-laying females showed that the two tissues shared a common polypeptide composition consisting of five major polypeptides with molecular weights ranging from 60-180 Kd. Following in vivo exposure to [(35)S]-methionine for up to 24 h, these polypeptides were labeled in a stage- and time-dependent manner, suggesting that they were transferred from the hemolymph to the oocyte during vitellogenesis. Fat body pulse-labeled with [(35)S]-methionine for up to 240 min and immunoprecipitated with an anti-yolk serum was labeled only in a fraction containing high molecular weight polypeptides. We presume these polypeptides to be VG precursors bearing a precursor-product relationship with the five major polypeptides of the hemolymph and developing ovarian follicles. Fat body exposed in vivo to [(3)H]-leucine for time intervals ranging from 20-240 min were processed for EM autoradiography. The results of this analysis showed that incorporated radioactivity was progressively transferred from the endoplasmic reticulum to the Golgi apparatus and from there to the composite granules. The data provided in this study are consonant with previous findings by which composite granules were shown to contain two compartments differing both in content and origin. In addition, the autoradiographical data of in vivo labeled fat body demonstrate that only the material partitioned into the electron-dense compartment of these granules is exocytosed.

Ultrastructure of the seminal vesicle of Phlebotomus perniciosus Newstead (Diptera, Psychodidae).

The seminal vesicles of Phlebotomus perniciosus were investigated by light microscopy, confocal scanning laser microscopy and by scanning and transmission electron microscopy. They have a complex structure, and three different morphological compartments called A, B and C are distinguished on the basis of their position and fine structure. Compartment A is continuous with the vasa deferentia and consists of a cylindrical wall limiting a lumen in which the spermatozoa are stored. Compartment B is hemispherical and surrounds compartment A like a muff. Compartment C constitutes an external coat surrounding A and B. The epithelial cells of each compartment are characterized by morphologically different secretory granules. The ultrastructural features of these cells are described and their role in sandfly reproductive biology is discussed.

Proteolytic release and partial characterization of human sperm-surface glycopeptides.

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.