Congenic Mice Provide Evidence for a Genetic Locus That Modulates Spontaneous Arthritis Caused by Deficiency of IL-1RA

To understand the role of genetic factors involved in the development of spontaneous arthritis in mice deficient in IL-1 receptor antagonist protein (IL_1RA), we have identified a genomic region containing a major quantitative trait locus (QTL) for this disease. The QTL is on chromosome 1 and appears to be the strongest genetic region regulating arthritis. To confirm the importance of the QTL and to identify potential candidate genes within it, we conducted speed congenic breeding to transfer the QTL region from DBA/1 mice that are resistant to spontaneous arthritis into BALB/c^−/− which are susceptible. Genetic markers along every chromosome were used to assist in the selection of progeny in each generation to backcross to BALB/c^−/−. By the 6th generation we determined that all of the chromosomes in the progeny were of BALB/c origin with the exception of portions of chromosome 1. At this stage we intercrossed selected mice to produce homozygous strains containing the genomic background of BALB/c^−/− except for the QTL region on chromosome 1, which was from DBA/1. We were able to establish two congenic strains with overlapping DBA/1 DNA segments. These strains were observed for the development of spontaneous arthritis. Both congenic strains were relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. The gene/s that regulates this major QTL would appear to be located in the region of the QTL that is shared by both strains. The common transferred region is between D1Mit110 and D1Mit209 on chromosome 1. We evaluated this region for candidate genes and have identified a limited number of candidates. Confirmation of the identity and precise role of the candidates will require additional study.     

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.     

Transient dynamics of genetic regulatory networks

We present an approximation scheme for deriving reaction rate equations of genetic regulatory networks. This scheme predicts the timescales of transient dynamics of such networks more accurately than does standard quasi-steady state analysis by introducing prefactors to the ODEs that govern the dynamics of the protein concentrations. These prefactors render the ODE systems slower than their quasi-steady state approximation counterparts. We introduce the method by examining a positive feedback gene regulatory network, and show how the transient dynamics of this network are more accurately modeled when the prefactor is included. Next, we examine the repressilator, a genetic oscillator, and show that the period, amplitude, and bifurcation diagram defining the onset of the oscillations are better estimated by the prefactor method. Finally, we examine the consequences of the method to the dynamics of reduced models of the phage lambda switch, and show that the switching times between the two states is slowed by the presence of the prefactor that arises from protein multimerization and DNA binding.     

Obesity potentiates development of fatty liver and insulin resistance, but not atherosclerosis, in high-fat diet-fed agouti LDLR-deficient mice

Obesity is increasing at an alarming rate, and its related disorders are placing a considerable strain on our healthcare system. Although they are not always coincident, obesity is often accompanied by hyperlipidemia. Both obesity and hyperlipidemia are independently associated with atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and insulin resistance (IR). Thus, we sought to determine the relative contributions of obesity and hyperlipidemia to these associated pathologies. Obese agouti (A(y)/a) mice and their littermate controls (a/a) were placed on an LDL receptor (LDLR)(-/-) background. At 4 mo of age, mice were either maintained on chow diet (CD) or placed on Western diet (WD) for 12 wk. These genetic and dietary manipulations yielded four experimental groups: 1) lean, a/a;LDLR(-/-)CD; 2) genetic-induced obesity (GIO), A(y)/a;LDLR(-/-)CD; 3) diet-induced obesity (DIO), a/a;LDLR(-/-)WD; and 4) genetic- plus diet-induced obesity (GIO/DIO), A(y)/a;LDLR(-/-)WD. Lipoprotein profiles revealed increased VLDL and LDL particles in WD-fed mice compared with CD-fed controls. The hyperlipidemia present in this mouse model was the result of both increased hepatic triglyceride production and delayed lipoprotein clearance from the plasma. Both WD-fed groups exhibited similar levels of atherosclerotic lesion area, with increased obesity in the GIO/DIO group having no impact on atherogenesis. However, the severe obesity in the GIO/DIO group did aggravate NAFLD and IR. These findings suggest that, although obesity and hyperlipidemia exert individual pathological effects, the combination of the two has the potential to exert an additive effect on NAFLD and IR but not atherosclerosis in this mouse model.     

Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Key words: calcium, calmodulin, cold stress, MAPK, Arabidopsis, protein phosphorylationCalcium, a universal second messenger in eukaryotic cells, mediates changes in external and internal signals leading to the physiological responses.^1–4 Calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinases (CaMKs) are very important players in calcium/calmodulin mediated signaling in mammalian cells.⁵ In plants, Ca²⁺/CaM-dependent protein phosphorylation was observed more than 25 years ago.⁶ Several calmodulin-regulated protein kinases have been identified and characterized.^7,8 For example, plants have a unique chimeric Ca²⁺/CaM-dependent protein kinase (CCaMK), which exhibits Ca²⁺-dependent autophosphorylation and Ca²⁺/CaM-dependent substrate phosphorylation.⁹ CCaMK is required for bacterial and fungal symbioses in plants.^10–12 Recently, we characterized a novel plant-specific calcium/CaM-regulated receptor-like kinase, CRLK1.¹³ Ca²⁺/CaM binds to CRLK1 and stimulates its kinase activity. Functional studies with CRLK1 indicate that CRLK1 acts as a positive regulator in plant response to chilling and freezing temperatures. To further define the CRLK1-mediated signal pathway, we isolated CRLK1 interacting proteins by co-immunoprecipitation using an anti-CRLK1 antibody. Since cold increases the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C for 1 hr before co-immunoprecipitation. The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We observed several bands of different sizes only in the wild-type but not in the crlk1 knockout mutant plants (Fig. 1A). Furthermore, the intensity of these bands increased upon cold treatment, suggesting that they are the putative partners or associated proteins of the CRLK1 immunocomplex.Open in a separate windowFigure 1CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CR LK associates with MEKK1 in vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.To determine the identities of these proteins, mass spectrometric analysis was performed with the total immunocomplex.¹⁴ In addition to CRLK1, there were 12 other proteins which matched the Arabidopsis database. Several of them appeared in the pull-down complex from WT, but not from crlk1 mutants. These putative interacting proteins included MEKK1, another unknown protein kinase, a type 2C phosphatase and CaM (Fig. 1B). MEKK1 is one of the 60 putative MAPKKKs in the Arabidopsis genome, and sits on the top of mitogen-activated protein kinase (MAPK) cascade. The MAPK signaling consists of a cascade of three consecutively acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase (MAPK). Plants possess multiple MAPKKKs, MAPKKs and MAPKs, which respond to different upstream signals and activate distinct downstream pathways.^15–17 The specific MAPK module responding to lower temperature has been determined in Arabidopsis.^18,19 MEKK1, a member of MAPKKKs, specifically interacts and phosphorylates MKK2 and regulates COR genes expression in response to cold stress.¹⁹ MEKK1 has been shown to play a role in mediating reactive oxygen species homeostasis.^20,21 Therefore we selected MEKK1 from the putative CRLK1 partners for further studies.     

Transcriptional activities of nuclear SREBP-1a, -1c,and -2 to different target promoters of lipogenic and cholesterogenic genes

Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.     

Molecular evolution of genes in avian genomes

Background

Obtaining a draft genome sequence of the zebra finch (Taeniopygia guttata), the second bird genome to be sequenced, provides the necessary resource for whole-genome comparative analysis of gene sequence evolution in a non-mammalian vertebrate lineage. To analyze basic molecular evolutionary processes during avian evolution, and to contrast these with the situation in mammals, we aligned the protein-coding sequences of 8,384 1:1 orthologs of chicken, zebra finch, a lizard and three mammalian species.

Results

We found clear differences in the substitution rate at fourfold degenerate sites, being lowest in the ancestral bird lineage, intermediate in the chicken lineage and highest in the zebra finch lineage, possibly reflecting differences in generation time. We identified positively selected and/or rapidly evolving genes in avian lineages and found an over-representation of several functional classes, including anion transporter activity, calcium ion binding, cell adhesion and microtubule cytoskeleton.

Conclusions

Focusing specifically on genes of neurological interest and genes differentially expressed in the unique vocal control nuclei of the songbird brain, we find a number of positively selected genes, including synaptic receptors. We found no evidence that selection for beneficial alleles is more efficient in regions of high recombination; in fact, there was a weak yet significant negative correlation between ω and recombination rate, which is in the direction predicted by the Hill-Robertson effect if slightly deleterious mutations contribute to protein evolution. These findings set the stage for studies of functional genetics of avian genes.