Faculty Bibliography

Seven carboxylic acid haptens of 8-prenylnaringenin (8-PN) were synthesized, coupled to cationized bovine serum albumin, and employed to raise specific antisera in rabbits. Two linkers of different lengths (C3H6COOH and C6H12COOH) were coupled to the C7-OH group and separated into their respective enantiomers yielding the first four haptens. Racemic derivatives with C4'-OH coupled linkers C5H10COOH and C9H18COOH were synthesized carrying a methylated C7-OH. Another racemic C4'-OH hapten (CH2COOH) was prepared starting from naringenin. The haptens elicited variable antibody titers dependent on linker lengths, with short linkers giving the best results. Three antisera were characterized in detail: anti-C7-carboxy-propyloxy-2S-(-)-8-PN (anti-H-11), anti-C7-carboxy-propyloxy-2R-(+)-8-PN (anti-H-10), and anti-C4'-carboxy-methoxy-rac-8-PN (anti-H-25). anti-H-10 and anti-H-11 showed about 9% enantiomeric cross-reactivity, and anti-H-11 did not discriminate between isoxanthohumol (IX) and 8-PN (84% cross-reactivity). For anti-H-10, cross-reactivities in the range of 2-5% were found for xanthohumol, IX, and 6-prenylnaringenin. Respective numbers for anti-H-25 were 0.02, 0.1, and 0.2%. Tritiated 8-PN was synthesized yielding a 3H-tracer of high specific radioactivity (2.22 GBq/mg). A radioimmunoassay using anti-H-25 and 3H-8-PN was established and used for the quantitative determination of 8-PN in various beer brands and in the urine of six men after the consumption of three different brands of beer. Furthermore, the dose-dependent excretion of 8-PN was tested after the consumption of a higher volume of a single beer brand with and without spiking with 8-PN and a small oral dose of authentic 8-PN, respectively. Conflicting results led to a pilot test on the in vivo conversion (demethylation) of IX into 8-PN in two men. Conversion rates of 1.9 and 4.4% were estimated. Thus, the total 8-PN dose in beer brands spiced with natural hop or hop products seems to be the sum of the 8-PN amount in a consumed volume and the amount arising from the conversion of IX.

The p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an alpha-secretase-mediated release of the extracellular domain followed by a gamma-secretase-mediated intramembrane cleavage. However, the specific determinants of p75(NTR) cleavage events are unknown. Many other substrates of gamma-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by gamma-secretase. Using a series of deletion mutations and chimeric receptors of p75(NTR) and the related Fas receptor, we have identified domains that are essential for p75(NTR) proteolysis. The initial alpha-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75(NTR), however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75(NTR). Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75(NTR) displaying only 15 extracellular amino acids in the stalk region

Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.

Terminal differentiation and mineralization are the final events in endochondral bone formation and allow the replacement of cartilage by bone. Retinoic acid (RA) stimulates these events, including upregulation of expression and activity of alkaline phosphatase (APase), expression of annexins II, V, and VI proteins, which bind to membranes and form Ca(2+) channels, expression of osteocalcin and runx2, another mineralization-related protein and terminal differentiation-related transcription factor, and ultimately mineralization. Chelating cytosolic Ca(2+) with BAPTA-AM, interfering with annexin Ca(2+) channel activities using K-201, a specific annexin Ca(2+) channel blocker, or suppression of annexin V expression using siRNA inhibited these events. Overexpression of annexin V in embryonic chicken growth plate chondrocytes resulted in an increase of cytoplasmic Ca(2+) concentration, [Ca(2+)](i) similar to [Ca(2+)](i) increase in RA-treated cultures. Overexpression of annexin V also resulted in upregulation of annexin II, annexin VI, osteocalcin, and runx2 gene expression, expression and activity of APase, and ultimately stimulation of mineralization. K-201 inhibited upregulation of osteocalcin and runx2 gene expression, APase expression and activity, and mineralization in annexin V-overexpressing growth plate chondrocytes. These findings indicate that annexins II, V, and VI alter Ca(2+) homeostasis in growth plate chondrocytes thereby regulating terminal differentiation and mineralization events. Overexpression of annexin V is sufficient to stimulate these terminal differentiation events in growth plate chondrocytes, whereas suppression of annexin V expression inhibits these events

The Hedgehog-GLI signaling pathway is important in animal development and tumorigenesis. Recent findings indicate that the growth and survival of human prostate cancer cells rely upon sustained signaling from the Hedgehog-GLI pathway. These findings have prompted a novel rational strategy for therapeutic treatment of prostate tumors, including metastatic tumors.

Until recently, stem cells were thought to be endowed with unlimited self-renewal capacity and, thus, assumed exempt from aging. But accumulating evidence over the past decade compellingly argues that a measurable and progressive replicative impairment in the hematopoietic, intestinal, and muscle stem cell activity exists from adulthood to old age, resulting in a decline in stem cell function and rendering stem cell aging as the possible link between cellular aging and organismal aging. By using a previously uncharacterized congenic animal model to study genetic regulation of hematopoietic stem cell aging, we have demonstrated definitively that a locus on murine chromosome 2 regulates hematopoietic stem cell aging. In addition to demonstrating that hematopoietic stem cell aging is regulated by a distinct genetic element, experimental evidence links the response of hematopoietic stem cells to DNA double-strand breaks to cellular aging, suggesting DNA integrity influences stem cell aging.

