Simultaneous determination of 33 amino acids and dipeptides in spent cell culture media by gas chromatography-flame ionization detection following liquid and solid phase extraction

A rapid, sensitive and reproducible gas chromatographic method with flame ionization detection is described for the simultaneous identification and quantification of 33 amino acids and dipeptides in spent cell culture media in under seven minutes. The method involves the use of the EZ:faast(Phenomenex) amino acid sample testing kit. Instrumental and assay precision, percent recovery, linear range, limit of detection and peak identity in highly complex cell culture media containing either soy hydrolysate or fetal bovine serum were validated using gas chromatography-flame ionization detector (GC-FID).     

Regulation of alternative macrophage activation by galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.     

Chronic oral exposure to the aldehyde pollutant acrolein induces dilated cardiomyopathy

Environmental triggers of dilated cardiomyopathy are poorly understood. Acute exposure to acrolein, a ubiquitous aldehyde pollutant, impairs cardiac function and cardioprotective responses in mice. Here, we tested the hypothesis that chronic oral exposure to acrolein induces inflammation and cardiomyopathy. C57BL/6 mice were gavage-fed acrolein (1 mg/kg) or water (vehicle) daily for 48 days. The dose was chosen based on estimates of human daily unsaturated aldehyde consumption. Compared with vehicle-fed mice, acrolein-fed mice exhibited significant (P < 0.05) left ventricular (LV) dilatation (LV end-diastolic volume 36 ± 8 vs. 17 ± 5 μl), contractile dysfunction (dP/dt(max) 4,697 ± 1,498 vs. 7,016 ± 1,757 mmHg/s), and impaired relaxation (tau 15.4 ± 4.3 vs. 10.4 ± 2.2 ms). Histological and biochemical evaluation revealed myocardial oxidative stress (membrane-localized protein-4-hydroxy-trans-2-nonenal adducts) and nitrative stress (increased protein-nitrotyrosine) and varying degrees of plasma and myocardial protein-acrolein adduct formation indicative of physical translocation of ingested acrolein to the heart. Acrolein also induced myocyte hypertrophy (~2.2-fold increased myocyte area, P < 0.05), increased apoptosis (~7.5-fold), and disrupted endothelial nitric oxide synthase in the heart. DNA binding studies, immunohistochemistry, and PCR revealed significant (P < 0.05) activation of nuclear factor-κB in acrolein-exposed hearts, along with upregulated gene expression of proinflammatory cytokines tumor necrosis factor-α and interleukin-1β. Long-term oral exposure to acrolein, at an amount within the range of human unsaturated aldehyde intake, induces a phenotype of dilated cardiomyopathy in the mouse. Human exposure to acrolein may have analogous effects and raise consideration of an environmental, aldehyde-mediated basis for heart failure.     

Patellar mechanics during simulated kneeling in the natural and implanted knee

Kneeling is required during daily living for many patients after total knee replacement (TKR), yet many patients have reported that they cannot kneel due to pain, or avoid kneeling due to discomfort, which critically impacts quality of life and perceived success of the TKR procedure. The objective of this study was to evaluate the effect of component design on patellofemoral (PF) mechanics during a kneeling activity. A computational model to predict natural and implanted PF kinematics and bone strains after kneeling was developed and kinematics were validated with experimental cadaveric studies. PF joint kinematics and patellar bone strains were compared for implants with dome, medialized dome, and anatomic components. Due to the less conforming nature of the designs, change in sagittal plane tilt as a result of kneeling at 90° knee flexion was approximately twice as large for the medialized-dome and dome implants as the natural case or anatomic implant, which may result in additional stretching of the quadriceps. All implanted cases resulted in substantial increases in bone strains compared with the natural knee, but increased strains in different regions. The anatomic patella demonstrated increased strains inferiorly, while the dome and medialized dome showed increases centrally. An understanding of the effect of implant design on patellar mechanics during kneeling may ultimately provide guidance to component designs that reduces the likelihood of knee pain and patellar fracture during kneeling.     

