Inactive GSK3ß is disturbed in the spinal cord during experimental autoimmune encephalomyelitis, but rescued by stem cell therapy.

Glycogen synthase kinase 3ß (GSK3ß) is known to control neuroinflammation, however the status of GSK3ß in multiple sclerosis, the most common inflammatory demyelinating disease of the CNS, and its animal model EAE, is unknown. In this study, we investigated the expression of phosphorylated GSK3ß, the inactive form of GSK3ß, in the spinal cords of EAE mice. We demonstrate that while the expression of phosphorylated GSK3ß was present in radial astrocytes and neurons of the control mice that received only complete Freund's adjuvant, it was absent in radial astrocytes and significantly lower in neurons of EAE animals. The loss of phosphorylated GSK3ß in radial glia and neurons in EAE spinal cords was concurrent with radial glia migration and astrogliosis. This disturbance in the expression of inactive GSK3ß was recovered in neurons, but not in the radial glia, after treatment of EAE mice with adipose-derived mesenchymal stem cells capable of inducing a Th2 shift. Collectively, our results suggest a link between inactive GSK3ß and modulation of the immune responses during EAE. Thus, we propose that maintenance of GSK3ß in its inactive status may play a role in preserving the normal physiology of the spinal cord and amelioration of EAE following stem cell therapy.

Pattern electroretinogram association with spectral domain-OCT structural measurements in glaucoma.

PURPOSE: To describe the association between pattern electroretinogram (PERG) amplitude and spectral domain-optical coherence tomography (SD-OCT) macular thickness, retinal nerve fibre layer (RNFL) thickness and optic disc topography measurements. SUBJECTS AND METHODS: Both eyes (n = 132) of 66 glaucoma patients (mean age = 67.9 years) enrolled in the University of California, San Diego, CA, USA, Diagnostic Innovations in Glaucoma Study (DIGS) were included. Eyes were tested with PERG (Glaid PERGLA, Lace Elettronica, Pisa, Italy), RTVue SD-OCT (Optovue Inc., Fremont, CA, USA) GCC, and NHM4 protocols on the same day. Of the 66 enrolled patients, 43 had glaucoma defined by repeated abnormal standard automated perimetry (SAP) results in at least one eye and 23 were glaucoma suspects defined by a glaucomatous-appearing optic disc by physicians' examination in at least one eye and normal SAP results in both eyes. Associations (R(2)) were determined between PERG amplitude (µV) and SD-OCT macular ganglion cell complex (GCC) thickness (µm), macular thickness (µm), macular outer retinal thickness (macular thickness minus GCC thickness) (µm), RNFL thickness (µm), neuroretinal rim area (mm(2)), and rim volume (mm(3)). RESULTS: PERG amplitude was significantly associated with GCC thickness (R(2) = 0.179, P < 0.001), RNFL thickness (R(2) = 0.174, P < 0.001), and macular thickness (R(2) = 0.095, P<0.001). R(2) associations with other parameters were not significant (all P > 0.624). Significant associations remained for GCC and average RNFL thickness when age and intraocular pressure at the time of testing were included in multivariate models (both P ≤ 0.030). CONCLUSIONS: PERG amplitude is significantly (but weakly) associated with macular GCC thickness, RNFL thickness, and macular thickness. The lack of association between PERG amplitude and macular outer retinal thickness supports previous results, possibly suggesting that that the PERG is driven primarily by retinal ganglion cell (inner retinal) responses.

Dunaliella biotechnology: methods and applications.

The microalga Dunaliella salina is the best commercial source of natural beta-carotene. Additionally, different species of Dunaliella can accumulate significant amounts of valuable fine chemicals such as carotenoids, glycerol, lipids, vitamins, minerals and proteins. They also have a large potential for biotechnological processes such as expressing of foreign proteins and treatment of wastewater. In this review, we discussed several biotechnological aspects of the mass cultivation of D. salina like strain selection, carotenoid induction, culture conditions, culture systems and downstream processes. We also discuss several traditional and new applications of the genus.

Isomalt production by cloning, purifying and expressing of the MDH gene from Pseudomonas fluorescens DSM 50106 in different strains of E. coli.

A NADH-dependent mannitol dehydrogenase gene (mtlD) was cloned from Pseudomonas fluorescens, subcloned into an expression vector (pDEST110) and entered into different strains of E. coli to compare their protein expression and the enzyme specific activity. Purifications were accomplished by Ni(2+)-NTA affinity chromatography. Using this approach, the efficiency of purification process significantly increased (up to 90%) so that the purified enzyme gave a sharp single band (55 kDa) in SDS-PAGE. The results showed that among the strains, BL21 (DE3) plysS exhibited the maximum expression level of MDH(mannitol dehydrogenase) (11 mg L(-1)). Results from activity assay with fructose as substrate also showed that in this strain the specific activity of 63 U mg(-1) protein monitored for the enzyme, the record not reported before. Resazurin staining also indicated that the enzyme reduced fructose, whereas oxidized other substrates including mannitol, sorbitol and arabitol under optimal assay condition. From HPLC analysis it was showed for the first time that the enzyme could convert substrate isomaltulose to the specific products, GPM and GPS. Interestingly, because of the high specificity of the enzyme for substrate, the method can be used as an alternative approach to substitute nonspecific conventional method of isomalt production.

Endogenous nerve growth factor and neurotrophin-3 act simultaneously to ensure the survival of postnatal sympathetic neurons in vivo.

For many years nerve growth factor was the only factor known to influence embryonic and postnatal development of sympathetic neurons. Its deprivation by antibody neutralization or gene mutation results in extensive neuron death. Recently it has been shown that these neurons also require neurotrophin-3 for survival in the late developmental period. Using neurotrophin-3 antiserum to neutralize endogenous factor in newborn rats. Our laboratory has shown that extensive numbers of neurons are lost from both pre- and paravertebral ganglia, indicating a continuing requirement for neurotrophin-3. In the present study we sought to determine whether neurons could survive in vivo in the presence of excess amounts of either nerve growth factor or neurotrophin-3 alone. Consistent with previous findings, administration of antiserum to nerve growth factor or neurotrophin-3 to newborn rats for eight days, resulted in an extensive loss of sympathetic neurons. Interestingly, administration of neurotrophin-3 together with nerve growth factor antiserum or nerve growth factor with neurotrophin-3 antiserum reversed this neuronal loss. However the latter combination was less effective than the former. Furthermore, the ability of exogenous nerve growth factor to increase both the number and size of sympathetic neurons was prevented by the simultaneous deprivation of endogenous neurotrophin-3. Unlike nerve growth factor, exogenous neurotrophin-3 failed to rescue the naturally occurring neuronal death in these newborn rats. Further evidence for a physiological role for both nerve growth factor and neurotrophin-3 was found by the detection of both trkA and trkC immunoreactivity in neurons of the superior cervical ganglion. Taken together, these results suggest that sympathetic neurons do not have an absolute requirement for either nerve growth factor or neurotrophin-3 and that the endogenous supply of either factor alone is insufficient to support neuronal survival postnatally. However, while each factor may play similar roles in the regulation of postmitotic neuronal function, some evidence for distinct functions has been identified.

Neurotrophic factors are required by mature sympathetic neurons for survival, transmission and connectivity.

1. Two neuronal growth factors, nerve growth factor (NGF) and neurotrophin 3 (NT3), have been studied for their action on the developing and mature sympathetic nervous system. 2. Antibodies to each factor have proved useful as reagents for the detection and quantification of NGF and NT3. They have also proved valuable in uncovering the functional roles of each factor by their ability to neutralize the endogenous molecules. 3. Nerve growth factor acts on postnatal neurons to control neurotransmission, connectivity and survival. Like NGF, NT3 is synthesized by effector tissues and is retrogradely transported by post-ganglionic neurons to prevent cell death. However, the two factors have been shown to have quite distinct functions in mature neurons, indicating the existence of different signalling pathways. This differential action extends to secondary influences on satellite glia. 4. Pathological consequences result from excessive growth factor synthesis leading, in the hypertensive rat, to hyperinnervation and elevated blood pressure. Satellite glial cell synthesis of the factors and their receptors following peripheral nerve damage appears to be responsible for the establishment of inappropriate neuronal connections between sympathetic nerve terminals and sensory somata. 5. It is concluded that these potent factors control, by both coincident and independent mechanisms, sympathetic neuronal function throughout the life of the animal.