Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes – in vitro study

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m² which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0–1250 Joules/m², whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m². Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m².     

Trypsin inhibition by a peptide hormone: crystal structure of trypsin-vasopressin complex

The large variety of serine protease inhibitors, available from various sources such as tissues, microorganisms, plants, etc., play an important role in regulating the proteolytic enzymes. The analysis of protease-inhibitor complexes helps in understanding the mechanism of action, as well as in designing inhibitors. Vasopressin, an anti-diuretic nonapeptide hormone, is found to be an effective inhibitor of trypsin, with a K(i) value of 5 nM. The crystal structure of the trypsin-vasopressin complex revealed that vasopressin fulfils all the important interactions for an inhibitor, without any break in the scissile peptide bond. The cyclic nature due to a disulfide bridge between Cys1 and Cys6 of vasopressin provides structural rigidity to the peptide hormone. The trypsin-binding site is located at the C terminus, while the neurophysin-binding site is at the N terminus of vasopressin. This study will assist in designing new peptide inhibitors. This study suggests that vasopressin inhibition of trypsin may have unexplored biological implications.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Microbial transformation of (-)-isolongifolol and butyrylcholinesterase inhibitory activity of transformed products

The microbial transformation of (-)-isolongifolol (1) by using the standard two-stage fermentation technique with Fusarium lini afforded polar oxygenated metabolites: 10-oxoisolongifolol (2), 10alpha-hydroxyisolongifolol (3), and 9alpha-hydroxyisolongifolol (4). Metabolites 3 and 4 were also formed with the incubation of 1 with Aspergillus niger. All three metabolites were found to be new. Compounds 3 and 4 inhibited butyrylcholinesterase enzyme in a concentration-dependent manner with IC50 values 13.6 and 299.5 microM, respectively. Compound 3 showed un-competitive mode of inhibition against butyrylcholinesterase with Ki value 15.0 microM. The structures of metabolites 2-4 were deduced on the basis of spectroscopic techniques and single-crystal X-ray diffraction techniques.     

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.     

Mechanisms of sex steroid effects on bone

Sex steroids play a major role in the regulation of bone turnover. Thus, gonadectomy in either sex is associated with an increase in bone remodeling, increased bone resorption, and a relative deficit in bone formation, resulting in accelerated bone loss. Recent physiological studies have established an important role for estrogen in regulating bone turnover not only in females, but also in males. Studies in mice with knock out of the estrogen receptor, aromatase, or androgen receptor have provided important insights into the in vivo mechanisms of sex steroid action on bone. The cellular and molecular mediators of sex steroid effects on the bone-forming osteoblasts and bone-resorbing osteoclasts are also being increasingly better defined. Estrogen inhibits bone remodeling by concurrently suppressing osteoblastogenesis and osteoclastogenesis from marrow precursors. Both estrogen and androgens inhibit bone resorption via effects on the receptor activator of NF-kappaB ligand (RANKL)/RANK/osteoprotegerin system, as well as by reducing the production of a number of pro-resorptive cytokines, along with direct effects on osteoclast activity and lifespan. Sex steroid effects on bone formation are also likely mediated by multiple mechanisms, including a prolongation of osteoblast lifespan via non-genotropic mechanisms, as well as effects on osteoblast differentiation and function. These pleiotropic actions of sex steroids on virtually all aspects of bone metabolism belie the importance of the skeleton not only in providing structural support for the body and in locomotion, but also as a dynamic tissue responsive, among other things, to the reproductive needs of the organism for calcium.     

A microarray study of MPP<Superscript>+</Superscript>-treated PC12 Cells: Mechanisms of toxicity (MOT) analysis using bioinformatics tools

Background

This paper describes a microarray study including data quality control, data analysis and the analysis of the mechanism of toxicity (MOT) induced by 1-methyl-4-phenylpyridinium (MPP⁺) in a rat adrenal pheochromocytoma cell line (PC12 cells) using bioinformatics tools. MPP⁺ depletes dopamine content and elicits cell death in PC12 cells. However, the mechanism of MPP⁺-induced neurotoxicity is still unclear.

Results

In this study, Agilent rat oligo 22K microarrays were used to examine alterations in gene expression of PC12 cells after 500 μM MPP⁺ treatment. Relative gene expression of control and treated cells represented by spot intensities on the array chips was analyzed using bioinformatics tools. Raw data from each array were input into the NCTR ArrayTrack database, and normalized using a Lowess normalization method. Data quality was monitored in ArrayTrack. The means of the averaged log ratio of the paired samples were used to identify the fold changes of gene expression in PC12 cells after MPP⁺ treatment. Our data showed that 106 genes and ESTs (Expressed Sequence Tags) were changed 2-fold and above with MPP⁺ treatment; among these, 75 genes had gene symbols and 59 genes had known functions according to the Agilent gene Refguide and ArrayTrack-linked gene library. The mechanism of MPP⁺-induced toxicity in PC12 cells was analyzed based on their genes functions, biological process, pathways and previous published literatures.

Conclusion

Multiple pathways were suggested to be involved in the mechanism of MPP⁺-induced toxicity, including oxidative stress, DNA and protein damage, cell cycling arrest, and apoptosis.

     

Fully degradable hydrophilic polyals for protein modification

Modification of proteins with hydrophilic polymers is an effective strategy for regulation of protein pharmacokinetics. However, conjugates of slowly or non-biodegradable materials, such as poly(ethylene glycol), are known to cause long-lasting cell vacuolization, in particular in renal epithelium. Conjugates of more degradable polymers, e.g., polysaccharides, have a significant risk of immunotoxicity. Polymers that combine complete degradability, long circulation in vivo, and low immuno and chemical toxicity would be most beneficial as protein conjugate components. This study explores new fully biodegradable hydrophilic polymers, hydrophilic polyals. They are nontoxic, stable at physiological conditions, and undergo proton-catalyzed hydrolysis at lysosomal pH. The model enzyme-polyal conjugates were prepared with 61-98% yield using conventional and novel conjugation techniques and retained 90-95% of specific activity. The model conjugates showed a significant prolongation of protein circulation in rodents, with a 5-fold reduction in the renal accumulation. The data suggests that hydrophilic polyals may be useful in designing protein conjugates with improved properties.     

A novel assay for protein phosphatase 2A (PP2A) complexes in vivo reveals differential effects of covalent modifications on different Saccharomyces cerevisiae PP2A heterotrimers

Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.     

Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans

(-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 angstrom resolution, respectively. The enzyme is a homotetramer, consisting of an alpha/beta single domain monomer containing seven parallel beta-strands flanked by eight alpha-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD(+) and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.