Faculty Bibliography

Transforming growth factor-betas (TGF-beta) are secreted as latent complexes consisting of the TGF-beta dimer, the TGF-beta propeptide dimer, and the latent TGF-beta binding protein (LTBP). Although the bonds between TGF-beta and its propeptide are cleaved intracellulary, the propeptide associates with TGF-beta by electrostatic interactions, thereby conferring latency to the complex. We reported that a specific sequence of LTBP-1 is required for latent TGF-beta activation by the integrin alphavbeta6. Here we describe a 24 amino acid sequence from the hinge domain required for activation. The LTBP-1 polypeptide rL1N, which includes the hinge, associates with fibronectin in binding assays. We present evidence that fibronectin null cells minimally activate latent TGF-beta and poorly incorporate the active hinge sequence into their matrix. In addition, cells missing the fibronectin receptor alpha5beta1 exhibit defective activation of latent TGF-beta by alphavbeta6 and decreased matrix incorporation. The results indicate specificity for integrin-mediated latent TGF-beta activation that include unique sequences in LTBP-1 and an appropriate matrix molecule

The TRIM/RBCC proteins are defined by the presence of the tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region. These proteins are involved in a plethora of cellular processes such as apoptosis, cell cycle regulation and viral response. Consistently, their alteration results in many diverse pathological conditions. The highly conserved modular structure of these proteins suggests that a common biochemical function may underlie their assorted cellular roles. Here, we review recent data indicating that some TRIM/RBCC proteins are implicated in ubiquitination and propose that this large protein family represents a novel class of 'single protein RING finger' ubiquitin E3 ligases.

PURPOSE:: We examined the immunolocalization of estrogen receptor (ER)alpha and ERbeta in the human fetal prostate. MATERIALS AND METHODS:: Tissue sections from human fetal prostates at 7 to 22 weeks of gestation were stained with antibodies to ERalpha, ERbeta, and cytokeratin 10 and 14. RESULTS:: ERalpha expression was not detected until 15 weeks of gestation with sparse staining in the utricle. By 19 weeks increased ERalpha expression was seen in the luminal cells of the ventral urogenital epithelium (UGE), basal cells of the dorsal UGE, utricle, distal periurethral ducts, peripheral stroma and posterior prostatic duct. K14 was detected in basal cells of the UGE and in several posterior acini. At 22 weeks ERalpha expression was more intense in all of these areas. ERbeta was expressed throughout the UGE, ejaculatory ducts, mullerian ducts and entire stroma at 7 weeks. Intense ERbeta staining was observed in these areas and in the prostatic buds by 8 weeks with persistent intense staining through 22 weeks. CONCLUSIONS:: To our knowledge we report the first immunolocalization of ERalpha in the human fetal prostate and the earliest demonstration of ERbeta expression in the prostate at 7 weeks of gestation. ERbeta expression is intense during ductal morphogenesis, suggesting a role in normal glandular growth and proliferation. The induction of squamous metaplasia in the UGE, distal periurethral ducts and utricle is associated with ERalpha expression in these areas, while the induction of squamous metaplasia in peripheral prostatic acini is associated with peripheral stromal ERalpha expression. This study suggests estrogen signaling pathways in the human fetal prostate via ERalpha that involve epithelial-epithelial and epithelial-stromal interactions

There is increasing evidence that neuron death in neurodegenerative diseases, such as Parkinson's disease, is due to the activation of programmed cell death. However, the upstream mediators of cell death remain largely unknown. One approach to the identification of upstream mediators is to perform gene expression analysis in disease models. Such analyses, performed in tissue culture models induced by neurotoxins, have identified up-regulation of CHOP/GADD153, a transcription factor implicated in apoptosis due to endoplasmic reticulum stress or oxidative injury. To evaluate the disease-related significance of these findings, we have examined the expression of CHOP/GADD153 in neurotoxin models of parkinsonism in living animals. Nuclear expression of CHOP protein is observed in developmental and adult models of dopamine neuron death induced by intrastriatal injection of 6-hydroxydopamine (6OHDA) and in models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). CHOP is a mediator of neuron death in the adult 60HDA model because a null mutation results in a reduction in apoptosis. In the chronic MPTP model, however, while CHOP is robustly expressed, the null mutation does not protect from the loss of neurons. We conclude that the role of CHOP depends on the nature of the toxic stimulus. For 6OHDA, an oxidative metabolite of dopamine, it is a mediator of apoptotic death.

There is a paradoxical relationship between nicotine and stress. To help elucidate their relationship on catecholamine biosynthesis, rats were infused with nicotine for 7-14 days before exposure to cold or restraint stress. Nicotine (5 mg/kg/day, 14 days) did not alter basal plasma corticosterone or its elevation with 24 h cold stress, but prevented corticosterone elevation following 2 h restraint stress. In adrenal medulla (AM), response of dopamine beta-hydroxylase (DBH), but not tyrosine hydroxylase (TH) mRNA, to both stressors was attenuated in nicotine-infused rats. In locus coeruleus (LC), restraint stress elevated TH and DBH mRNA in saline-, but not in nicotine-infused rats. Cold stress triggered a similar response of TH and DBH mRNAs in LC with and without nicotine infusion. With shorter nicotine infusion (8 mg/kg/day, 7 days), TH mRNA in AM was not induced by restraint stress on one (1x) or two (2x) consecutive days nor was DBH mRNA in AM or LC by 2x. The findings demonstrate that constant release of nicotine can modulate, or even prevent, some stress responses at the level of the HPA axis and gene expression of catecholamine biosynthetic enzymes in LC and AM.

Plant secondary metabolites with estrogenic activity (phyto-estrogens) have been studied in the past as a potential alternative to classical hormone-replacement therapy (HRT) in menopausal women. No final verdict on the efficacy of soy or red clover based pharmaceutical preparations has been reached despite numerous clinical studies. We have studied the novel and most potent phyto-estrogen 8-prenylnaringenin (8-PN) in adult ovariectomized rats, an established animal model to mimic hormone dependent osteoporosis in menopausal women. Our results demonstrate that 8-PN can completely protect from ovariectomy induced bone-loss while exhibiting minimal, (dose independent) trophic effects on uterus and endometrium. It is estimated that at equivalent bone protective doses of 17beta-estradiol and 8-PN, the phyto-estrogen has a 10-fold lower stimulatory effect on uterus and endometrium. The bone tissue specific effect of 8-PN was confirmed in a transgenic reporter mouse model (ERE-Luc mice). Here we also found pronounced estrogenic activity in prostate. Present results add important aspects to the pharmacological profile of 8-PN and position this compound as an interesting alternative new candidate for treatment of peri- and postmenopausal symptoms.

Vascular complications in diabetes are a significant source of human morbidity and mortality, affecting multiple organ systems and persisting despite tight glucose control. Many of these complications can be linked to impairments in vasculogenesis, the process by which circulating and bone marrow-derived endothelial progenitor cells (EPCs) contribute to new vessel formation. Recent evidence suggests that hyperglycemia alone, through the mitochondrial overproduction of reactive oxygen species (ROS), can induce changes in gene expression and cellular behavior in diabetes. In this review, we examine how hyperglycemia-induced overproduction of ROS could explain EPC impairments observed in diabetes. Experimentally, impairments in EPC function prevent new blood vessel growth and are potentially reversible by manipulations to decrease ROS. Novel strategies aimed at reducing hyperglycemia-induced ROS may be a useful adjuvant to antihyperglycemic therapies in the restoration of vasculogenesis and the prevention of diabetic complications

Chronic wounds present a significant challenge, because there are few available treatment options for timely healing. Topical negative pressure devices have been used in a number of different types of wounds, including chronic wounds. They are believed to hasten wound healing by (1) maintaining a moist environment, (2) removing wound exudates, (3) increasing local blood flow, (4) increasing granulation tissue formation, (5) applying mechanical pressure to promote wound closure, and (6) reducing bacterial loads in the wound. Multiple nonrandomized, noncontrolled studies have reported that the use of these devices results in faster healing times and more successful closures. Five small randomized, controlled trials have also shown favorable outcomes with the use of topical negative pressure devices compared with conventional treatment. Adverse effects include discomfort, pain, and excessive tissue growth into the dressing. Complications are limited if the device is used properly. In light of the current treatment options, topical negative pressure devices may be considered useful as adjuvant therapy for chronic wounds; however, there is inadequate definitive evidence that wound healing is substantially better with these devices than with traditional therapy

PURPOSE: This study determines the analytic accuracy of a Luminex bead-based commercial analyte-specific reagent for the simultaneous analysis of 30 mutations prevalent in Ashkenazi Jews at eight genetic disease loci. METHODS: DNA from 20 samples with known abnormal genotypes were run a total of 109 times. DNA from 820 patients with unknown genotypes submitted for Ashkenazi Jewish testing panels were analyzed using our current laboratory techniques. The 820 samples were then stripped of identifiers, coded, and reanalyzed using the Tm Biosciences (Toronto, Canada) Ashkenazi Jewish panel analyte-specific reagent in a blinded fashion. For the controls, comparisons were made with their known genotypes. For the patient samples, the results of the Tm assay were compared with the results of our current assay. For 24 of the 30 mutations, we had genomic DNA controls or detected patients' samples heterozygous for these mutations. RESULTS: There were no discrepant results in the control or patient samples. In the patient samples, 19,680 genotyping reactions were performed without error in both our laboratory-developed single-disease assays and the Tm multiplex assay. Including the controls, 22,296 genotypes were determined without error. CONCLUSION: The Tm Biosciences Ashkenazi Jewish analyte-specific reagent is capable of performing accurate analyses of 24 different mutations in eight different genes in a single multiplex reaction and can be used with confidence in the clinical molecular genetics laboratory.