Faculty Bibliography

There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

Transforming growth factor-beta (TGFbeta) is a well-known master regulator of cellular proliferation and is a critical factor in the maintenance of tissue homeostasis. TGFbeta is classically defined as a tumor suppressor that functions in the early stages of carcinogenesis, yet paradoxically it functions as a tumor promoter in established cancers. Less well studied is its role in maintaining genomic stability through its participation in the DNA damage response (DDR). Deletion of Tgfb1 in murine epithelium increases genomic instability (GIN) as measured by gene amplification, aneuploidy, and centrosome aberrations; likewise, GIN is increased by depleting the TGFbeta ligand or inhibiting TGFbeta pathway signaling in human epithelial cells. Subsequent studies demonstrated that TGFbeta depletion compromises cell survival in response to radiation and impairs activation of the DDR because of severely reduced activity of ataxia telangiectasia mutated (ATM), a serine/threonine protein kinase that is rapidly activated by DNA double-strand breaks. The SMAD transcription factors are intermediaries in the crosstalk between the TGFbeta and ATM pathways in the DDR. Recent studies have shown that SMAD2 and SMAD7 participate in the DDR in a manner dependent on ATM or TGFbeta receptor type I, respectively, in human fibroblasts and epithelial cells. Understanding the role of TGFbeta in the DDR and suppressing GIN is important to understanding its seemingly paradoxical roles in tumorigenesis and thus has therapeutic implications for improving the response to DNA damage-inducing therapy.

When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

STRUCTURED ABSTRACT: Study Design. Animal modelObjective. Determine whether Amicar and TXA inhibit spine fusion volumeSummary of Background Data. Amicar and TXA are antifibrinolytics used to reduce perioperative bleeding. Prior in vitro data showed that antifibrinolytics reduce osteoblast bone mineralization. This study tested whether antifibrinolytics Amicar and TXA inhibit spine fusion.Methods. Posterolateral L4-L6 fusion was performed in fifty mice, randomized into groups of ten, that received the following treatment before and after surgery: (1) Saline; (2) TXA 100mg/kg; (3) TXA 1000mg/kg; (4) Amicar 100 mg/kg; (5) Amicar 1000 mg/kg. High-resolution plane radiography was performed after 5 weeks and micro-CT was performed at the end of the 12-week study. Radiographs were graded using the Lenke scale. Micro-CT was used to quantify fusion mass bone volume. One-way analysis of variance (ANOVA) by ranks with Kruskal-Wallis testing was used to compare the radiographic scores. One-way ANOVA with least-significant differences (LSD) post-hoc testing was used to compare the micro-CT bone volume.Results. The average (+/- SD) bone volume/total volume (%) measured in the saline, TXA 100 mg/kg, TXA 1000 mg/kg, Amicar 100 mg/kg and Amicar 1000 mg/kg groups were 10.8+/-2.3, 9.7+/-2.2, 13.4+/-3.2, 15.5+/-5.2 and 17.9+/-3.5%, respectively. There was a significant difference in the Amicar 100 mg/kg (p < 0.05) and Amicar 1000 mg/kg (p < 0.001) groups compared to saline. There was greater bone volume in the Amicar groups compared to the TXA group (p < 0.001). There was more bone volume in the TXA 1000 mg/kg group compared to TXA 100 mg/kg (p < 0.05) but the bone volume in neither of the TXA groups was different to saline (p = 0.49). There were no between-group differences observed using plane radiographic scoring.Conclusions. Amicar significantly enhanced the fusion bone mass in a dose-dependent manner while TXA did not have a significant effect on fusion compared to saline control.These data are in contrast to prior in vitro data that antifibrinolytics inhibit osteoblast bone mineralization.

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Forster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

BACKGROUND: The American Heart Association recommends targeting 7 cardiovascular (CV) health metrics to reduce morbidity and mortality. Control of these targets in patients undergoing CV intervention is uncertain. METHODS: We prospectively studied patients undergoing elective percutaneous coronary or peripheral intervention from November 2010 to May 2012. We recorded data on patient demographics, clinical characteristics, and social history. Risk factor control was categorized as ideal, intermediate, or poor according to the 7 American Heart Association-defined CV health metrics (smoking status, body mass index, physical activity, diet, cholesterol, blood pressure, and metabolic control). Linear regression model was used to evaluate the association between baseline characteristics and poor CV health. RESULTS: Among 830 consecutive patients enrolled, mean age is 67.3 +/- 10.8 years, 74.2% are male, and 62.1% are white. The adequacy of achievement of ideal CV health is suboptimal in our cohort; the mean number of ideal CV metrics is 2.15 +/- 1.06. Less than 1 in 10 (9.7%) met >/=4 ideal CV health metrics. After multivariate analysis, male sex (P = .04), nonwhite race (P = .01), prior coronary artery disease (P < .01), prior peripheral arterial disease (P < .01), and history of depression (P = .01) were significantly associated with poor CV health. CONCLUSIONS: Among patients referred for elective CV intervention, achievement of ideal CV health is poor. Elective interventions represent an opportunity to identify and target CV health for risk factor control and secondary prevention.

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan-collagen hydrogel scaffold for improving bone marrow-derived mesenchymal stem cell survival within ischemic skin wounds by creating a "stem cell niche" that enhances regenerative cytokine secretion. Adipose-derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel-based therapies. To optimize hydrogel cell seeding, a rapid, capillary force-based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis-related genes, including Oct4, Vegf, Mcp-1, and Sdf-1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC-seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan-collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC-seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signalling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signalling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood-brain barrier. Local insulin/IGF signalling from glial cells, the blood-brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signalling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Alex Schier looks back at the life and research of his graduate mentor and friend Walter Gehring.