Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema.

Transgenic mice were generated that expressed a human collagenase transgene in their lungs under the direction of the haptoglobin promoter. Histological analysis demonstrated disruption of the alveolar walls and coalescence of the alveolar spaces with no evidence of fibrosis or inflammation. This pathology is strikingly similar to the morphological changes observed in human emphysema and therefore implicates interstitial collagenase as a possible etiological agent in the disease process. Although elastase has been proposed as the primary enzyme responsible for emphysematous lung damage, this study provides evidence that other extracellular matrix proteases could play a role in emphysema. In addition, these transgenic mice are a defined genetic animal model system to study the pathogenesis of emphysema.     

Collagenase expression in transgenic mouse skin causes hyperkeratosis and acanthosis and increases susceptibility to tumorigenesis.

In a series of transgenic mice, the human tissue collagenase gene was expressed in the suprabasal layer of the skin epidermis. Visually, the mice had dry and scaly skin which upon histological analysis revealed acanthosis, hyperkeratosis, and epidermal hyperplasia. At the ultrastructural level, intercellular granular materials were absent in the transgenic skin epidermis but contact was maintained through the intact desmosomes. Despite a diversity of underlying etiologies, similar morphological hyperproliferative changes in the epidermis are observed in the human skin diseases of lamellar ichthyosis, atopic dermatitis, and psoriasis. Subsequent experiments demonstrate that when the transgenic mouse skin was treated once with an initiator (7,12-dimethyl-benz[a]anthracene) and then twice weekly with a promoter (12-O-tetradecanoylphorbol-13-acetate), there was a marked increase in tumor incidence among transgenic mice compared with that among control littermates. These experiments demonstrate that by overexpressing the highly specific proteolytic enzyme collagenase, a cascade of events leading to profound morphological changes which augment the sensitivity of the skin towards carcinogenesis is initiated in the epidermis.     

Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Superoxide-independent reduction of vanadate by rat liver microsomes/NAD(P)H: vanadate reductase activity.

It has been reported that vanadate-stimulated oxidation of NAD(P)H by microsomal systems can proceed anaerobically, in contrast to the general notion that the oxidation proceeds exclusively by an O(2-)-dependent free radical chain mechanism. The current study indicates that microsomal systems are endowed with a vanadate-reductase property, involving a NAD(P)H-dependent electron transport cytochrome P450 system. Our ESR measurements demonstrated the formation of a vanadium(IV) species in a mixture containing vanadate, rat liver microsomes, and NAD(P)H. This vanadium(IV) species was identified as the vanadyl ion (VO2+) by comparison with the ESR spectrum of VOSO4. The initial rate of vanadium(IV) formation depends linearly on the concentration of microsomes. The Michaelis-Menten constants were found to be: km = 1.25 mM and Vmax = 0.066 mumol (min)-1 (mg microsomes)-1, respectively. Pretreatment of the microsomes with carbon monoxide or K3Fe(CN)6 reduced vanadium(IV) generation, suggesting that the NAD(P)H-dependent electron transport cytochrome P450 system plays a significant role in the microsomal reduction of vanadate. Measurements under argon or in the presence of superoxide dismutase caused only minor (less than 10%) reductions in vanadium(IV) generation. The VO2+ species was also detected in NAD(P)H oxidation by fructose plus vanadate, a reaction known to proceed via an O(2-)-mediated chain mechanism. However, the amount of vanadium(IV) generated by this reaction was an order of magnitude smaller than that by the microsomal system and was inhibitable by superoxide dismutase, affirming the conclusion that the microsomal/NAD(P)H system is endowed with the (O(2-)-independent) vanadium(V) reductase property.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

On the formation of oxygenated radicals by fredericamycin A and implications to its anticancer activity: an ESR investigation

It has been recently suggested that the exceptionally high antitumor and antibacterial activity of natural fredericamycin A (FMA) is related to its ability to spontaneously generate the superoxide anion (O2-) and hydroxyl (.OH) radicals in aerobic solutions [Hilton, B. D., Misra, R., & Zweier, J. L. (1986) Biochemistry 25, 5533]. With a view to understand the mechanistic details, attempts were made to reproduce earlier electron spin resonance (ESR) evidence for the oxygenated free radical formation in well-aerated solutions of natural FMA in dimethyl sulfoxide and dilute H2O2. Little or no evidence was obtained for the formation of the O2- and methoxy (.OCH3) radicals, while the detected formation of the .OH and methyl (.CH3) radicals was attributable largely to mechanisms not involving FMA. These results thus reopen the question regarding the mechanism of its exceptionally high tumoricidal-bacteriocidal activity.     

Esr Spin Trapping and Cytotoxicity Investigations of Freshly Fractured Quartz: Mechanism of Acute Silicosis

Electron spin resonance (ESR) measurements show that grinding of quartz particles in air produces silicon-based (Si· and SiO·) radicals which decay with aging in air. ESR spin trapping measurements provide evidence for the generation of hydroxyl and possibly superoxide radicals from a suspension of fresh quartz particles. The hydroxyl radical generation potential of the fresh quartz particles decreases on storing in ambient air and on the addition of catalase, superoxide dismutase, desferroxamine. or DMSO. Silica-induced lipid peroxidation also decreases on storing the fresh particles in ambient air. These findings suggest that oxygenated radicals play a role in the biochemical mechanism of pneumoconiosis in general and acute silicosis in particular.     

ACE inhibitors hypothesis generation for selective design,synthesis and biological evaluation of 3-mercapto-2-methyl-propanoyl-pyrrolidine-3-imine derivatives as antihypertensive agents

A series of new 3-mercapto-2-methyl-propanoyl-pyrrolidine derivatives (V, VIa–e) were designed. A new validated ACE inhibitors pharmacophore model (hypothesis) was generated for the first time in this research from the biologically active (frozen) conformation of Lisinopril–Human ACE complex that was downloaded from PDB, using stepwise technique of CATALYST modules. The molecular modeling compare–fit study of the designed molecules (V, VIa–e), with such ACE inhibitors hypothesis was fulfilled, and several compounds showed significant high simulation fit values. The compounds with high fit values were synthesized and biologically evaluated in vivo as hypotensive agents. It appears that the in vivo hypotensive activity of compounds V, VIa, VIb, and VIe was consistent with their molecular modeling results, and compound VIe showed the highest activity in comparison to Captopril.