Faculty Bibliography

Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in atherosclerotic lesions. We investigated the extent of the colocalization of SAP with apolipoprotein A-I (apoA-I), apoB, apoC-II, and apoE in human coronary arteries and explored potential roles for SAP in these regions, specifically the effect of SAP on the rate of formation and macrophage recognition of amyloid fibrils composed of apoC-II. Analysis of 42 human arterial sections by immunohistochemistry and double label fluorescence microscopy demonstrated that SAP and apoA-I, apoB, apoC-II, and apoE were increased significantly in atherosclerotic lesions compared with nonatherosclerotic segments. SAP colocalized with all four apolipoproteins to a similar extent, whereas plaque macrophages were found to correlate most strongly with apoC-II and apoB. In vitro studies showed that SAP accelerated the formation of amyloid fibrils by purified apoC-II. Furthermore, SAP strongly inhibited the phagocytosis of apoC-II amyloid fibrils by primary macrophages and macrophage cell lines and blocked the resultant production of reactive oxygen species. The ability of SAP to accelerate apoC-II amyloid fibril formation and inhibit macrophage recognition of apoC-II fibrils suggests that SAP may modulate the inflammatory response to amyloid fibrils in atherosclerosis

The development of cerclage systems for fixation of greater trochanteric osteotomies has progressed from monofilament wires to multifilament cables to cable grip and cable plate systems. Cerclage wires and cables have various clinical indications, including fixation for fractures and for trochanteric osteotomy in hip arthroplasty. To achieve stable fixation and eventual union of the trochanteric osteotomy, the implant must counteract the destabilizing forces associated with pull of the peritrochanteric musculature. The material properties of cables and cable grip systems are superior to those of monofilament wires; however, potential complications with the use of cables include debris generation and third-body polyethylene wear. Nevertheless, the cable grip system provides the strongest fixation and results in lower rates of nonunion and trochanteric migration. Cable plate constructs show promise but require further clinical studies to validate their efficacy and safety

Complex primary total hip arthroplasty (THA) is defined as primary THA in patients with compromised bony or soft-tissue states, including but not limited to dysplastic hip, ankylosed hip, prior hip fracture, protrusio acetabuli, certain neuromuscular conditions, skeletal dysplasia, and previous bony procedures about the hip. Intraoperatively, provisions must be made for the possible use of modular implants and/or bone grafts. In this article, we review the principles of preoperative, intraoperative, and postoperative management of patients requiring a complex primary THA

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein. R6IP1 also binds to Rab11A in its GTP-bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6-dependent manner but can associate with Rab11-positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.

In the present study, we utilised an in vitro digestion procedure to deliver molecules contained in tomatoes to cultured cells and to analyse potential mechanisms underlying the antitumoural effects of tomatoes reported in the literature. Ripe tomatoes underwent in vitro simulated digestion and the aqueous fraction obtained was delivered to HT-29 and HCT-116 colon adenocarcinoma cells. The amount of lycopene released during digestion and transferred to the aqueous fraction during digestion was 10-fold lower than that present in tomato homogenate before digestion. The carotenoid was accumulated by colon adenocarcinoma cells in a dose-dependent manner after the addition of tomato digestate (20-100 ml/l) for 24 h. Tomato digestate inhibited the growth of HT-29 and HCT-116 cells in a dose-dependent manner. Growth inhibition resulted from an arrest of cell cycle progression at the G0/G1 phase and by apoptosis induction. A down regulation of cyclin D1, Bcl-2 and Bcl-xL expression was also observed, without apparent changes in p53, p21, p27 and Bax. In conclusion, the present data demonstrate that the in vitro digestion procedure represents a useful approach to supply tomato to colon cultured cells. Moreover, we have shown that tomato digestate is able to inhibit the growth of colon cancer cells by modulating the expression of regulators of the cell cycle and apoptosis.

Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one-week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20-fold) and cellular density (20-fold). The increased cellularity is accompanied by a four-fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53-null mice, with down-regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII-null mice. We conclude that mechanical loading early in the proliferative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt-dependent mechanism

Beta2 adrenergic receptors were identified in keratinocytes more than 30 years ago, but their function in the epidermis continues to be elucidated. Abnormalities in their expression, signaling pathway, or in the generation of endogenous catecholamine agonists by keratinocytes have been implicated in the pathogenesis of cutaneous diseases such as atopic dermatitis, vitiligo, and psoriasis. New studies also indicate that the beta2AR also modulates keratinocyte migration, and thus can function to regulate wound reepithelialization. This review focuses on the function of these receptors in keratinocytes and their contribution to cutaneous physiology and disease

OBJECTIVE: To explore an association between the use of beta-adrenergic receptor agonists or antagonists and the onset of venous leg ulcers (VLUs). DESIGN: Retrospective cohort study. SETTING: Ambulatory setting of general practice in the United Kingdom. Patients Patients followed by participating physicians. MAIN OUTCOME MEASURE: Onset of VLU. RESULTS: A total of 414 887 patients registered in the General Practice Research Database met our study criteria for eligibility. Of these individuals, 62 886 were exposed to a beta-adrenergic receptor agonist and 54 861 were exposed to a beta-adrenergic receptor antagonist (6620 used both beta-adrenergic receptor antagonists and agonists). Of those exposed to a beta-adrenergic receptor agonist, 15.5% developed a VLU, whereas 18.4% of those who were not exposed developed a VLU. Of those exposed to a beta-adrenergic receptor antagonist, 18.2% developed a VLU, whereas 19.9% of those not exposed developed a VLU. The odds ratio (OR) of association between beta-adrenergic receptor antagonist and VLUs was 1.02 (95% confidence interval [CI], 0.99-1.04); for the association between beta-adrenergic receptor agonist and VLUs, 0.84 (95% CI, 0.82-0.86). The fully adjusted ORs were 1.04 (95% CI, 0.98-1.11) and 0.44 (95% CI, 0.42-0.45), respectively. Furthermore, using propensity score models, we were able to confirm the association for beta-adrenergic receptor agonist users. In addition, beta-adrenergic receptor antagonist users in many of the propensity score quintiles were also protected from developing VLUs. CONCLUSIONS: A protective association between beta-adrenergic receptor agonists and perhaps beta-adrenergic receptor antagonists and VLUs exists. There is strong laboratory evidence to support these epidemiologic findings. The evidence in this study should not be used as a rationale for treatment of VLUs with beta-adrenergic receptor agents but should be compelling for the consideration of a randomized clinical trial

Microtubule-based vesicular transport is well documented in epithelial cells, but the specific motors involved and their regulation during polarization are largely unknown. We demonstrate that KIF5B mediates post-Golgi transport of an apical protein in epithelial cells, but only after polarity has developed. Time-lapse imaging of EB1-GFP in polarized MDCK cells showed microtubule plus ends growing toward the apical membrane, implying that plus end-directed N-kinesins might be used to transport apical proteins. Indeed, time-lapse microscopy revealed that expression of a KIF5B dominant negative or microinjection of function-blocking KIF5 antibodies inhibited selectively post-Golgi transport of the apical marker, p75-GFP, after polarization of MDCK cells. Expression of other KIF dominant negatives did not alter p75-GFP trafficking. Immunoprecipitation experiments demonstrated an interaction between KIF5B and p75-GFP in polarized, but not in subconfluent, MDCK cells. Our results demonstrate that apical protein transport depends on selective microtubule motors and that epithelial cells switch kinesins for post-Golgi transport during acquisition of polarity.

Glucose regulatory protein (GRP58) is known to mediate mitomycin C (MMC)-induced DNA cross-linking. However, the mechanism remains elusive. We hypothesized that thioredoxin-like domains, one at NH2 terminus and another at COOH terminus, are required for GRP58-mediated MMC reductive activation leading to DNA cross-linking. Site-directed mutagenesis mutated cysteines in thioredoxin domains to serines. Wild-type (WT) and mutant GRP58 were cloned in pcDNA to produce GRP58 V5-tagged WT and mutant proteins on transfection in mammalian cells. Human colon carcinoma (HCT116) cells transiently expressing and Chinese hamster ovary cells stably expressing WT and mutant GRP58 were analyzed for MMC-induced DNA cross-linking. WT GRP58 was highly efficient in MMC-induced DNA cross-linking. However, both NH2- and COOH-terminal thioredoxin mutants showed significant reduction in MMC-induced DNA cross-linking. The coexpression of GRP58 with thioredoxin reductase 1 and/or treatment of cells with NADPH increased MMC-induced DNA cross-linking from the WT GRP58. In similar experiments, siRNA inhibition of thioredoxin reductase 1 led to decreased MMC-induced DNA cross-linking. Further experiments revealed that mutations in thioredoxin domains led to significant decrease in metabolic reductive activation of MMC. These results led to conclusion that GRP58, through its two thioredoxin-like domains, functions as a reductase leading to bioreductive drug MMC activation and DNA cross-linking.