Analysis of glycated insulin by MALDI-TOF mass spectrometry

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.     

CD28 signals in the immature immunological synapse

T cell recognition of peptide-MHC complexes on APCs results in the aggregation of TCRs at a central supramolecular activation complex (c-SMAC) within a mature immunological synapse. T cells require a second "costimulatory" signal for activation, the most important of which, for naive T cells, is from CD28. However the time at which CD28-derived signals are induced relative to c-SMAC formation is not well understood. In this study, we have assessed the kinetics of CD28 localization and function relative to well-established aspects of c-SMAC formation. CD28 accumulates at the immature synapse alongside the TCR and is likewise enriched at the synapse at the onset of the calcium signal. In addition, using CD28 deficient or reconstituted murine cells in a single-cell recording approach shows that CD28 regulates this signal within seconds of a TCR-mediated rise in intracellular calcium levels. Finally, CD28 exerts effects on both the initiation and stabilization of the synapse in parallel with its effects on the downstream proliferation of T cells. Together, the data show that CD28 functions in the immunological synapse before the formation of the c-SMAC.     

CD4+CD25+ cells controlling a pathogenic CD4 response inhibit cytokine differentiation, CXCR-3 expression, and tissue invasion

It is well established that CD4(+)CD25(+) regulatory T cells (Tregs) inhibit autoimmune pathology. However, precisely how the behavior of disease-inducing T cells is altered by Tregs remains unclear. In this study we use a TCR transgenic model of diabetes to pinpoint how pathogenic CD4 T cells are modified by Tregs in vivo. We show that although Tregs only modestly inhibit CD4 cell expansion, they potently suppress tissue infiltration. This is associated with a failure of CD4 cells to differentiate into effector cells and to up-regulate the IFN-gamma-dependent chemokine receptor CXCR-3, which confers the ability to respond to pancreatic islet-derived CXCL10. Our data support a model in which Tregs permit T cell activation, yet prohibit T cell differentiation and migration into Ag-bearing tissues.     

Coordination chemistry of half-open trozircenes: Adduct formation of [(η-C7H7)Zr(η-2,4-C7H11)] with isocyanides, phosphines and N-heterocyclic carbenes

The reactions of the half-open trozircene [(η⁷-C₇H₇)Zr(η⁵-2,4-C₇H₁₁)] (1) with the two-electron donor ligands tert-butyl isocyanide (CN-tBu), 1,2-bis(dimethylphosphino)ethane (dmpe), trimethylphosphine (PMe₃) and 1,3,4,5-tetramethylimidazolin-2-ylidene (IMe, :C[N(Me)C(Me)]₂) have led to the 1:1 adducts 3, 4, 5 and 6, respectively. The latter three were structurally characterized by X-ray diffraction analysis. Additionally, the stability of the adducts was probed by DFT calculations employing the B3LYP and M05-2X functionals showing that the strongly σ-basic N-heterocyclic carbene forms a thermodynamically much more stable adduct than the other three.     

Effects of ocean acidification on the growth and biochemical composition of a green alga (Ulva fasciata) and its associated microbiota

In marine ecosystems, fluctuations in surface-seawater carbon dioxide (CO₂), significantly influence the whole metabolism of marine algae, especially during the early stages of macroalgal development. In this study, the response of the green alga Ulva fasciata for elevating ocean acidification was investigated using four levels of pCO₂ ~ 280, 550, 750 and 1050 µatm. Maximum growth rate (6.6% day⁻¹), protein (32.43 %DW) and pigment (2.9 mg/g) accumulation were observed at pCO₂-550 with an increase of ~2-fold compared to control. On the other hand, lipid and carbohydrate contents recorded their maximum production (4.23 and 46.96 %DW, respectively) at pCO₂-750 while control showed 3.70 and 42.37 %DW, respectively. SDS-PAGE showed the presence of unique bands in response to pCO_2, especially at 550 µatm. Dominant associated bacteria was shifted from Halomonas hydrothermalis of control to Vibrio toranzoniae at pCO₂-1050. These findings suggest that ocean acidification at 550 µatm might impose noticeable effects on growth, protein, pigments, and protein profile of U. fasciata, which could be a good source for fish farming. While, pCO₂-750 was recommended for energetic purpose, due to its high lipid and carbohydrate contents.     

Clozapine but not haloperidol suppresses the changes in the levels of neuropeptides in MK-801-treated rat brain regions

Noncompetitive NMDA receptor antagonist (+)MK-801 is known to induce neurotoxicity and schizophrenia-like symptomatology where atypical neuroleptic clozapine is effective in contrast to typical neuroleptic, haloperidol. Although neuropeptides are implicated in memory and cognition, their roles in schizophrenia are not well understood. In the present study, we therefore examined the possible roles of neuropeptides, cholecystokinin (CCK) and somatostatin (SS) in the posterior cingulate/retrosplenial cortices (PC/RSC), frontal cortex, and hippocampus of a MK-801-induced schizophrenia-like model rat brain. This study further investigated the pretreated effect of atypical versus typical neuroleptics on the peptidergic system. SS mRNA and peptide levels significantly decreased in the PC/RSC and hippocampus but not in the frontal cortex 3 days after 0.5 mg/kg MK-801 treatment whereas CCK mRNA and peptide levels significantly decreased in all of the brain regions examined. Pretreatment with clozapine but not haloperidol completely recovered the changes in both mRNA and peptide levels of SS and CCK in those brain regions. These data suggest that peptidergic system in the brain presumably plays an important role in the control of negative schizophrenia.     

PLGA-microsphere mediated clearance of bilirubin in temporarily hyperbilirubinemic rats: an alternate strategy for the treatment of experimental jaundice

In the present study, we have demonstrated the suitability of microspheres in removal of plasma bilirubin from systemic circulation of hyperbilirubinemic rats. Poly (lactide co-glycolide) microspheres (PLGA microspheres) have been shown to bind with bilirubin in both a concentration and time dependent manner. The binding affinity of bilirubin to microspheres was enhanced when rat serum albumin (RSA) was loaded into the microspheres. On evaluating the potential of microspheres in elimination of bilirubin from the systemic circulation, RSA bearing microspheres were found to be competent in both removing bilirubin from the systemic circulation and controlling elevated plasma levels of liver function enzymes in temporarily hyperbilirubinemic rats. On the basis of results of the present study, we suggest that microsphere-based delivery system may help in development of safe, effective and alternate strategy for the treatment of hyperbilirubinemic conditions in model animals.     

A coiled-coil domain of melanophilin is essential for Myosin Va recruitment and melanosome transport in melanocytes

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.