Preparation and Specificity of 11 Monoclonal Antibodies to GM1 Ganglioside

Eleven monoclonal antibodies to GM1 ganglioside were prepared from hybridoma clones obtained by fusion of spleen cells from mice immunized with GM1 with mouse myeloma cells. When the reactivities of these 11 monoclonal antibodies were determined by enzyme-linked immunosorbent assay with six glycosphingolipids (GM1, GD1a, GD1b, GT1b, GM2, and asialo-GM1), they showed different degrees of specificity. From their reactivity patterns, they could be divided into three groups: Group 1, those that react only with GM1 (C3 and D3); Group 2, those that react predominantly with GM1 (C6, B6, D1, e1, g1, g9, and e12); and Group 3, those that show poor discrimination (h2 and A4). The clones differed in their biological activities.     

Antipsychotic drugs differentially modulate apolipoprotein D in rat brain

Apolipoprotein-D (apoD), a member of the lipocalin family of proteins, binds to arachidonic acid and cholesterol among other hydrophobic molecules. Recently, elevated apoD levels have been reported in the post-mortem brains, as well as plasma, of schizophrenic patients and in rodent brains after chronic treatment with clozapine (CLOZ). These findings and the evidence for altered membrane lipid metabolism in schizophrenia suggest that apoD may have a role in the pathophysiology of illness, and also in the differential clinical outcome following treatment with typical and atypical antipsychotic drugs. Here, we compared the effects of these antipsychotics on the expression of apoD in rat brain. Chronic treatment with typical antipsychotic, haloperidol (HAL) reduced apoD expression in hippocampus, piriform cortex and caudate-putamen (p = 0.027-0.002), whereas atypical antipsychotics, risperidone (RISP) and olanzapine (OLZ) increased (p = 0.051 to < 0.001 and p = 0.048 to < 0.001, respectively) apoD expression. In hippocampus, HAL-induced changes were present in CA1, CA3 and dentate gyrus, however, apoD levels in motor cortex were unchanged. There were also very dramatic effects of HAL on the neuronal morphology, particularly, cellular shrinkage and disorganization with the loss of neuropil. Post-treatment, either with RISP or OLZ, was very effective in restoring the HAL-induced reduction of apoD, as well as cellular morphology. Similarly, pre-treatments were also effective, but slightly less than post-treatment, in preventing HAL-induced reduction of apoD. The increased expression of apoD by atypical antipsychotics may reflect a novel molecular mechanism underlying their favorable effects compared with HAL on cognition, negative symptoms and extra-pyramidal symptoms in schizophrenia.     

Mechanisms of angiogenic incompetence in Hutchinson–Gilford progeria via downregulation of endothelial NOS

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. The majority of HGPS cases are caused by a de novo point mutation in the LMNA gene (c.1824C>T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes normal angiogenic function in HGPS. Here, we use human iPSC‐derived endothelial cell (iPSC‐EC) models to study angiogenesis in HGPS. We cultured normal and HGPS iPSC‐ECs under both static and fluidic culture conditions. HGPS iPSC‐ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared with normal controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary‐like microvascular network formation. Furthermore, the expression of matrix metalloproteinase 9 (MMP‐9) was reduced in HGPS iPSC‐ECs, while the expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) was upregulated relative to healthy controls. Finally, we used an adenine base editor (ABE7.10max‐VRQR) to correct the pathogenic c.1824C>T allele in HGPS iPSC‐ECs. Remarkably, ABE7.10max‐VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized the misregulated TIMPs, and restored angiogenic competence in HGPS iPSC‐ECs. Together, these results provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE could be a promising therapeutic approach for correcting HGPS‐related cardiovascular phenotypes.     

Exploration of new scaffolds as potential MAO-A inhibitors using pharmacophore and 3D-QSAR based in silico screening

Monoamine oxidase-A (MAO-A) inhibitors are of particular importance in the treatment of depressive disorders. Herein described is pharmacophore generation and atom-based 3D-QSAR analysis of previously reported pyrrole based MAO-A inhibitors in order to get insight into their structural requirements responsible for high affinity. The best pharmacophore model generated consisted of four features DHHR: a hydrogen bond donor (D), two hydrophobic groups (H) and an aromatic ring (R). Based on model generated, a statistically valid 3D-QSAR with good predictability was developed. Derived pharmacophore was used as a query to search Zinc ‘clean drug-like’ database. Hits retrieved were passed progressively through filters like fitness score, predicted activity and docking scores. The survived hits present new scaffolds with a potential for MAO-A inhibition.     

