Global and 3D Spatial Assessment of Neuroinflammation in Rodent Models of Multiple Sclerosis

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.     

In vivo effects of vanadate on hepatic glycogen metabolizing and lipogenic enzymes in insulin-dependent and insulin-resistant diabetic animals

The insulin-mimetic action of vanadate is well established but the exact mechanism by which it exerts this effect is still not clearly understood. The role of insulin in the regulation of hepatic glycogen metabolizing and lipogenic enzymes is well known. In our study, we have, therefore, examined the effects of vanadate on these hepatic enzymes using four different models of diabetic and insulin-resistant animals. Vanadate normalized the blood glucose levels in all animal models. In streptozotocin-induced diabetic rats, the amount of liver glycogen and the activities of the active-form of glycogen synthase, both active and inactive-forms of phosphorylase, and lipogenic enzymes like glucose 6-phosphate dehydrogenase and malic enzyme were decreased and vanadate treatment normalized all of these to near normal levels. The other three animal models (db/db mouse, sucrose-fed rats and fa/fa obese Zucker rats) were characterized by hyperinsulinemia, hypertriglyceridemia, increases in activities of lipogenic enzymes, and marginal changes in glycogen metabolizing enzymes. Vanadate treatment brought all of these values towards normal levels. It should be noted that vanadate shows differential effects in the modulation of lipogenic enzymes activities in type I and type II diabetic animals. It increases the activities of lipogenic enzymes in streptozotocin-induced diabetic animals and prevents the elevation of activities of these enzymes in hyperinsulinemic animals. The insulin-stimulated phosphorylation of insulin receptor subunit and its tyrosine kinase activity was increased in streptozotocin-induced diabetic rats after treatment with vanadate. Our results support the view that insulin receptor is one of the sites involved in the insulin-mimetic actions of vanadate.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Design and synthesis of tricyclic cores for kinase inhibition

Interest in therapeutic kinase inhibitors continues to grow beyond success in oncology. To date, ATP-mimetic kinase inhibitors have focused primarily on monocyclic and bicyclic heterocyclic cores. We sought to expand on the repertoire of potential cores for kinase inhibition by exploring tricyclic variants of classical bicyclic hinge binding motifs such as pyrrolopyridine and pyrrolopyrazine. Herein we describe the syntheses of eight alternative tricyclic cores as well as in vitro screening results for representative kinases of potential therapeutic interest.     

Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.     

A Statistical Thermodynamic Model for Investigating the Stability of DNA Sequences from Oligonucleotides to Genomes

We describe the development and testing of a simple statistical mechanics methodology for duplex DNA applicable to sequences of any composition and extensible to genomes. The microstates of a DNA sequence are modeled in terms of blocks of basepairs that are assumed to be fully closed (paired) or open. This approach generates an ensemble of bubblelike microstates that are used to calculate the corresponding partition function. The energies of the microstates are calculated as additive contributions from hydrogen bonding, basepair stacking, and solvation terms parameterized from a comprehensive series of molecular dynamics simulations including solvent and ions. Thermodynamic properties and nucleotide stability constants for DNA sequences follow directly from the partition function. The methodology was tested by comparing computed free energies per basepair with the experimental melting temperatures of 60 oligonucleotides, yielding a correlation coefficient of −0.96. The thermodynamic stability of genic/nongenic regions was tested in terms of nucleotide stability constants versus sequence for the Escherichia coli K-12 genome. It showed clear differentiation of the genes from promoters and captures genic regions with a sensitivity of 0.94. The statistical thermodynamic model presented here provides a seemingly new handle on the challenging problem of interpreting genomic sequences.     

A biomimetic robotic jellyfish (Robojelly) actuated by shape memory alloy composite actuators

An analysis is conducted on the design, fabrication and performance of an underwater vehicle mimicking the propulsion mechanism and physical appearance of a medusa (jellyfish). The robotic jellyfish called Robojelly mimics the morphology and kinematics of the Aurelia aurita species. Robojelly actuates using bio-inspired shape memory alloy composite actuators. A systematic fabrication technique was developed to replicate the essential structural features of A. aurita. Robojelly's body was fabricated from RTV silicone having a total mass of 242 g and bell diameter of 164 mm. Robojelly was able to generate enough thrust in static water conditions to propel itself and achieve a proficiency of 0.19 s(-1) while the A. aurita achieves a proficiency of around 0.25 s(-1). A thrust analysis based on empirical measurements for a natural jellyfish was used to compare the performance of the different robotic configurations. The configuration with best performance was a Robojelly with segmented bell and a passive flap structure. Robojelly was found to consume an average power on the order of 17 W with the actuators not having fully reached a thermal steady state.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.