Characterization and Translation of Poly(A)+RNA from Wounded and Crown Gall Tissues of Potato Tubers

Poly(A)⁺ and poly(A)^–RNA from wounded potato tuber tissuesand crown gall tumors were separated from total RNA by oligodeoxythymidylicacid-cellulose affinity chromatography. The poly(A)⁺RNA wascharacterized by sucrose density gradient centrifugation, hybridizationwith ³(H)polyuridylic acid [Poly(U)] and in vitro translationin a rabbit reticulocyte lysate system. The tumor poly(A)⁺RNAwas a heterodisperse mixture from 3.5S to 35S. Upon poly(U)hybridization of the gradient fractions two major hybridizationpeaks at 7S and 21S and two peaks at 11S and 16S appeared. Inan in vitro translation system the poly(A)⁺RNA programmed thesynthesis of 23 different polypeptides of 9,000 to 79,800 daltonsmolecular weight as determined by SDS-polyacrylamide gel electrophoresis.The 21S poly(A)+RNA was about 5 times more active in in vitroprotein synthesis than the 7S poly(A)⁺RNA. The poly(A)⁺RNA from wounded tissues was also heterodisperse(from 4.5S to 31S) with a modal peak at 18S. This RNA codedfor at least 28 polypeptides, which were different from thoseof crown gall tumor tissues. On a per unit poly(A)⁺RNA basis the tumor RNA was slightly moreactive in translation than that from wounded tissues. The translationof tumor poly(A)⁺RNA was completely blocked by 0.5 mM 7-methylguanosine5'-phosphate, but not by 7-methylguanosine, suggesting the presenceof a 5'-cap structure. (Received May 15, 1982; Accepted June 30, 1982)     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

Synthesis and pharmacological evaluation of some dual-acting amino-alcohol ester derivatives of flurbiprofen and 2-[1,1'-biphenyl-4-yl]acetic acid: a potential approach to reduce local gastrointestinal toxicity

The search for safer non-steroidal anti-inflammatory drugs (NSAIDs) continues with the failure of anticipated 'ideal' anti-inflammatory agents, the coxibs, on long-term usage. Increased gastric motility and acidity due to the free carboxy group are involved in the etiology of gastric toxicity, common to conventional NSAIDs. Keeping this fact in mind, it was planned to modify some of the conventional NSAIDs to amino-alcohol ester derivatives, which satisfied the structural requirements for these compounds to possess anticholinergic activity in the intact form. Besides blocking the acidic carboxylic group, incorporation of anticholinergic acivity in these molecules was expected to reduce the gastric toxicity by decreasing gastric acid secretion and motility. Synthesis and pharmacological evaluation of six different N,N-disubstituted amino-ethyl ester derivatives, structurally resembling the amino-alcohol ester class of anticholinergic agents, each for [1,1'-biphenyl]-4-acetic acid (3) and flurbiprofen (10), have been reported as potential substitutes for these NSAIDs, with improved therapeutic profile. All the ester derivatives were found to have sufficient chemical stability in buffers (pH 2.0 and 7.4), ensuring them to be absorbed as intact moieties from the gastrointestinal tract. A significant reduction in ulcerogenic potency in comparison to the parent drugs with a slightly higher anti-inflammatory potency suggests that the majority of these candidates have an improved therapeutic profile over their parent drugs. Hence, a promising novel approach, different from the conventional prodrug concept, has been successfully worked out to overcome the local gastric toxicity, yielding therapeutically better compounds for long-term oral anti-inflammatory therapy.     

Proteomic analysis of circulating human monocytes in coronary artery disease

Monocytes play an important role in inflammation and atherosclerosis; however, the molecular details underlying these diverse functions are not completely understood. Proteomic analysis of monocytes can provide new insights into their biological role in coronary artery disease (CAD). Twenty angiographically confirmed male, CAD patients (≥50% stenosis) attending cardiology clinic of Nehru Hospital, PGIMER, Chandigarh, and who were not receiving any lipid lowering therapy and 20 TMT negative subjects who served as controls were enrolled in the study. Circulating monocytes isolated from overnight fasting blood samples were analyzed by 2D gel electrophoresis (pH 4-7), and differentially expressed protein spots were subjected to mass spectrometry and identification of proteins. We observed 333 ± 40 protein spots in monocytes from patients and 312 ± 20 in controls; out of which 63 protein spots showed altered intensity in CAD patients. Thirteen spots showed fivefold increased and two protein spots showed fivefold decreased expression in CAD group as compared to control group, respectively. Two proteins showing decreased expression in monocytes from CAD patients were identified as: (i) glutathione transferase and (ii) heat shock protein 70 KDa. Proteins showing increased expression in CAD patients were identified as: (i) vimentin, (ii) mannose binding lectin receptor protein, and (iii) S100A8 calcium-binding protein. The results of our study offer identification of several proteins in monocytes which can provide new perspectives in role of monocytes in pathogenesis of atherosclerosis.     

