Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases,in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Thermal diffusion of a stiff rod-like mutant Y21M fd-virus

We investigated the thermal diffusion phenomena of a rodlike mutant filamentous fd-Y21M virus in the isotropic phase by means of an improved infrared thermal-diffusion-forced Rayleigh scattering (IR-TDFRS) setup optimized for measurements of slowly diffusing systems. Because this is the first thermal diffusion study of a stiff anisotropic solute, we investigate the influence of the shape anisotropy on the thermal diffusion behavior. The influence of temperature, fd-Y21M concentration, and ionic strength in relation with the thermodiffusion properties is discussed. We characterize and eliminate the effect of these parameters on the absolute diffusion of the rods and show that diffusion determines the behavior of the Soret coefficient because the thermal diffusion coefficient is constant in the investigated regime. Our results indicate that for the thermal diffusion behavior structural changes of the surrounding water are more important than structural changes between the charged macroions. In the investigated temperature and concentration range, the fd-Y21M virus is thermophobic for the low salt content, whereas the solutions with the high salt content change from thermophobic to thermophilic behavior with decreasing temperature. A comparison with recent measurements of other charged soft and biological matter systems shows that the shape anisotropy of the fd-virus becomes not visible in the results.     

Biodegradable Ocular Inserts for Sustained Delivery of Brimonidine Tartarate: Preparation and <Emphasis Type="Italic">In Vitro</Emphasis>/<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation

The bioavailability of therapeutic agents from eye drops is usually limited due to corneal barrier functions and effective eye protective mechanisms. Therefore, the current study aims to enhance ocular bioavailability of brimonidine, a potent antiglaucoma drug, through the preparation of ocular inserts. Solvent casting technique was employed to prepare the inserts using polyvinylpyrrolidone K-90 (PVP K-90) as film-forming polymer blended with different viscosity grades of bioadhesive polymers namely hydroxypropyl methycellulose, carbopol, sodium alginate, and chitosan. The prepared ocular inserts were evaluated for various physicochemical parameters, swelling behavior, and in vitro release patterns. Sodium alginate-based ocular inserts revealed the most sustainment in drug release (99% at 6 h), so it was selected for further modifications via coating it, on one side or dual sides, using hydrophobic film composed of either ethylcellulose or Eudragit RSPO. The obtained in vitro release results for the modified ocular inserts revealed that ethylcellulose is superior to Eudragit RSPO in terms of brimonidine release sustainment effect. Ocular inserts composed of 7% PVP K-90, 1.5% low molecular weight sodium alginate with or without ethylcellulose coat were able to sustain the in vitro release of brimonidine. Their therapeutic efficacy regarding intraocular pressure (IOP) lowering effect when inserted in albino rabbits eyes showed superior sustainment effect compared with that of brimonidine solution. Furthermore, due to both the mucoadhesive property and the drug sustainment effect, the one-side-coated ocular insert showed more IOP lowering effect compared with that of its non-coated or dual-side-coated counterpart.     

FTIR Assessment of the Effect of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> Leave Extract (EGb 761) on Mammalian Retina

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this study, we investigated the effect of prolonged administration of EGb 761, up to 10 weeks, on mammalian retina using Fourier transform infrared spectroscopy (FTIR). Two main groups were involved in this study: the normal group (n = 10); and EGb-administrated group (n = 50) that received—orally—a dose of 40 mg/kg/day EGb 761. The results demonstrated that EGb administration was associated with different beneficial effects on the retinal constituents especially the underlying amide I protein secondary structure components as well as the NH-OH region. It concluded that the optimum daily administration period of EGb (40 mg/kg) for ophthalmic applications that targeting the retina ranges from 5 to 8 weeks.     

Effects of immunomodulatory drugs on plasma inflammatory markers in a rabbit model of atherosclerosis

Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive immuno-inflammatory mechanisms are involved. Inflammatory cytokines are implicated in the development and progression of atherosclerotic lesions. Immunomodulatory therapies have been proposed for the treatment of atherosclerosis. Therefore, the aim of this study was to investigate the systemic anti-inflammatory and immunomodulatory effects of atorvastatin, cyclosporine A (CsA), and tacrolimus (FK506) on plasma inflammatory markers in atherosclerotic rabbits. Male New Zealand rabbits were randomized into five groups each of 12 animals. Standard diet-fed group served as control, and the cholesterol-fed group received a diet supplemented with 1% cholesterol alone, cholesterol + atorvastatin, cholesterol + FK506, and cholesterol + CsA. Serum levels of lipid profile parameters (triglycerides, cholesterol, and high-density lipoprotein) were measured using colorimetric methods. Serum levels of C-reactive protein (CRP), interleukin-6 (Il-6), and interferon-gamma (INF-γ) were measured in all studied groups using ELISA techniques. Our results revealed a significant decrease (p < 0.001) in the serum levels of lipid profile parameters, CRP, Il-6, and INF-γ in atorvastatin-treated group compared with the cholesterol-fed group. On the other hand, a non-significant difference was observed for the same parameters in either FK506- or CsA-treated groups compared with the cholesterol-fed group. In conclusion, atorvastatin has a systemic anti-inflammatory role that far surpassed the cholesterol reduction effect alone. FK506 or CsA failed to suppress elevated plasma inflammatory markers. Thus, low doses of these two immunomodulating drugs could not have generalized systemic anti-inflammatory or immunosuppressive effects.