Differential metabolism of benzo[alpha]pyrene in vitro by human placental tissues exposed to active maternal cigarette smoke

OBJECTIVES: Generation of different metabolites and DNA-adduct(s) of metabolites of benzo[alpha]pyrene (B[alpha]P) in vitro by placental tissues (microsomes) of mothers who actively smoked cigarettes (tobacco) and those who did not smoke were analyzed to determine the variability in metabolism of the B[alpha]P substrate among individual placental samples. METHODS: Overall B[alpha]P metabolism was assayed by alkaline aqueous extraction of metabolites, and reactive metabolites by DNA adducts of B[alpha]P-metabolites produced by salmon sperm DNA added to the incubation mixtures of the substrate and microsomes of exposed- and unexposed-placentas to maternal cigarette smoke. Array of B[alpha]P-metabolites produced by the same incubations were identified by high pressure liquid chromatography of the aqueous extracts. RESULTS: Subsets of smoke-exposed placentas assessed by cluster analysis had augmented metabolic activity, others did not respond to smoke exposure. CYP1A1 expression in trophoblast cells analyzed by immunohistochemistry did not correlate with smoke exposure. The DNA-adducts generated was variable, regardless of verbally reported levels of maternal exposure. The amounts of different B[alpha]P-metabolites produced by individual samples matched for similar levels of exposure during pregnancy by self-reported smoking (1 pack/day) were also not comparable. Metabolism of B[alpha]P into different metabolites, and production of toxic DNA adducts from metabolites in vitro by human placenta were variable and unrelated to the extent of smoke exposure. CONCLUSIONS: The metabolic characteristic of human placenta for xenobiotic exposure substrates is based on the expression and function of diverse enzymes, and such metabolism exhibited inter-individual variation for toxic metabolite production or detoxification of the substrates in response to maternal smoke exposure.     

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy

Background

Human α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α₁-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.

Methods

Five variants of plant-expressed recombinant α₁-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α₁-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.

Results

Urea-induced unfolding of recombinant α₁-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~ 5-fold at 54 °C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α₁-PI but provided enhanced resistance to oxidative inactivation.

Conclusions

Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α₁-PI protein, without inflicting the inhibitory activity of the protein.

General significance

Our results demonstrate the significance of engineered modifications in plant-derived recombinant α₁-PI protein molecule for further therapeutic development.     

Downregulation of NF-κB and PCNA in the regulatory pathways of apoptosis by cyclooxygenase-2 inhibitors in experimental lung cancer

Non-steroidal anti-inflammatory drugs (NSAIDs) are emerging as novel chemopreventive agents against a variety of cancers owing to their capability in blocking the tumor development by cellular proliferation, angiogenesis and by promoting apoptosis. The present study further explored the comparative role of a traditional NSAID, indomethacin and a newly developed coxib, etoricoxib against 9,10-dimethylbenz(a)anthracene (DMBA)-induced lung carcinogenesis in rats. Morphological and histological analysis revealed the occurrence of tumors and lesions along with constricted alveolar spaces in the DMBA treated animals which were largely corrected both by indomethacin and etoricoxib. COX-1 was found to be uniformly expressed in all the groups while COX-2 levels were raised prominently in the DMBA treated animals. Proliferation, as studied by PCNA expression was found to be markedly increased in the DMBA group as compared to the others. Increased NF-κB expression in the DMBA group was found to correct with the co-administration of NSAIDs. Also, fluorescent co-staining of the isolated lung cells revealed a significantly decreased apoptosis and altered mitochondrial membrane potential. In conclusion, these parameters indicate to the chemopreventive action of the two NSAIDs studied in lung cancer and as their mechanism of action suggests, can be achievable both by COX-dependent and COX-independent pathways.     

Methoxylated isoflavones,cajanin and isoformononetin,have non‐estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling

