Inducible cytochrome P450 activities in renal glomerular mesangial cells: biochemical basis for antagonistic interactions among nephrocarcinogenic polycyclic aromatic hydrocarbons

BACKGROUND: Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes in vitro and in vivo. While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons. METHODS: To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 - 30 microM) or in the presence of ANTH (3 microM) or CHRY (3 microM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC). RESULTS: Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism. CONCLUSION: These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential.     

Mouse linkage cytogenetics (L-C) probes

We have identified 149 hybridization probes at 10-cM intervals in the mouse and have confirmed their order and linkage by fluorescence in situ hybridization. These probes represent a new resource for mapping in the mouse and can be used to correlate linkage and cytogenetic maps, to map novel sequences to within a few centimorgans, to relate cytogenetic abnormalities to the genetic map, and to make cross-species comparisons.     

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.     

Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

Strain degeneration in solventogenic clostridia is a known problem in the technical acetone–butanol fermentation bioprocess, especially in the continuous process mode. Clostridial strain degeneration was studied by Fourier transform infrared (FT-IR) spectroscopy of the bacterial cells. Degenerative variant formation in two strains, Clostridium beijerinckii NCIMB 8052 and Clostridium species AA332, was detected spectroscopically. Colonies on solid media were sampled, or assayed directly in situ by IR microscopy. It has previously been shown that the distinctive acidogenic and solventogenic physiological phases of Clostridium acetobutylicum in liquid medium can be discriminated by FT-IR spectroscopy. This was confirmed here for C. beijerinckii NCIMB 8052. The proportion of degenerate cells in a mixed population in liquid medium could be quantified, as the spectral features change in different ways during the normal growth cycle of wild type organisms and degenerate variants in batch culture. This opens a new perspective for physiology-based process monitoring and control, especially of the continuous acetone–butanol fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 314–321. Received 06 October 2000/ Accepted in revised form 20 April 2001     

Physiological roles of taurine in heart and muscle

Taurine (aminoethane sulfonic acid) is an ubiquitous compound, found in very high concentrations in heart and muscle. Although taurine is classified as an amino acid, it does not participate in peptide bond formation. Nonetheless, the amino group of taurine is involved in a number of important conjugation reactions as well as in the scavenging of hypochlorous acid. Because taurine is a fairly inert compound, it is an ideal modulator of basic processes, such as osmotic pressure, cation homeostasis, enzyme activity, receptor regulation, cell development and cell signalling. The present review discusses several physiological functions of taurine. First, the observation that taurine depletion leads to the development of a cardiomyopathy indicates a role for taurine in the maintenance of normal contractile function. Evidence is provided that this function of taurine is mediated by changes in the activity of key Ca²⁺ transporters and the modulation Ca²⁺ sensitivity of the myofibrils. Second, in some species, taurine is an established osmoregulator, however, in mammalian heart the osmoregulatory function of taurine has recently been questioned. Third, taurine functions as an indirect regulator of oxidative stress. Although this action of taurine has been widely discussed, its mechanism of action is unclear. A potential mechanism for the antioxidant activity of taurine is discussed. Fourth, taurine stabilizes membranes through direct interactions with phospholipids. However, its inhibition of the enzyme, phospholipid N-methyltransferase, alters the phosphatidylcholine and phosphatidylethanolamine content of membranes, which in turn affects the function of key proteins within the membrane. Finally, taurine serves as a modulator of protein kinases and phosphatases within the cardiomyocyte. The mechanism of this action has not been studied. Taurine is a chemically simple compound, but it has profound effects on cells. This has led to the suggestion that taurine is an essential or semi-essential nutrient for many mammals.     

Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.     

Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair

Fetal wound healing is characterized by minimal inflammation and scarless repair. IL-6 stimulates inflammation in postnatal wound healing. We hypothesized that fetal skin has a diminished IL-6 response and that exogenous IL-6 will result in scar formation. Human adult or fetal skin was placed subcutaneously in SCID mice and incisionally wounded. Wounds were excised after 4, 12, 24 or 72 h for IL-6 mRNA quantification by RT-PCR. In other grafts, 5 microgram of IL-6 was injected at wounding and then harvested at 7 days for analysis of scar formation. IL-6 production was examined in primary cultures of human fetal or adult dermal fibroblasts incubated for 8 h with 0, 0.1, 1 or 10 ng/ml of PDGF-BB. IL-6 mRNA was detected 4 h after wounding in fetal and adult wounds, but by 12 h there was no IL-6 mRNA in the fetal wounds. Adult wounds had IL-6 mRNA persisting to 72 h. IL-6 administration to fetal wounds resulted in scar formation. Fetal fibroblasts produced less IL-6 protein and mRNA at all points examined (P<0.01 vs adult). Diminished production of inflammatory cytokines such as IL-6 may be responsible for the lack of inflammation seen during fetal wound healing. Diminished inflammation may provide a permissive environment for scarless wound healing.     

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Mutations on the Switch III region and the alpha3 helix of Galpha16 differentially affect receptor coupling and regulation of downstream effectors

Background

Gα₁₆ can activate phospholipase Cβ (PLCβ) directly like Gα_q. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα₁₆. Previous studies on Gα_q have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα₁₆ might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.

Results

Alanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα₁₆. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα₁₆ was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G₁₆ and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.

Conclusion

The integrity of the switch III region and α3 helix of Gα₁₆ is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα₁₆ to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G₁₆. The studied region was shown to bear multiple functionally important roles of G₁₆.