AN ANTIGENIC POLYPEPTIDE FRAGMENT ISOLATED FROM TETANUS TOXIN: CHEMICAL CHARACTERIZATION, BINDING TO GANGLIOSIDES AND RETROGRADE AXONAL TRANSPORT IN VARIOUS NEURON SYSTEMS

Abstract— The isolation and purification of an antigenic polypeptide fragment from tetanus toxin is described. The main physico-chemical, chemical, immunological, and pharmacological characteristics of this fragment, designated as B-II_b fraction, are reported. It is a polypeptide with a molecular weight close to 46,000. Its amino acid composition is on the whole comparable with that of the toxin. It contains one disulphide link and two free sulphhydryl groups which are not directly available for reaction. Tyrosine and lysine were found to be the two N -terminal groups. However, that B-II_b fraction has a subchain structure is still to be demonstrated. There is some evidence that B-II_b fraction may consist of 'isofragments'. This toxin fragment shows a cross-reaction with intact toxin and a specific flocculating activity of about three times that of the latter. In contrast, however, B-II_b fraction exhibits a specific toxicity approximately one hundred thousand times lower than the intact toxin. Although practically atoxic, this toxin fragment is still able to bind to gangliosides with an affinity which is even greater than that of the toxin. It is also capable of migrating towards the central nervous system by a mechanism of retrograde axonal transport as shown in peripheral adrenergic, sensory and motoneurons.
These unique features of B-II_b fraction are discussed in relation to the use of such fragments both for competing in vivo with the attachment of the toxin in the central nervous system and for specifically carrying therapeutic and pharmacological drugs into the central nervous system by neural route.     

Mutations in Endothelin 1 Cause Recessive Auriculocondylar Syndrome and Dominant Isolated Question-Mark Ears

Auriculocondylar syndrome (ACS) is a rare craniofacial disorder with mandibular hypoplasia and question-mark ears (QMEs) as major features. QMEs, consisting of a specific defect at the lobe-helix junction, can also occur as an isolated anomaly. Studies in animal models have indicated the essential role of endothelin 1 (EDN1) signaling through the endothelin receptor type A (EDNRA) in patterning the mandibular portion of the first pharyngeal arch. Mutations in the genes coding for phospholipase C, beta 4 (PLCB4) and guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3 (GNAI3), predicted to function as signal transducers downstream of EDNRA, have recently been reported in ACS. By whole-exome sequencing (WES), we identified a homozygous substitution in a furin cleavage site of the EDN1 proprotein in ACS-affected siblings born to consanguineous parents. WES of two cases with vertical transmission of isolated QMEs revealed a stop mutation in EDN1 in one family and a missense substitution of a highly conserved residue in the mature EDN1 peptide in the other. Targeted sequencing of EDN1 in an ACS individual with related parents identified a fourth, homozygous mutation falling close to the site of cleavage by endothelin-converting enzyme. The different modes of inheritance suggest that the degree of residual EDN1 activity differs depending on the mutation. These findings provide further support for the hypothesis that ACS and QMEs are uniquely caused by disruption of the EDN1-EDNRA signaling pathway.     

Penetration of resveratrol into bovine aortic endothelial cells (BAEC): a possible passive diffusion

Several studies have demonstrated that, in a context of oxidative stress, resveratrol, a polyphenol found in wine, could act as a protective agent on endothelial cells by various mechanisms but without showing that it could penetrate inside the cell. The aim of this study was to detect for the first time resveratrol inside bovine endothelial aortic cells and to determine which kind of transport mechanism was involved. Intracellular and membrane concentrations of resveratrol have been measured by high performance liquid chromatography after incubation of several concentrations of resveratrol with endothelial cells for 24h. Concentrations of resveratrol in the culture media have been determined by UV spectrophotometry and experiments of transport mechanisms have been performed. Our results showed that, for the concentrations tested (1, 5, 10 and 50 μM), resveratrol was detected inside the cells and suggested that it was able to penetrate into the cells through a passive diffusion mechanism.     

Predation risk and the structure of freshwater zooplankton communities

Summary Many predators inflict substantial mortality on their prey. The prey respond to these selective pressures with changes in their spatial and temporal overlap with the predator (density risk responses), or with changes in their vulnerability to the predator (prey vulnerability responses). Here we develop a simple predation model that permits quantification of the basic response types of the prey in nature. We then test the hypothesis that prey response will be proportional to the intensity of the predation mortality relative to all other sources of mortality and decreased natality acting on the prey. A significant regression relationship is obtained for the prey vulnerability response but not for any of the density risk responses. The individual response values and regression statistics are used to interpret the relative importance of the different response types and to assess the predator's influence on prey community structure.Supported by Lehigh University Environmental Studies Center     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Endothelin-1 binding sites and inositol-monophosphate formation in kidney medulla from adult and juvenile SHR and WKY rats

High resolution autoradiography at the cellular level localized endothelin-1 binding sites to the collecting ducts in the rat renal papilla. These receptors were functionally coupled to the phosphatidyl-inositol system since endothelin-1 stimulated the accumulation of IP₁ in a concentration-dependent manner in cross chopped slices from renal papillae. The concentration-effect curves in 4 week old normotensive Wistar-Kyoto rats (WKY) lay to the right of curves from 4 week old and adult spontaneously hypertensive rats (SHR) and adult WKY (20–24 week old) animals; the EC₅₀ values in the 4 week old animals were 17 ± 5 and 8 ± 1 nM (P < 0.05, N = 5; mean ± SE) for the normotensive and hypertensive animals, respectively. Autoradiographic studies showed that the density and distribution of binding sites for [¹²⁵I]endothelin-1 in the kidneys did not differ between the groups; receptor densities in the renal papillae were 461 ± 37 (fmol/mg protein) in the 4 week old WKY, and 443 ± 27 (fmol/mg protein) in the 4 week old SHR. The plasma levels of immunoreactive endothelin-1 were also similar between groups; 4 week old SHR (39 ± 3 pg/ml) and 4 week old WKY (36 ± 1 pg/ml). The increased response to endothelin-1 may be related to the development of the hypertensive state in the SHR.