Development of an Argopecten-Specific 18S rRNA Targeted Genetic Probe

Comparison of 18S ribosomal RNA gene sequences between diverse bivalve species, including eight scallop species, allowed the design of an 18S rRNA targeted oligonucleotide probe (BS-1364) that was specific for scallops belonging to the genus Argopecten (bay and calico scallops). The high sequence similarity of the 18S rRNA gene between Argopecten irradians and Argopecten gibbus (98.8%) prevented the design of an A. irradians species-specific probe. Hybridization studies using amplified 18S rDNA from a diverse collection of bivalve species demonstrated that the specificity of the digoxygenin-labeled probe was consistent with the predicted specificity indicated by sequence comparison. Hybridization studies using laboratory-spawned bay scallop veligers indicated that a single veliger could be detected by probe hybridization in a blot format, and that probe hybridization signal was proportional (r ²= .99) to the abundance of veligers. Methods for rRNA extraction and blotting were developed that allowed bay scallop veligers to be specifically and quantitatively identified in natural plankton samples. Preliminary studies conducted in Tampa Bay, Florida, suggest that introduced scallops can successfully spawn and produce veligers under in situ conditions. The Argopecten-specific probe and methods developed in this study provide the means to study the production and fate of bay scallop larvae in nature and provide evidence that scallops introduced into Tampa Bay have the potential for successful reproduction and enhancement of scallop stocks. Received January 25, 1999; accepted May 7, 1999.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Importance of sucking stimulation in lactation: histological aspects of the mammary gland in the rat

The Authors refer the results of a study concerning the mammary gland of lactating rats subjected to suctional stimuli of different intensities. We made used of Wistar rats subdivided in 3 groups: Group A: each lactating 8 pups; Group B: 8 days before the birth we provided to hide the nipples of a half of the breasts with sutures like tobacco-pouch. The animals were anesthetized with ether. Each rat lactates 4 pups. Group C: They do not lactate. In the 6th day of suckling we took away the newborn from theirs mothers for 8 Hours and then we put them to lactate for other 4 hours. After this period we removed the mammary glands. We prepared this material as the routine methods for optical microscopy and after wards we stained with Hematoxylin-Eosin. Direct relationship is demonstrated between the intensity of suctional stimulus and morphologically evaluated synthetic activity.     

Line-tension controlled mechanism for influenza fusion

Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension), which is essential to (i) stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii) drive its subsequent evolution. Such line-tension controlled fusion event does not proceed along the hypothesized standard stalk-hemifusion pathway. In modeled influenza fusion, single point mutations in the influenza fusion peptide either completely inhibit fusion (mutants G1V and W14A) or, intriguingly, specifically arrest fusion at a hemifusion state (mutant G1S). Our simulations demonstrate that, within a line-tension controlled fusion mechanism, these known point mutations either completely inhibit fusion by impairing the peptide's ability to stabilize the required peptide bundle (G1V and W14A) or stabilize a persistent bundle that leads to a kinetically trapped hemifusion state (G1S). In addition, our results further suggest that the recently discovered leaky fusion mutant G13A, which is known to facilitate a pronounced leakage of the target membrane prior to lipid mixing, reduces the membrane integrity by forming a 'super' bundle. Our simulations offer a new interpretation for a number of experimentally observed features of the fusion reaction mediated by the prototypical fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions.     

Immunological resistance to L1210 leukemia induced by viable L1210/DTIC cells

Summary Permanent, drug-induced antigenic alterations, not detectable in parental cells and transmissible after the withdrawal of treatment with the drug, have been obtained in mouse lymphoma. Viable L1210/DTIC cells, because they are rejected by syngeneic animals and carry L1210-associated TAA, can elicit host resistance to a subsequent inoculum of parental L1210. Mice challenged with viable L1210/DTIC cells, following rejection, were more resistant than mice immunized with inactivated parental cells. Resistance was specific and related to the immunogenicity of the TAA of the original tumor line employed.Active immunization was potentiated by adoptive transfer of immune lymphocytes, as evidenced by marked improvement in animal survival. Also, the treatment of tumor-bearing animals with anticancer compounds in conjunction with immunological alteration may result in an improved therapeutic response. BCNU administered to immunized animals 6 days after challenge with parental tumor cells resulted in augmented host survival, possibly attributable to partial resistance of a secondary immune response to the drug and a late nadir of immunosuppression, occurring after the completion of therapeutic action. Cyclophosphamide given before immunization enhanced host survival to a subsequent challenge of L1210 leukemia, conceivably as the result of preferential inhibition of T suppressor cells.     

Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications

Tissue engineering of multilayered constructs that model complex tissues poses a significant challenge for regenerative medicine. In this study, a three-layered scaffold consisting of an electrospun silk fibroin (SF) mat sandwiched between two dense collagen (DC) layers was designed and characterized. It was hypothesized that the SF layer would endow the DC-SF-DC construct with enhanced mechanical properties (e.g., apparent modulus, tensile strength, and toughness), while the surrounding DC layers provide an extracellular matrix-like environment for mesenchymal stem cell (MSC) growth. MSC-seeded DC-SF-DC hybrids were produced using the plastic compression technique and characterized morphologically, chemically, and mechanically. Moreover, MSC viability was assessed for up to 1 wk in culture. Scaffold analyses confirmed compaction and integration of the meso-scaled multilayered DC-SF-DC hybrid, which was reflected in a significantly higher toughness value when compared to DC and SF alone. MSCs directly incorporated into the DC layers remained viable for up to day 7. The ease of multilayered construct fabrication, enhanced biomechanical properties, along with uniformity of cell distribution confirmed the possibility for the incorporation and segregation of different cell types within distinct layers for the regeneration of complex tissues, such as skin, or central nervous system dura mater.