Appraisal of Transdermal Water-in-Oil Nanoemulgel of Selegiline HCl for the Effective Management of Parkinson’s Disease: Pharmacodynamic,Pharmacokinetic, and Biochemical Investigations

In the present study, the potential of transdermal nanoemulsion gel of selegiline hydrochloride for the treatment of Parkinson’s disease was investigated. Water-in-oil nanoemulsions were developed by comparing low- and high-energy methods and were subjected to thermodynamic stability tests, in vitro permeation, and characterization studies. In vitro studies indicated that components of nanoemulsion acted as permeation enhancers with highest flux of 3.531 ± 1.94 μg/cm²/h from nanoemulsion SB6 containing 0.5 mg selegiline hydrochloride, 3% distilled water, 21% S _mix (Span 85, Tween 80, PEG 400), and 76% isopropyl myristate by weight. SB6 with the least droplet size of 183.4 ± 0.35 nm, polydispersity index of 0.42 ± 0.06 with pH of 5.9 ± 0.32 and viscosity of 22.42 ± 0.14 cps was converted to nanoemulsion gel NEGS4 (viscosity = 22,200 ± 400 cps) by addition of Viscup160® for ease of application and evaluated for permeation, safety, and pharmacokinetic profile in Wistar rats. It provided enhancement ratio 3.69 times greater than conventional gel. NEGS4 showed 6.56 and 5.53 times increase in bioavailability in comparison to tablet and conventional gel, respectively, along with sustained effect. Therefore, the developed water-in-oil nanoemulsion gel promises to be an effective vehicle for transdermal delivery of selegiline hydrochloride.     

Impact of ivermectin on the ultrastructure of the testis of <Emphasis Type="Italic">Argas (Persicargas) persicus</Emphasis> (Ixodoidea: Argasidae)

Mated male Argas persicus were dissected 1 and 2 weeks after feeding on untreated and ivermectin (IVM)-treated pigeons. One week after feeding, testes of untreated ticks were filled with rounded spermatids with subplasmalemmal vesicles and cytoplasmic organelles, but lacking in treated ticks. Two weeks after feeding, testes were crowded with elongated spermatozoa supported by double-walled cisternal tubes. The tubes consisted of two opposite walls, each with outer-fringed processes and inner elongated cisternae. Both were supported with electron dense striated plates in the middle of the spermatozoon. Internally, the cisternal tubes contained mitochondria and vacuoles. The nuclei were elongated dense masses between the tubes and the cell membranes. Subcutaneous inoculation of IVM at the dose 400 μg/kg pigeon resulted in extensive alterations in the testis of A. persicus. IVM prevented the development of new spermatids. There was a break down of cell membranes and cytoplasmic organelles of spermatozoa. Multivesicular bodies and numerous vacuoles were noticed in their cytoplasm. Double membranes of elongated cisternae and striation of electron dense plates became indistinct. IVM caused granulation and vacuolization of the nucleus as well as injury of mitochondrial cristae. The results suggest that IVM may bind to the neurotransmitter or the hormone involved in the process of sperm development or may be toxic to the germinal cells of A. persicus testis.     

Effect of bromocriptine,a dopamine receptor agonist,on the experimentally induced gastric ulcers in albino rats

The effect of bromocriptine, a dopamine receptor agonist, has been studied on the aspirin, phenylbutazone and reserpine induced gastric ulcers in rats. A single dose of bromocriptine 4 mg/kg s.c. produced a significant exacerbation of gastric ulcers induced by all the three ulcerogenic drugs, whereas in the same dose administered once daily for 5 consecutive days, it produced a marked protective effect in all the models. A review of the literature shows that different mechanisms may be involved in the opposite effects of acutely and chronically administered bromocriptine observed in this study. The study also points towards a role of dopamine in the pathogenesis of gastroduodenal ulceration.     

HGF converts ErbB2/Neu epithelial morphogenesis to cell invasion

Activation of the hepatocyte growth factor receptor Met induces a morphogenic response and stimulates the formation of branching tubules by Madin-Darby canine kidney (MDCK) epithelial cells in three-dimensional cultures. A constitutively activated ErbB2/Neu receptor, NeuNT, promotes a similar invasive morphogenic program in MDCK cells. Because both receptors are expressed in breast epithelia, are associated with poor prognosis, and hepatocyte growth factor (HGF) is expressed in stroma, we examined the consequence of cooperation between these signals. We show that HGF disrupts NeuNT-induced epithelial morphogenesis, stimulating the breakdown of cell-cell junctions, dispersal, and invasion of single cells. This correlates with a decrease in junctional proteins claudin-1 and E-cadherin, in addition to the internalization of the tight junction protein ZO-1. HGF-induced invasion of NT-expressing cells is abrogated by pretreatment with a pharmacological inhibitor of the mitogen-activated protein kinase kinase (MEK) pathway, which restores E-cadherin and ZO-1 at cell-cell junctions, establishing the involvement of MEK-dependent pathways in this process. These results demonstrate that physiological signals downstream from the HGF/Met receptor synergize with ErbB2/Neu to enhance the malignant phenotype, promoting the breakdown of cell-cell junctions and enhanced cell invasion. This is particularly important for cancers where ErbB2/Neu is overexpressed and HGF is a physiological growth factor found in the stroma.