Small-animal imaging using clinical positron emission tomography/computed tomography and super-resolution

Considering the high cost of dedicated small-animal positron emission tomography/computed tomography (PET/CT), an acceptable alternative in many situations might be clinical PET/CT. However, spatial resolution and image quality are of concern. The utility of clinical PET/CT for small-animal research and image quality improvements from super-resolution (spatial subsampling) were investigated. National Electrical Manufacturers Association (NEMA) NU 4 phantom and mouse data were acquired with a clinical PET/CT scanner, as both conventional static and stepped scans. Static scans were reconstructed with and without point spread function (PSF) modeling. Stepped images were postprocessed with iterative deconvolution to produce super-resolution images. Image quality was markedly improved using the super-resolution technique, avoiding certain artifacts produced by PSF modeling. The 2 mm rod of the NU 4 phantom was visualized with high contrast, and the major structures of the mouse were well resolved. Although not a perfect substitute for a state-of-the-art small-animal PET/CT scanner, a clinical PET/CT scanner with super-resolution produces acceptable small-animal image quality for many preclinical research studies.     

Induction of genomic instability in cultured human colon epithelial cells following exposure to isocyanates

The toxic response of cultured human colon epithelial-FHC cells to methyl isocyanate was investigated with regard to genomic instability. Qualitative and quantitative assessments of the extent of phosphorylation of DNA damage signaling factors such as ATM, γH2AX and p53, was increased in treated cells compared to controls. At the same time, many treated cells were arrested at the G2/M phase of the cell cycle, and had an elevated apoptotic index and increased inflammatory cytokine levels. Cytogenetic analyses revealed varied chromosomal anomalies, with abnormal expression of pericentrin protein. Analysis through ISSR PCR demonstrated increased microsatellite instability. The results imply that isocyanates can cause genomic instability in colonocytes.     

Rat hepatic natural killer cells (pit cells) express mRNA and protein similar to in vitro interleukin-2 activated spleen natural killer cells

To address how FasL-expressing tumors induce neutrophil emigration and abrogate tumorigenicity, we investigated the behavior of FasLcDNA-transfected hepatoma MH134 (G2) cells injected into wild-type (+) mice, lpr(cg)/lpr(cg) (lpr(cg)) mice with death domain (DD)-mutated Fas, and gld/gld lpr/lpr (gld/lpr) mice with defects in FasL/Fas. G2 cells were eradicated after extensive infiltration of neutrophils around them in + mice but formed tumors without such infiltration in lpr(cg) and gld/lpr mice. Abundant cell debris suggestive of apoptosis of infiltrating neutrophils was found among G2 tumor cells in + mice but a few neutrophils infiltrating among G2 cells were intact in lpr(cg) and gld/lpr mice. Collectively, these results indicate the crucial role of Fas DD in Fas-mediated apoptosis of neutrophils and suggest that apoptosis of neutrophils with FasL-expressing tumors may trigger the extensive infiltration of neutrophils, resulting in violent inflammation and ultimately in the eradication of tumor cells.     

Th1- and Th2-dependent endothelial progenitor cell recruitment and angiogenic switch in asthma

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.     

Synthesis, docking and in vitro antimalarial evaluation of bifunctional hybrids derived from β-lactams and 7-chloroquinoline using click chemistry

1,2,3-Triazole tethered β-lactam and 7-chloroquinoline bifunctional hybrids were synthesized and evaluated as potential antimalarial agents. Activity against cultured Plasmodium falciparum was dependent on the N-substituent of the β-lactam ring as well as the presence of bis-triazole at the C-3 position. The observed activity profiles were further substantiated by docking studies via inhibition of P. falciparum dihydrofolate reductase (PfDHFR), a potential target for the development of new anti-malarials.