Faculty Bibliography

PURPOSE OF REVIEW: Paradoxically, many well-established components of the heart-healthy lifestyle are pro-oxidant, including polyunsaturated fat and moderate alcohol consumption. Moreover, antioxidant supplements have failed to decrease cardiovascular risk in extensive human clinical trials to date. Recent progress in understanding the roles of oxidants in regulating VLDL secretion and as essential signaling molecules supports the concept that oxidation may be beneficial in certain circumstances but damaging in others. We summarize recent data on the roles played by oxidative metabolism in different tissues and pathways, and address whether it is currently advisable to use antioxidant supplements to reduce cardiovascular risk. RECENT FINDINGS: Our recent study reported that in liver cells, polyunsaturated fatty acids increased reactive oxygen species, which in turn lowered the secretion of the atherogenic lipoprotein, VLDL, in vitro and in vivo. Antioxidant treatments prevented VLDL-lowering effects of polyunsaturated fatty acids in vitro, suggesting that supplemental antioxidants could either raise apolipoprotein-B-lipoprotein plasma levels in vivo, or impair the response to lipid-lowering therapies. The failure of antioxidants to decrease cardiovascular disease risk in many trials is also discussed in the context of current models for atherosclerosis progression and regression. SUMMARY: Oxidation includes distinct biochemical reactions, and it is overly simplistic to lump them into a unitary process that affects all cell types and metabolic pathways adversely. Guidelines for diet should adhere closely to what has been clinically proved, and by this standard there is no basis to recommend antioxidant use, beyond what is inherent to the 'heart healthy' diet in order to benefit cardiovascular health.

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family with an important role in cholesterol metabolism. PCSK9 expression is regulated by dietary cholesterol in mice and cellular sterol levels in cell culture via the sterol regulatory element binding protein transcription factors, and mutations in PCSK9 are associated with a form of autosomal dominant hypercholesterolemia. Overexpression of PCSK9 in mice leads to increased total and low-density lipoprotein (LDL) cholesterol levels because of a decrease in hepatic LDL receptor (LDLR) protein with normal mRNA levels. To study the mechanism, PCSK9 was overexpressed in human hepatoma cells, HepG2, by adenovirus. Overexpression of PCSK9 in HepG2 cells caused a decrease in whole-cell and cell-surface LDLR levels. PCSK9 overexpression had no effect on LDLR synthesis but caused a dramatic increase in the degradation of the mature LDLR and a lesser increase in the degradation of the precursor LDLR. In contrast, overexpression of a catalytically inactive mutant PCSK9 prevented the degradation of the mature LDLR; whereas increased degradation of the precursor LDLR still occurred. The PCSK9-induced degradation of the LDLR was not affected by inhibitors of the proteasome, lysosomal cysteine proteases, aspartic acid proteases, or metalloproteases. The PCSK9-induced degradation of the LDLR was shown to require transport out of the endoplasmic reticulum. These results indicate that overexpression of PCSK9 induces the degradation of the LDLR by a nonproteasomal mechanism in a post-endoplasmic reticulum compartment.

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oocyte, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. Here, we report the identification of twin, a homolog of the yeast CCR4 deadenylase. We show that twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, our data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects we observe in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. We propose that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation

Periprosthetic fracture after total joint replacement predominantly occurs at the stem tip. In this study, the effects of gap size, stem stability and cortical thickness between two press-fit, ipsilateral intramedullary stems on the tensile stresses created in the femur were investigated using finite-element models. The findings were confirmed with strain-gauge tests using a composite Sawbone femur. Gap size did not affect the level of stress on the femur. Cortical thickness had an important effect on stress distribution: peak stresses increased as bone cortical thickness decreased. Irrespective of gap size, the tips of loose stems acted as stress risers particularly with thinner cortices; the tips of well-fixed stems, however, did not

Invasive cell migration in both normal development and metastatic cancer is regulated by various signaling pathways, transcription factors and cell-adhesion molecules. The coordination between these activities in the context of cell migration is poorly understood. During Drosophila oogenesis, a small group of cells called border cells exit the follicular epithelium to perform a stereotypic, invasive migration. We find that the ETS transcription factor Yan is required for border cell migration and that Yan expression is spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor Tyrosine Kinase pathways in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. Our results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells

Follicular papilla (FP) cells, but not their closely related dermal fibroblasts, can maintain hair growth suggesting cell type-specific molecular signals. To define the molecular differences between these two cell types, we generated a subtraction complementary DNA (cDNA) library highly enriched in FP-specific cDNA. Differential screening identified FP-1 as the most abundant cDNA sequence in this subtraction library. FP-1 message RNA is highly abundant in cultured rat vibrissa FP cells, can be detected at very low levels in the stomach and the ovary, and is undetectable in cultured dermal fibroblasts and in 16 rat non-follicular tissues. The full-length, 2.3 kb FP-1 cDNA encodes a protein of 549 amino acids harboring a signal peptide, collagen triple helix repeats, and an olfactomedin-like domain. Monospecific rabbit antibodies to FP-1 recognize in cultured FP cells a single approximately 72 kDa glycoprotein with a approximately 60 kDa protein core. FP-1 protein is expressed in vivo in a hair cycle-dependent manner, as it can be detected in FP during anagen, but not in catagen and telogen phases of the hair cycle. FP-1 is presumably a highly specific extracellular matrix protein synthesized by FP cells and may be involved in the organization of FP during certain phases of normal or pathological hair growth

Monitoring Editor: Jeffrey Brodsky The apical surface of mammalian urothelium is covered by 16-nm protein particles packed hexagonally to form 2D crystals of asymmetric unit membranes (AUM) that contribute to the remarkable permeability barrier function of the urinary bladder. We have shown previously that bovine AUMs contain four major integral membrane proteins, i.e., uroplakins Ia, Ib, II and IIIa, and that UPIa and Ib (both tetraspanins) form heterodimers with UPII and IIIa, respectively. Using a panel of antibodies recognizing different conformational states of uroplakins, we demonstrate that the UPIa-dependent, furin-mediated cleavage of the prosequence of UPII leads to global conformational changes in mature UPII, and that UPIb also induces conformational changes in its partner UPIIIa. We further demonstrate that tetraspanins CD9, CD81 and CD82 can stabilize their partner protein CD4. These results indicate that tetraspanin uroplakins, and some other tetraspanin proteins, can induce conformational changes leading to the ER-exit, stabilization and cell surface expression of their associated, single-transmembrane-domained partner proteins, and thus can function as 'maturation-facilitators.' We propose a model of AUM assembly in which conformational changes in integral membrane proteins induced by uroplakin interactions, differentiation-dependent glycosylation and the removal of the prosequence of UPII play roles in regulating the assembly of uroplakins to form AUM

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species