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.     

Increasement of Heterologous Expression of Recombinant Vit v 1 in Pichia pastoris KM71 by Nonionic Detergents as a Cost-effective Approach

Applied Biochemistry and Microbiology - Vit v 1 as a lipid-transfer protein is a major allergen of grapes (Vitis vinifera) that elicits food allergy in many patients in Iran. Todays, recombinant...     

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.     

Gonadotropin-Releasing Hormone Agonists Sensitize,and Resensitize,Prostate Cancer Cells to Docetaxel in a p53-Dependent Manner

Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. We previously showed that GnRH agonists exert an antitumor activity in CRPC cells; however, a link between GnRH receptors and the apoptotic machinery remains to be defined. Aim of this study was to evaluate whether, in CRPC cells, GnRH agonists might affect the expression/activity of apoptosis-related proteins and might sensitize, or resensitize, cancer cells to chemotherapeutics. We demonstrated that, in p53-positive DU145 cells, GnRH agonists: a) increase the expression of the proapoptotic protein Bax; this effect is mediated by the phosphorylation (activation) of p53, triggered by the p38 MAPK; b) potentiate the antiproliferative/proapoptotic activity of docetaxel; c) resensitize docetaxel-resistant cells to the antitumor activity of the cytotoxic drug. These data indicate that GnRH agonists sensitize and, more importantly, resensitize DU145 CRPC cells to chemotherapy in a p53-dependent manner. To confirm the crucial role of p53 in the activity of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein.