Comparison of the Temporary Dynamics of NGF and BDNF Gene Expression in Rat Hippocampus,Frontal Cortex,and Retina Under Semax Action

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation, and maintenance of function of different neuron populations. Some peptides are able to affect the production and activity of neurotrophins. One of these synthetic peptides is heptapeptide Semax, an analog of the N-terminal adrenocorticotropic hormone fragment 4-10. It is known that Semax has effects on learning and memory formation and exerts some neuroprotective effects in rodents and humans. Male Wistar rats were treated for 20 min, 40 min, 90 min, 3 h, 8 h, and 24 h with Semax. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) gene expression in rat brain and retina was analyzed by real-time polymerase chain reaction. It was revealed that after Semax administration the multidirectional activation of the expression of the genes under investigation in the hippocampus, frontal cortex, and retina was observed. The expression of both neurotrophin genes was decreased in rat hippocampus and retina 20 min after Semax administration and was increased in the frontal cortex. The expression levels of NGF remained practically constant in the retina at the initial stage, whereas the expression levels of BDNF were significantly increased 90 min after Semax administration.     

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Background

Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky.

Design and Methods

Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed.

Results

In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO₃-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na⁺/K⁺ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K⁺ uptake. Unidirectional K⁺ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K⁺,Cl⁻ co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors.

Conclusions

These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.     

Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases and mitogen activated kinase phosphatase‐1 in microglial cells

Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK‐3/6, MKK‐1/2 and MKK‐4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP‐1. Exposure of N9 microglia to Mn (250 µm ), LPS (100 ng ml^?1) or Mn + LPS increased MKK‐3/6 and MKK‐4 activity at 1 h; the effect of Mn + LPS on MKK‐4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK‐3/6 and MKK‐1/2 phosphorylation, whereas MKK‐4 was activated only by Mn and Mn + LPS. Besides activating MKK‐4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK‐4's phosphorylation on Ser80, which negatively regulates MKK‐4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP‐1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF‐α, IL‐6 and Cox‐2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK‐1/2, MKK‐3/6 and MKK‐4, are activated by Mn suggests that Mn‐induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn‐induced activation was persistent at least for 4 h, indicating a long‐term effect. Copyright © 2010 John Wiley & Sons, Ltd.     

Humoral and cell-mediated immunity following vaccination with synthetic Candida cell wall mannan derived heptamannoside-protein conjugate: Immunomodulatory properties of heptamannoside-BSA conjugate

Chemically defined glycoprotein conjugate composed of synthetically prepared mannan-derived heptamannoside with terminal β-1,2-linked mannose residue attached to the α-1,3-linked mannose residues and BSA as carrier protein (M7-BSA conjugate) was analysed for the capacity to induce protective humoral immunity and appropriate alteration cellular immunity. To identify protective antigenic structure of Candida cell wall mannan M7-BSA conjugate was used for BALB/c mice immunization. The obtained results were compared with placebo group and with heat-inactivated C. albicans whole cells immunization. The administration route of M7-BSA conjugate secondary booster injection significantly affected the intensity of humoral immune response and the specificity of produced antibodies. All prepared sera were able to elevate candidacidal activity of polymorphonuclear leukocytes (PMN) in cooperation with complement. Moreover, polyclonal sera obtained after secondary subcutaneous (s.c.) booster injection of M7-BSA conjugate were able to induce candidacidal activity of PMN also in complement independent manner. M7-BSA conjugate immunization induced increases of phagocytic activity and respiratory burst of granulocytes, caused a raise of the proportion of CD3(+) T lymphocytes and increased the CD4(+)/CD8(+) T lymphocyte ratio. We observed also an increasing proportion of CD4(+)CD25(+) T cells compared to immunization with heat inactivated whole C. albicans cells, which in turn promoted an increase of the CD8(+)CD25(+) cell proportion. Immunization with M7-BSA conjugate induced Th1, Th2 and Th17 immune responses as indicated by the elevation of relevant cytokines levels. These data provide some insights on the immunomodulatory properties of oligomannosides and contribute to the development of synthetic oligosaccharide vaccines against fungal diseases.     

Protected Graft Copolymer (PGC) Basal Formulation of Insulin as Potentially Safer Alternative to Lantus® (Insulin-Glargine): A Streptozotocin-Induced,Diabetic Sprague Dawley Rats Study

Purpose

To develop a long-acting formulation of native human insulin with a similar pharmacodynamics (PD) profile as the insulin analogue insulin glargine (Lantus®, Sanofi-Aventis) with the expectation of retaining native human insulin’s superior safety profile as insulin glargine is able to activate the insulin-like growth factor 1 (IGF-1) receptor and is linked to a number of malignancies at a higher rate than regular human insulin.

Methods

Development of protected graft copolymer (PGC) excipients that bind native human insulin non-covalently and testing blood glucose control obtained with these formulations in streptozotocin-induced diabetic Sprague Dawley rats compared to equally dosed insulin glargine.