Following a lead obtained from stem‐bark extract of Butea monosperma, two structurally related methoxyisoflavones; cajanin and isoformononetin were studied for their effects in osteoblasts. Cajanin had strong mitogenic as well as differentiation‐promoting effects on osteoblasts that involved subsequent activation of MEK‐Erk and Akt pathways. On the other hand, isoformononetin exhibited potent anti‐apoptotic effect in addition to promoting osteoblast differentiation that involved parallel activation of MEK‐Erk and Akt pathways. Unlike genistein or daidzein, none of these two compounds appear to act via estrogen receptors in osteoblast. Once daily oral (by gavage) treatment for 30 consecutive days was given to recently weaned female Sprague–Dawley rats with each of these compounds at 10.0 mg kg^?1 day^?1 dose. Cajanin increased bone mineral density (BMD) at all skeletal sites studied, bone biomechanical strength, mineral apposition rate (MAR) and bone formation rate (BFR), compared with control. BMD levels at various anatomic positions were also increased with isoformononetin compared with control however, its effect was less potent than cajanin. Isoformononetin had no effect on the parameters of bone biomechanical strength although it enhanced MAR and BFR compared with control. Isoformononetin had very mild uterotrophic effect, whereas cajanin was devoid of any such effect. Our data suggest that cajanin is more potent than isoformononetin in accelerating peak bone mass achievement. To the best of our knowledge, this work represents the first attempt to elucidate structure‐activity relationship between the two methoxylated isoflavones regarding their effects in osteoblasts and bone formation. J. Cell. Biochem. 108: 388–399, 2009. © 2009 Wiley‐Liss, Inc.     

Patterns of morphological traits shaping the feeding guilds in the intertidal mudflat fishes of the Indian Sundarbans

Broad-scale patterns of resource utilization and the corresponding morphological evolution is a result of an integral relationship among form and function. In addition, there is also an inherent role of the latter in determining species co-interaction and assemblage pattern that forms an integral aspect of ecological research. The present study aimed to evaluate the ecomorphological relationship among 37 fish species inhabiting the intertidal mudflats of the Indian Sundarbans by outlining the following objectives: (i) identifying and characterizing feeding guilds/groups and (ii) understanding the inter-relationship between morphometry with (a) the established feeding guild classifications and (b) observed prey taxa (that characterizes these feeding groups) for determining the role of morphometry in prey acquisition followed by (iii) the evaluation of their potential phylogenetic convergence among the species. For the first objective, two approaches for feeding guild classification were made (3-Guild and 8-Guild) for assessing the prediction accuracy of morphological characters in identifying the different guilds. While the former was based on troph values, the latter classification mode relied on the similarities in diet composition among the different fish species. For addressing the second objective, we employed two different models namely, linear discriminant (LDA) and redundancy analysis (RDA). While the LDA model tested the prediction accuracy of morphological traits in classifying the different feeding guilds, RDA was applied to model the correlation between the morphological traits and the prey categories. In the LDA model, morphological characters showed higher accuracy (78.4%) in classifying three feeding groups rather than eight feeding groups (73%). Following this, the RDA model (explaining 79.78% of constrained variance) showed gill raker intensity, protrusion length, head depth, caudal peduncle, eye diameter and inter-orbital distance to be highly associated with selection of specific prey types by species, thereby characterizing a particular feeding guild. However, generalized linear models testing for correlation between troph value and feeding groups showed substantial variation (90.35%) in the dietary index being explained by the 8-Guild classification. Hence, our study maintains the assumption that broad morphological differentiation acts as one of the underlying processes resulting in dietary variations that results in the varying modes of resource utilization by the coexisting species, thereby determining the structure of a trophic guild. Furthermore, it also suggests that in terms of prey abundance or selectivity, the 8-Guild model is much more conducive in representing the feeding habits of the species while the morphological traits reflected a relatively broader scheme of classification, (i.e., 3-Guild model) with certain traits being phylogenetically conserved within these groups.     

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV “O” 100 GPID₅₀ challenge virus.

     

Crystal structure of BMP-9 and functional interactions with pro-region and receptors

Bone morphogenetic proteins (BMPs), a subset of the transforming growth factor (TGF)-beta superfamily, regulate a diverse array of cellular functions during development and in the adult. BMP-9 (also known as growth and differentiation factor (GDF)-2) potently induces osteogenesis and chondrogenesis, has been implicated in the differentiation of cholinergic neurons, and may help regulate glucose metabolism. We have determined the structure of BMP-9 to 2.3 A and examined the differences between our model and existing crystal structures of other BMPs, both in isolation and in complex with their receptors. TGF-beta ligands are translated as precursors, with pro-regions that generally dissociate after cleavage from the ligand, but in some cases (including GDF-8 and TGF-beta1, -2, and -3), the pro-region remains associated after secretion from the cell and inhibits binding of the ligand to its receptor. Although the proregion of BMP-9 remains tightly associated after secretion, we find, in several cell-based assays, that the activities of BMP-9 and BMP-9.pro-region complex were equivalent. Activin receptor-like kinase 1 (ALK-1), an orphan receptor in the TGF-beta family, was also identified as a potential receptor for BMP-9 based on surface plasmon resonance studies (BIAcore) and the ability of soluble ALK-1 to block the activity of BMP-9.pro-region complex in cell-based assays.     

Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii

The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.