Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.     

Fungal mutualists enhance growth and phytochemical content in Echinacea purpurea

An emerging paradigm in sustainable biotechnique is the use of mutualists to enhance plant growth and secondary metabolism. Our objective was to determine impact of two groups of fungal mutualists on growth and phytochemistry of Echinacea purpurea. Growth, development, and phytochemical concentration were measured in greenhouse-grown 12-week-old plants colonized by arbuscular mycorrhizal fungi (AMF) (Rhizophagus intraradices and Gigaspora margarita) or the endophytic entomopathogen, Beauveria bassiana. In one experiment, all measured growth parameters were increased in mycorrhizal plants. Biomass of AMF-colonized plants was over 13-fold greater than non-mycorrhizal controls receiving the same levels of phosphorous, and over 4-fold greater than non-mycorrhizal controls given additional phosphorous. Endophytic colonization by B. bassiana had minor effects on growth. Colonization by AMF and B. bassiana alone or in combination altered concentrations of phytochemicals (pigments, polyphenolics, alkylamides, and terpenes). Mycorrhizal plants produced up to 4.6-fold higher concentration of polyphenolics. Specific alkylamides increased 1.7 fold in plants colonized only with B. bassiana and up to a 2.4-fold increase in plants colonized by both mutualists. Changes in other phytochemical classes were related to differences in plant size induced by AMF. Phytochemical content (concentration × biomass) was increased up to 30-fold in mycorrhizal plants. Phytochemical relationships to plant biomass were confirmed in a second experiment in which non-mycorrhizal plants were fertilized to produce biomass equivalent to that of mycorrhizal plants. Based on this study, mycorrhizal colonization of E. purpurea enhances phytochemical content; this has major implications for the natural product industries and growers of E. purpurea.     

Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

Strategies for imaging mitophagy in high-resolution and high-throughput

The selective autophagic removal of mitochondria called mitophagy is an essential physiological signaling for clearing damaged mitochondria and thus maintains the functional integrity of mitochondria and cells. Defective mitophagy is implicated in several diseases, placing mitophagy as a target for drug development. The identification of key regulators of mitophagy as well as chemical modulators of mitophagy requires sensitive and reliable quantitative approaches. Since mitophagy is a rapidly progressing event and sub-microscopic in nature, live cell image-based detection tools with high spatial and temporal resolution is preferred over end-stage assays. We describe two approaches for measuring mitophagy in mammalian cells using stable cells expressing EGFP-LC3 – Mito-DsRed to mark early phase of mitophagy and Mitochondria-EGFP – LAMP1-RFP stable cells for late events of mitophagy. Both the assays showed good spatial and temporal resolution in wide-field, confocal and super-resolution microscopy with high-throughput adaptable capability. A limited compound screening allowed us to identify a few new mitophagy inducers. Compared to the current mitophagy tools, mito-Keima or mito-QC, the assay described here determines the direct delivery of mitochondrial components to the lysosome in real time mode with accurate quantification if monoclonal cells expressing a homogenous level of both probes are established. Since the assay described here employs real-time imaging approach in a high-throughput mode, the platform can be used both for siRNA screening or compound screening to identify key regulators of mitophagy at decisive stages.     

Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs). Discovery of capromorelin

Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituicyte EC(50)=3 nM) and the des-methyl analogue 5c (hGHS-R1a K(i)=17 nM, rat pituicyte EC(50)=3 nM), increased plasma GH levels in an anesthesized rat model, with ED(50) values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-duration GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal frailty in elderly adults.     

Tests of pre- and postpollination barriers to hybridization between sympatric species of Ipomopsis (Polemoniaceae)

The Ipomopsis aggregata species complex (Polemoniaceae) includes species pairs that hybridize readily in nature as well as pairs that meet along contact zones with no apparent hybridization. Artificial hybrids can be made between I. aggregata and I. arizonica, yet morphological intermediates between these two species have not been observed in natural populations. This apparent lack of hybridization is perplexing given that plants of the two species often grow within a few metres of each other and both species have red flowers visited by the same species of hummingbirds. We used trained hummingbirds to examine pollen transfer within and between species. We also hand-pollinated flowers to examine paternal success of heterospecific and conspecific pollen, testing paternity with electrophoretic examination of seeds. Hummingbirds were not simply better at transferring pollen within than between species. Instead, I. arizonica was a better pollen donor so that considerable pollen transfer was observed from I. arizonica to I. aggregata, but very little in the opposite direction. Conversely, once pollen arrived at stigmas, I. arizonica pollen performed very poorly on I. aggregata pistils. However, pollen from I. aggregata could, in some cases, sire seeds on I. arizonica. We hypothesize that hybrids are scarce in nature, in part, because of asymmetric barriers to reproduction: little pollen transfer in one direction and poor pollen performance in the other.