Mammalian telomeres end in single-stranded, G-rich 3' overhangs resulting from both the 'end-replication problem' (the inability of DNA polymerase to replicate the very end of the telomeres) and postreplication processing. Telomeric G-rich overhangs are precisely defined in ciliates; the length and the terminal nucleotides are fixed. Human telomeres have very long overhangs that are heterogeneous in size (35-600 nt), indicating that their processing must differ in some respects from model organisms. We developed telomere-end ligation protocols that allowed us to identify the terminal nucleotides of both the C-rich and the G-rich telomere strands. Up to approximately 80% of the C-rich strands terminate in CCAATC-5', suggesting that after replication a nuclease with high specificity or constrained action acts on the C strand. In contrast, the G-terminal nucleotide was less precise than Tetrahymena and Euplotes but still had a bias that changed as a function of telomerase expression

The currently approved therapies for Alzheimer's disease (AD) in the US are designed to modify the function of specific neurotransmitter systems in the brain. While these palliative treatments can benefit some patients for a period of time, they do not halt the relentless cognitive and behavioral deterioration that characterize this neurodegenerative disorder. Consequently, much current research on AD is directed toward illuminating the disease process itself, particularly the abnormal accumulation of certain proteins in brain: the amyloid-beta protein (Abeta) in senile plaques and cerebral blood vessels, and the tau protein in neurofibrillary tangles. Genetic, biochemical and pathologic evidence now favors a primary role of Abeta aggregation in the Alzheimer proteopathic cascade, and studies in mice indicate that lowering the amount of this protein in brain can be beneficial. Recently, Abeta-immunization therapy has emerged as a particularly promising therapeutic option for treating Alzheimer's disease, but unexpected treatment-related side-effects are an overriding issue. These adverse events were not anticipated from preclinical studies with rodents; hence, more biologically relevant models, such as nonhuman primates, are needed to test the safety and efficacy of novel therapies for Alzheimer's disease

Tissue function requires coordinated multicellular behavior as a consequence of diverse signals integrated through the tissue microenvironment; importantly, these cell-cell and cell-microenvironment interactions also actively suppress cancer. Ionizing radiation (IR) elicits a well-defined cellular response to DNA damage that mediates the fate of the individual cell, concomitantly with a less well-characterized overarching tissue stress response that coordinates the response of multiple cell types via microenvironment signaling. We have now shown that these programs to reestablish homeostasis intersect via mutual regulation by transforming growth factor beta1 (TGF beta 1), which acts as an extracellular sensor and signal of stress. In this review, the concept that this type of functional integration of cell and tissue stress response programs is essential to cancer suppression will be discussed. Our experiments using IR, and several recent studies that experimentally manipulate stromal TGF beta, show that disruption of microenvironment signaling actively promotes malignant progression. Understanding the dynamic interactions between tissue and cell stress responses will be necessary for an accurate assessment of cancer risk and may also provide targets for prevention

BACKGROUND: The impact of strict enforcement of Section 405 of the New York State Public Health Code to restrict resident work to eighty hours per week and the adoption of a similar policy by the Accreditation Council on Graduate Medical Education in 2002 for orthopaedic residency training have not been evaluated. Adoption of these rules has created accreditation as well as staffing problems and has generated controversy in the surgical training community. The purposes of this study were (1) to evaluate the attitudes of orthopaedic residents and attending surgeons toward the Code 405 work-hour regulations and the effect of those regulations on the perceived quality of residency training, quality of life, and patient care and (2) to quantify the effect of the work-hour restrictions on the actual number of hours worked. METHODS: We administered a thirty-four-question Likert-style questionnaire to forty-eight orthopaedic surgery residents (postgraduate years [PGY]-2 through 5) and a similar twenty-nine-question Likert-style questionnaire to thirty-nine orthopaedic attending surgeons. All questionnaires were collected anonymously and analyzed. Additionally, resident work hours before and after strict enforcement of the Code 405 regulations were obtained from resident time sheets. RESULTS: The average weekly work hours decreased from 89.25 to 74.25 hours for PGY-2 residents and from 86.5 to 73.25 hours for PGY-3 residents, and they increased from 61.5 to 68.5 hours for PGY-4 residents. Residents at all levels felt that they had increased time available for reading. There was general agreement between attending and resident surgeons that their operating experience had been negatively impacted. Senior residents thought that their education had been negatively affected, while junior residents thought that their operating experience in general had been negatively affected. Senior residents and attending surgeons felt that continuity of care had been negatively impacted. All agreed that quality of life for the residents had improved and that residents were more rested. CONCLUSIONS: On the basis of the survey data, the implementation of the new work-hour restrictions was found to result in a decrease in the number of hours worked per week for PGY-2 and PGY-3 residents and in an increase in work hours for PGY-4 residents. This could explain the definite difference between the attitudes expressed by the senior residents and those of the junior residents. Senior residents felt that their education was negatively impacted by the work rules, while junior residents expressed a more neutral view. However, senior residents did not believe that their operative experience was as negatively impacted as did junior residents. Although junior and senior residents and attending surgeons agreed that resident quality of life had improved, we were not able to determine whether this offset the perceived negative impact on education, continuity of care, and operative experience