Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

Novel fatty acid chain modified GLP-1 derivatives with prolonged in vivo glucose-lowering ability and balanced glucoregulatory activity

Glucagon-like peptide-1 is a potent hypoglycemic hormone with beneficial properties for the treatment of diabetes. However, its half-life is short because the rapid metabolic degradation. This study aims to prolong the half-life of glucagon-like peptide-1 through conjugation with the fatty acid side chain which helps the conjugates to interact with the albumin. Firstly, we chose two optimized polypeptide chains which have tremendous hypoglycemic effect named Cys₁₇-Gly₈-GLP-1(7-36)-NH₂ and Cys₃₇-Gly₈-GLP-1(7-37)-NH₂, and various fatty acid chains were modified. All conjugates preserved relatively strong GLP-1R activation and I-6 behaved best in glucose-lowering ability. The prolonged antidiabetic effects of I-6 were further confirmed by hypoglycemic efficacy test in vivo. Meanwhile, once daily injection of I-6 to diabetic mice achieved long-term beneficial effects on glucose tolerance, body weight and blood chemistry. It is concluded that I-6 is a promising agent for further investigation of its potential to treat obese patients with diabetes.     

MicroRNAs and epigenetic regulation in the mammalian inner ear: implications for deafness

Sensorineural hearing loss is the most common sensory disorder in humans and derives, in most cases, from inner-ear defects or degeneration of the cochlear sensory neuroepithelial hair cells. Genetic factors make a significant contribution to hearing impairment. While mutations in 51 genes have been associated with hereditary sensorineural nonsyndromic hearing loss (NSHL) in humans, the responsible mutations in many other chromosomal loci linked with NSHL have not been identified yet. Recently, mutations in a noncoding microRNA (miRNA) gene, MIR96, which is expressed specifically in the inner-ear hair cells, were linked with progressive hearing loss in humans and mice. Furthermore, additional miRNAs were found to have essential roles in the development and survival of inner-ear hair cells. Epigenetic mechanisms, in particular, DNA methylation and histone modifications, have also been implicated in human deafness, suggesting that several layers of noncoding genes that have never been studied systematically in the inner-ear sensory epithelia are required for normal hearing. This review aims to summarize the current knowledge about the roles of miRNAs and epigenetic regulatory mechanisms in the development, survival, and function of the inner ear, specifically in the sensory epithelia, tectorial membrane, and innervation, and their contribution to hearing.     

Paenibacillus dendritiformis Bacterial Colony Growth Depends on Surfactant but Not on Bacterial Motion

Most research on growing bacterial colonies on agar plates has concerned the effect of genetic or morphotype variation. Some studies have indicated that there is a correlation between microscopic bacterial motion and macroscopic colonial expansion, especially for swarming strains, but no measurements have been obtained for a single strain to relate the microscopic scale to the macroscopic scale. We examined here a single strain (Paenibacillus dendritiformis type T; tip splitting) to determine both the macroscopic growth of colonies and the microscopic bacterial motion within the colonies. Our multiscale measurements for a variety of growth conditions revealed that motion on the microscopic scale and colonial growth are largely independent. Instead, the growth of the colony is strongly affected by the availability of a surfactant that reduces surface tension.Bacteria are able to colonize many different surfaces through collective behavior such as swarming and biofilm formation. Studies of such behavior (10, 18, 26, 31) have revealed cooperative phenomena on both microscopic and colonial scales (4, 5, 7, 8, 20), including production of extracellular “lubricant-wetting” fluid for movement on medium and hard surfaces (19, 22, 25), chemical signaling such as quorum sensing and chemotactic signaling (1, 12, 27), and the secretion of inhibiting and killing factors (2, 9, 11, 14, 15, 17).Research has suggested possible links between the microscopic behavior of a colony and the rate at which the colony expands (12, 23, 24, 29). For Pseudomonas aeruginosa, increased reversal rates for flagella lead to hyperswarming (a larger colony) (26). Similar flagellar modulation affects Escherichia coli (32); if the bacteria never tumble (flagella rotate only counterclockwise) or only tumble (flagella rotate only clockwise), the final colony is much smaller than a colony formed when the bacteria both swim and tumble. For Rhizobium etli, a correlation has been observed between microscopic swarming motion and expansion of the colony, and an acylhomoserine lactone molecule has been found to be a swarming regulator, as well as a biosurfactant that controls surface activity (12). These studies suggest that there is a correlation between microscopic activity and colonial expansion; however, a mutation may be pleiotropic, affecting both motility and surfactant production. Further, there may be additional, unidentified differences between mutant and wild-type strains. For example, the failure of Bacillus subtilis laboratory strains to swarm is caused by a mutation in a gene (sfp) needed for surfactin synthesis and a mutation(s) in an additional unknown gene(s) (21). Experiments that avoid this ambiguity by studying the response of a single strain exposed to changing physical environments have not been performed. Further, except for measurements of the size of an expanding colony as a function of time (3, 6), no detailed time development studies of a growing bacterial colony have been reported.Here we exposed a single bacterial strain, Paenibacillus dendritiformis type T (tip splitting) (4), to different substrate hardnesses, nutrition levels, and surfactant concentrations to identify the parameters that determine colonial growth. P. dendritiformis is a gram-positive rod-shaped (4 μm by 1 μm) bacterium that swims on top of an agar gel in a thin layer (a few micrometers thick) of fluid, presumably secreted by the bacterial cells. The bacteria develop complex colonial (bush-like) branching patterns that are sensitive to small changes in the environment when the bacteria are grown on nutrient-limited surfaces (low peptone levels [approximately 1 g/liter]) (6). The colonies grow slowly (0.1 mm/h) so the microscopic motion can be followed with a microscope for about 10 min without moving the field of view. Also, this strain shows swarming-like microscopic motion where the bacteria move collectively in whirls and jets. This makes this bacterium well suited for studying simultaneously the development of a colony and the internal structure of branches. We constructed a novel setup to observe 10 growing P. dendritiformis colonies in each experiment, and complementary microscopic measurements were obtained for the velocity field of individual bacteria or small groups of cells within the colonies. Specifically, we measured the “bacterial speed,” which was the average of the values for the velocity vectors for the bacteria in a region near the edge of a growing colony, and the “tip velocity,” which was the speed of the moving growth front at the edge of a colony. We also quantified the collective bacterial motion within the colonies by computing spatial and temporal velocity autocorrelation functions.     

Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca²⁺ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca²⁺ influx in hα3β4 AChRs with ～9-fold higher potency than that for PCP, (b) [³H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (K_d = 0.46 ± 0.06 μM), and ibogaine inhibits [³H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [³H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time.     

Development of biosensors for cancer clinical testing

Biosensors are devices that combine a biochemical recognition/binding element (ligand) with a signal conversion unit (transducer). Biosensors are already used for several clinical applications, for example for electrochemical measurement of blood glucose concentrations. Application of biosensors in cancer clinical testing has several potential advantages over other clinical analysis methods including increased assay speed and flexibility, capability for multi-target analyses, automation, reduced costs of diagnostic testing and a potential to bring molecular diagnostic assays to community health care systems and to underserved populations. They have the potential for facilitating Point of Care Testing (POCT), where state-of-the-art molecular analysis is carried out without requiring a state-of-the-art laboratory. However, not many biosensors have been developed for cancer-related testing. One major challenge in harnessing the potential of biosensors is that cancer is a very complex set of diseases. Tumors vary widely in etiology and pathogenesis. Oncologists rely heavily on histological characterization of tumors and a few biomarkers that have demonstrated clinical utility to aid in patient management decisions. New genomic and proteomic molecular tools are being used to profile tumors and produce "molecular signatures." These signatures include genetic and epigenetic signatures, changes in gene expression, protein profiles and post-translational modifications of proteins. These molecular signatures provide new opportunities for utilizing biosensors. Biosensors have enormous potential to deliver the promise of new molecular diagnostic strategies to patients. This article describes some of the basic elements of cancer biology and cancer biomarkers relevant for the development of biosensors for cancer clinical testing, along with the challenges in using this approach.