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of β-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1β, and tumor necrosis factor alpha (TNF-α). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.     

Simultaneous determination of cinnamaldehyde, eugenol and piperine by HPTLC densitometric method

An HPTLC densitometric method for the simultaneous determination of cinnamaldehyde and eugenol as well as trace amounts of piperine in pepper-contaminated cinnamon was developed. The applicability of the method was tested with cinnamon bark powder adulterated with pepper powder, cinnamon oil, clove powder, clove oil and a commercial preparation containing cinnamaldehyde and eugenol. The method was validated for specificity, precision, accuracy and robustness. The method was found to be precise for different concentrations of cinnamaldehyde, eugenol and piperine. The accuracy of the method was checked by conducting a recovery study at three different levels. The linearity was found to be in the ranges 52.54-735.56, 533.2-8531.2 and 50-300 ng/spot, respectively, with correlation coefficients of 0.9985 +/- 0.04, 0.9982 +/- 0.06 and 0.9937 +/- 0.11 for cinnamaldehyde, eugenol and piperine.     

Filtration and agar embedding applied to fine needle aspirates for tumor diagnosis by electron microscopy. A combined technique.

OBJECTIVE: To develop a novel method for processing of fine needle aspirates subjected to electron microscopic (EM) study. STUDY DESIGN: Included 70 cases of poorly differentiated malignant tumors in which a definitive diagnosis was not possible on light microscopic (LM) examination and that thus required application of an ancillary technique such as FNA/EM, for diagnosis. We have established a novel method of processing, a technique of filtration through nylon mesh filters to eliminate red blood cells (RBCs) and necrotic debris, followed by agar well embedding to avoid loss of diagnostic material during processing without centrifugation at later steps after agar embedding, thus minimizing the time required for processing. It was successfully carried out in 70 cases. RESULTS: The combined technique was extremely effective in eliminating RBCs and necrotic debris. It also avoided further loss of valuable diagnostic material. An accurate diagnosis was rendered in 70 cases; that was not possible by LM alone. The whole procedure saves two to three hours of processing as centrifugation is not required after the agar embedding step. CONCLUSION: This technique was found to be cost- and time-effective, particularly suitable for developing countries, where financial resources are limited.     

Effect of system variables involved in packed column SFC of nevirapine as model analyte using response surface methodology: application to retention thermodynamics, solute transfer kinetic study and binary diffusion coefficient determination

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of nevirapine analysis by packed column supercritical fluid chromatography (PC-SFC). The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. The method is based on methanol-modified carbon dioxide as the mobile phase at flow rate of 3.0 ml/min with elution through a JASCO Finepak SIL-5, [C18 (5-micron, 25 cm x 4.6 mm, i.d.)] column using photodiode array detection. The method has been successfully used to analyze commercial solid dosage form to assess the chromatographic performance of SFC system. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of nevirapine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.     

Differential effects of haloperidol and olanzapine on the expression of erythropoietin and its receptor in rat hippocampus and striatum

Compared with first-generation antipsychotics (FGAs), second-generation antipsychotics (SGAs) seem to be neuroprotective and trigger neuroplasticity. Because neuroplasticity is regulated by a variety of neurotrophic factors we studied differential effects of haloperidol (HAL, a FGA) and olanzapine (OLZ, a SGA) on temporal expression of erythropoietin (EPO), a potent neuroprotective factor and its receptor (EPOr) in rat brain. Rats (8-10/group) were treated with HAL or OLZ for 14 days (HAL-14 or OLZ-14) or 45 days (HAL-45 or OLZ-45). Animals were killed by decapitation or by perfusion to collect brains for immunoblotting and immunohistochemical analysis respectively. In hippocampus, the levels of both EPO and EPOr were significantly increased in HAL-14 (p < 0.001) and OLZ-14 (p < 0.001) groups. Their levels decreased in HAL-45 compared with levels in HAL-14 (EPO, p < 0.001; EPOr, p < 0.05), whereas the levels were further increased (EPO, p < 0.05) in OLZ-45 compared with OLZ-14. In striatum, the levels of both EPO and EPOr were unchanged in HAL-14 and EPO levels significantly decreased in HAL-45 (p < 0.05), whereas their levels were significantly increased in OLZ-14 and OLZ-45 compared with the vehicle-treated control (p < 0.001). Both EPO and EPOr were primarily expressed by neurons and endothelial cells. These data suggest that SGAs such as OLZ may have neuroprotective effects through expression of EPO that may be clinically relevant for long-term safe and beneficial management of psychotic patients.