Construction of streptavidin-luciferase fusion protein for ATP sensing with fixed form

A fusion protein consisting of streptavidin and firefly luciferase was constructed to establish an accurate measuring technique of local ATP concentration. The fusion protein retained the binding ability of streptavidin and enzymatic activity of luciferase. Also, it could detect the concentration of antigens and could determine nanomolar concentrations of ATP in its fixed form via interactions with biotin-conjugated antibodies.     

Estrogen increases messenger RNA and immunoreactivity of aryl-hydrocarbon receptor nuclear translocator 2 in the rat mediobasal hypothalamus

We examined the effect of estrogen on the expression of aryl-hydrocarbon receptor (AhR) and two types of AhR nuclear translocator (Arnt1 and Arnt2) mRNAs in the hypothalamus of ovariectomized rats. Northern blotting demonstrated that, in the mediobasal hypothalamus, a subcutaneous injection of 20 microg estradiol benzoate (E(2)) significantly increased the expression of Arnt2 mRNA, but induced no significant changes in the expression of AhR and Arnt1 mRNAs. The expression of Arnt2 mRNA was significantly increased at 4, 24, and 72h after the injection. Immunocytochemical study revealed that the number of Arnt2 immunoreactive cells was also significantly increased at 72h after the injection. Conversely, in the preoptic area, injection of E(2) did not cause significant changes in the expression of any of the three mRNAs. These observations suggest that estrogen regulates Arnt2 expression in the mediobasal hypothalamus and modulates the toxic action of dioxins in rats.     

Effects of 5alpha-androst-16-en-3alpha-ol on the pulsatile secretion of luteinizing hormone in human females

We examined the effects of 5alpha-androst-16-en-3alpha-ol (3alpha-androstenol) on pulsatile luteinizing hormone (LH) secretion in human females. The frequency of the LH pulse in the follicular phase was decreased by exposing the women to 3alpha-androstenol.     

The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc

IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

Delivery of antibody-captured proteins into living cells using PTD-fused protein A

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.     

Intracerebroventricular administration of ghrelin rapidly suppresses pulsatile luteinizing hormone secretion in ovariectomized rats.

Ghrelin, an endogenous growth hormone (GH) secretagogue, is shown to increase food intake, which action is similar to that of orexin, also a hypothalamic peptide. Since orexin suppresses pulsatile LH secretion in ovariectomized (OVX) rats, the present study was undertaken to investigate whether ghrelin also suppresses LH secretion. Effects of intracerebroventricularly injected ghrelin (0.1 nmol/0.3 microl) were examined in OVX rats treated with a small dose of 17beta-estradiol (E(2)). After ghrelin injection, pulsatile LH secretions which were ongoing in these E(2)-treated OVX rats were significantly suppressed for about 1 h, whereas GH secretion increased, peaking at 30 min. The main parameter suppressed by ghrelin was the pulse frequency, not the pulse amplitude, suggesting the hypothalamus as the site of ghrelin action. This study provides evidence that ghrelin acts not only in the control of food intake but also in the control of LH secretion.     

Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.