Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Impact of PCB on resistance to Flavobacterium psychrophilum after experimental infection of rainbow trout Oncorhynchus mykiss eggs by nanoinjection

The effects of sublethal exposure of a commercial blend of polychlorinated biphenyls (PCB), i.e. Clophen A50, on disease resistance to the aetiological agent of rainbow trout fry syndrome, Flavobacterium psychrophilum, were investigated. Newly fertilised rainbow trout Oncorhynchus mykiss eggs were nanoinjected with 2 doses of Clophen A50 (0.4 or 2 microg egg(-1)) and/or 100 colony forming units of F. psychrophilum. The mean cumulative mortality in control groups, and groups exposed to the lower dose of Clophen A50 (0.4 microg egg(-1)) was below 5.0%. The mean cumulative mortality in groups exposed to the higher dose of Clophen A50 (2.0 microg egg(-1)) was 5.8%, which was not significantly different from the control groups. In all groups infected with F. psychrophilum, with or without exposure to Clophen A50, significantly higher cumulative mortalities compared with control groups were recorded. No differences in mortality were recorded between groups exposed to bacteria alone or bacteria in combination with the higher dose of Clophen A50 (21.6 and 20.4%, respectively). Decreased disease resistance was recorded in groups exposed to F. psychrophilum and the lower dose of Clophen A50, with a mean cumulative mortality of 56.0%. These results could be due to non dose-dependent effects on the immune system, or toxic effects of PCB or their metabolites on the bacteria in groups exposed to the higher dose of Clophen A50. The present study indicates that maternal transfer of PCB might affect disease resistance to vertically transmitted F. psychrophilum.     

Thyroid hormone inhibits slow skeletal TnI expression in cardiac TnI-null myocardial cells

A cardiac troponin I (cTnI) gene knockout mouse model has been created and the phenotype of the cTnI null mice is an acute heart failure resulting from the deficiency of TnI and a diastolic dysfunction. Two isoforms of TnI (the fetal form ssTnI and the adult form cTnI) are mainly expressed in the heart under a developmentally regulated program. In our previous studies, we demonstrated that thyroid hormone could alter the time course of ssTnI gene expression in the heart. In the present study, we have successfully cultured neonatal cardiac myocytes from wild type and cTnI null mouse hearts. The ssTnI gene expression pattern has been investigated in these cells. By using Western blotting assays, a TnI isoform switching has been observed in the wild type cardiac myocytes. The pattern of TnI isoform switching is very similar to that of in vivo study we reported previously. In cTnI null cardiac myocytes cultured from day 1 to day 7, there is a continuous decline in ssTnI concentration in the cells. The time course of ssTnI decline in cTnI null cells is similar to that of wild type cardiac myocytes, suggesting that there is no significant compensation of ssTnI gene expression for the absence of the cTnI. This observation is different from what we found previously at a whole heart level. In addition, when thyroid hormone T3 (20 ng/ml) is added to cultured cTnI null cardiac myocytes, the decline of ssTnI concentration occurs earlier. This is inconsistent with our observations from previous in vivo studies. The data demonstrate that thyroid hormone can alter the time course of ssTnI gene expression in cultured cardiac myocytes and TnI gene regulation is also controlled by some unknown programmed events inside of cardiac myocytes.     

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.     

Aspiration cytology of neuroendocrine (Merkel-cell) carcinoma of the skin. Report of a case

An 89-year-old female with a two-year history of a growing tumor of the skin on her left thigh was subjected to fine needle aspiration. Cytologic examination of the aspirate revealed a small-cell carcinoma, and a Merkel-cell carcinoma was suggested. The diagnosis was confirmed by histopathologic and electron microscopic analysis of the removed tumor. The cytologic findings are presented and correlated with the light and electron microscopic pictures of the operative specimen.     

Kinetics of long-term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin-activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen-stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H-thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.     

Peroxiredoxin-linked detoxification of hydroperoxides in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii is highly susceptible to oxidative stress caused by tert-butyl-hydroperoxide, juglone, and phenazine methylsulfate with IC(50) in the nanomolar range. Using dichlorofluorescein diacetate, a detector of endogenous oxidative stress, it was shown that juglone and phenazine methylsulfate are potentially toxic to the parasites without affecting the host cells. These results demonstrate that T. gondii is vulnerable to oxidative challenge that results from disruption of its redox balance and so this could be an effective approach to therapeutic intervention. This study has characterized redox active and antioxidant peroxidases belonging to the class of 1-Cys and 2-Cys peroxiredoxins that play crucial roles in maintaining redox balance. The tachyzoite stages of T. gondii express thioredoxin (TgTrx), 1-Cys peroxiredoxin (TgTrx-Px2), and a 2-Cys peroxiredoxin (TgTrx-Px1) and immunofluorescent studies revealed that all three proteins are located in the cytosol of the parasite confirming previous studies on TgTrx-Px1 (Kwok, L.Y., Schluter, D., Clayton, C., and Soldati, D. (2004) Mol. Microbiol. 51, 47-61). TgTrx-Px1 showed K(m) values for H(2)O(2) and tert-butyl hydroperoxide in the nanomolar range, emphasizing the great affinity of the protein for theses substrates. Moreover, the catalytic efficiency of TgTrx-Px1 for these substrates at 10(6)-10(7) M(-1) s(-1) is unusually high, which qualifies the enzyme as an extremely potent antioxidant. Kinetic analyses revealed that TgTrx-Px1 is inhibited by tert-butyl hydroperoxide, and apparent inhibition constants were determined to be between 33 and 35.6 microm depending on the concentration of the non-inhibitory substrate thioredoxin. TgTrx-Px2 protected glutamine synthetase from inactivation by Fe(3+)/DTT, showing that it is an active peroxiredoxin.     

Flow cytometric DNA ploidy analysis of soft tissue sarcomas. A comparative study of preoperative fine needle aspirates and postoperative fresh tissues and archival material

Flow cytometric (FCM) DNA ploidy measurements on frozen fresh samples of soft tissue sarcomas were compared with the corresponding analyses on preoperative fine needle aspirates and postoperative formalin-fixed archival tissues from the same tumors. A concordance in ploidy status (diploid versus non-diploid) was obtained for 63% of the fresh tissue-fine needle aspiration (FNA) sample comparisons and for 85% of the fresh tissue-archival material comparisons. The majority of discordances in the fresh tissue-FNA sample comparisons could be explained by FNA sampling errors. In the remaining discordant cases (3 of 27 FNA sample comparisons and 6 of 40 archival material comparisons), sampling errors could not explain the differences in ploidy status. The discordant cases were evenly distributed among the different sampling methods. Method reproducibility was not responsible for the differences in ploidy determinations; tumor heterogeneity may be an explanation for the discrepancies. This study showed that archival soft tissue sarcoma samples are as well suited for DNA ploidy analysis as are fresh frozen tissues.