Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

Mitochondrial haplotype diversity in the tortoise species <Emphasis Type="Italic">Testudo graeca</Emphasis> from North Africa and the Middle East

Background  

To help conservation programs of the endangered spur-thighed tortoise and to gain better insight into its systematics, genetic variation and evolution in the tortoise species Testudo graeca (Testudines: Testudinidae) was investigated by sequence analysis of a 394-nucleotide fragment of the mitochondrial 12S rRNA gene for 158 tortoise specimens belonging to the subspecies Testudo graeca graeca, Testudo graeca ibera, Testudo graeca terrestris, and a newly recognized subspecies Testudo graeca whitei. A 411-nucleotide fragment of the mitochondrial D-loop was additionally sequenced for a subset of 22 T. graeca, chosen because of their 12S gene haplotype and/or geographical origin.     

The role of interferon-alpha in a successful murine tumor therapy

Combination therapy using reovirus type 3 and the chemo-therapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is sufficient to cure approximately 80% of EL-4 lymphoma tumor-bearing BD2F1 male mice. Cured animals can be challenged with the EL-4 tumor, in the absence of the therapy, to yield 100% survival, whereas those challenged with heterologous tumor produce 0% survival. These results strongly suggest that a host-immune response is responsible for the observed therapeutic effect. Reovirus, a double-stranded RNA virus, is an efficient inducer of type I interferon. In an effort to determine the role of virus in this therapy, we substituted interferon-alpha (IFN-alpha) for reovirus in the therapy. Doses of IFN-alpha from 1000-10,000 U were capable of replacing reovirus to produce cure rates similar to reovirus. Spleen cells isolated from therapy-treated animals demonstrated high levels of cytotoxicity against the natural killer cell-sensitive cell line YAC-1, but not against EL-4 tumor. In vitro stimulation of isolated spleen cells by IFN-alpha resulted in a high level of natural killer cell activity, but no cytotoxicity against the EL-4 tumor. A significant antiproliferative effect against the EL-4 tumor in cell culture was demonstrated by IFN-alpha. Finally, therapy-treated, tumor-bearing mice that were injected with anti-IFN-alpha + -beta antibodies had similar survival levels as control mice, indicating that other cytokines might also play a role in promoting tumor killing. These investigations suggest that IFN-alpha may be a mediator of antitumor activity in the reovirus therapy system.     

Biochemical and cell-based assays for characterization of BACE-1 inhibitors

The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.     

Three approaches for estimating the elastic modulus of the tympanic membrane

The function of the middle ear is to resolve the acoustic impedance mismatch between the air in the ear canal and the fluid of the inner ear. Without this impedance matching, very little acoustic energy would be absorbed into the cochlea. The first step in this process is the tympanic membrane (TM) converting sound in the ear canal into vibrations of the middle ear bones. Understanding how the TM manages its task so successfully over such a broad frequency range should lead to more satisfactory and less variable TM repairs (myringoplasty). In addition, understanding the mechanics of the TM is necessary to improve the coupling between ossicular prostheses and the TM. Mathematical models have played a central role in helping the research community understand the mechanics of the eardrum. However, all models require parameters as inputs. Unfortunately, most of the parameters needed for modeling the TM are not well known. In this work, several approaches for inferring the material properties of the TM are explored. First, constitutive modeling is used to estimate an elastic modulus based on the elastic modulus of collagen and experimentally observed fiber densities. Second, experimental tension and bending test results from the literature are re-interpreted using composite laminate theory. Lastly, dynamic measurements of the cat TM are used in conjunction with a composite shell model to bound the material parameters. Values from the literature, both measurement and modeling efforts, and from the present analysis are brought together to form a coherent picture of the TM's material properties. In the human, the data bound the elastic modulus between 0.1 and 0.3 GPa. In the cat, the data suggest a range of 0.1-0.4 GPa. These values are significantly higher than previous estimates.     

Nonmyeloablative bone marrow transplantation of BXSB lupus mice using fully matched allogeneic donor cells from green fluorescent protein transgenic mice

Male BXSB mice, a mouse model of systemic lupus erythematosus, were given bone marrow transplants (BMT) at 20 wk of age using MHC-matched donor cells and nonmyeloablative conditioning (550 cGy irradiation). Transplanted mice and irradiation controls were followed for a period of 20 wk. Mice transgenic for green fluorescent protein were used as donors to allow tracking of donor cells and a determination of chimerism. Radiation controls had reduced renal pathology at 10 wk posttransplant, but not at 20 wk compared with untreated mice, while nonmyeloablative BMT mice had significantly reduced pathology at both time intervals. The monocytosis characteristic of older BXSB mice was also reduced by BMT, but the treatment did not prevent production of Ab to dsDNA. A stable chimerism of 24-40% donor CD45-positive cells was achieved in spleen and bone marrow, and there was no evidence of clinical graft vs host disease. Donor cells were detected in most recipient organs, notably the thymus and renal glomeruli. The results suggest that complete depletion of mature lymphocytes or of progenitor stem cells is not required to control lupus nephritis in BXSB mice.     

Nutritional composition and digestibility by ocelots (Leopardus pardalis) of whole animals and a commercial diet

Felids are adapted to eat whole prey, but in North American zoos are usually fed processed diets based on muscle meat. We analyzed proximate nutrient composition and digestibility by ocelots of a commercial processed diet and whole animals of five species. The processed diet did not differ significantly from the whole animals in proximate composition, although it was at one end of the range of results for all nutrients. Domestic chicks were significantly lower than all other dietary items tested in digestibility of energy and fat, and lower than rabbits and quail in digestibility of dry matter. There were no other significant differences. These results suggest that the commercial diet tested provides an appropriate nutritional environment for ocelots with respect to proximate constituents. Studies of vitamin and mineral composition and digestibility and comparisons to wild prey species should be conducted to permit a full evaluation. Zoo Biol 29:753–759, 2010. © 2009 Wiley‐Liss, Inc.