Results

PGC-formulations of native human insulin are able to control blood glucose to the same extent and for the same amount of time after s.c. injection as the insulin analogue insulin glargine. No biochemical changes were made to the insulin that would change receptor binding and activation with their possible negative effects on the safety of the insulin.

Conclusion

Formulation with the PGC excipient offers a viable alternative to biochemically changing insulin or other receptor binding peptides to improve PD properties.

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Figure Blood glucose development in STZ-diabetic Sprague Dawley rats after s.c. injection of 1 mg/kg regular human insulin formulated with formulations 605c, 421a, and 421b, or an equivalent dose of insulin glargine.

     

Role of glial cells in manganese neurotoxicity

The objectives of this focused review are to (i) provide a systematic overview of recent advances pertaining to the role of glia, namely microglia and astrocytes, in the neuropathology associated with excessive exposure to manganese (Mn), (ii) highlight possible mechanisms and factors involved in Mn-modulated, glia-derived neuroinflammation, and (iii) discuss the implications of excessive neuroinflammation on neuronal injury within the context of Mn overexposure. As this is not meant to be a comprehensive review on the topic of Mn neurotoxicity, the reader may wish to refer to several broader and more comprehensive reviews. After a brief introduction to Mn neurotoxicity, we first discuss the role of glial cells in neurodegeneration. Next, we review existing in vitro and in vivo studies that implicate Mn as a modulator of glial activation and ensuing neuroinflammation. This is followed by an examination of recognized and potential mechanisms that are involved in the modulation of glial inflammatory output by Mn; here the common pathways activated by Mn in glial and neuronal cells, including outcomes of such activation, are also addressed. We finish with a discussion of the implications of Mn-modulated glial activation for neuronal survival and with a list of data gaps in the topic that need to be filled in the future.     

Aggregation behavior and in vitro biocompatibility study of octopus-shaped macromolecules based on tert-butylcalix[4]arenes

A series of products based on tert-butylcalix[4]arene have been synthesized by anionic polymerization of ethylene oxide. The resulting products are amphiphilic octopus-shaped macromolecules, consisting of a hydrophobic calix[4]arene core and four arms of hydrophilic poly(ethylene oxide) chains. In aqueous solutions the polyoxyethylated tert-butylcalix[4]arenes were found to self-associate above certain CMC determined by dye solubilization technique. The light scattering study reveals that the polyoxyethylated tert-butylcalix[4]arenes form aggregates of narrow size distribution and hydrodynamic diameters ranging from about 155 to 245nm and aggregation numbers from tens to hundreds macromolecules per particle depending on the degree of polymerization of the PEO chains. An in vitro biocompatibility study showed that the tested compounds are practically devoid of intrinsic cytotoxic and hemolytic effects and moreover they failed to modulate the mitogen-induced interleukin-2 release from the human T-lymphocyte cell line Jurkat E6-1. Taken together the excellent in vitro biocompatibility profile and the favorable physicochemical characteristics of the tested polyoxyethylated calix[4]arenes give us reason to consider them as promising for further evaluation as drug delivery platforms.     

Pathways of ion–molecular interactions of nucleogenic phenyl cations with the nucleophilic centers of picolines

Background

The nuclear-chemical method brought unique opportunity for synthesis of unknown and hardly available organic compounds. Presence of tritium labeling allows one-step preparation of radioactive markers for the investigation of chemical and biological processes.

Methods

The ion–molecular reactions of nucleogenic phenyl cations with 4-picoline have been carried out. The phenyl cations were generated by spontaneous tritium β-decay within the tritium-labeled benzene. Both additions to the nitrogen and substitutions about the aromatic ring were able to be studied simultaneously.

Results

Unusual substitutions on both the α- and β-positions of the ring system have been revealed.

Conclusion

By unknown direct phenylation of nitrogen atom tritium-labeled N-phenylpicolinium derivatives, perspective biological markers have been synthesized.     

Molecular characterization of infectious bronchitis virus isolates from Russia and neighbouring countries: identification of intertypic recombination in the S1 gene

Infectious bronchitis virus (IBV) isolates recovered in Russia, Ukraine, and Kazakhstan between 2007 and 2010 were subjected to molecular characterization and compared with those isolated a decade ago. The IBV genome was detected in 202 out of 605 field samples from chickens with various clinical signs. Partial sequencing of the S1 gene revealed 153 vaccine strains and 49 field isolates of several genetic groups. Massachusetts, 793/B and D274 remained the predominant IBV genotypes along with QX, whereas B1648, Italy-02, Arkansas and variants accounted for about 12% of the total number. Three IBVs contained recombinant S1 gene sequences comprising genome fragments of QX-type field isolates and vaccine strains H120 (UKR/02/2009) or 4/91 (RF/03/2010), and vaccine strains H120 and D274 (RF/01/2010). The results of the present study showed a significant decline in prevalence of variant IBVs and a further spread of QX-type isolates in commercial chicken flocks in Russia as compared with the 1998 to 2002 data.     

RNA chaperone activity of the tombusviral p33 replication protein facilitates initiation of RNA synthesis by the viral RdRp in vitro

Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 replication protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity.