Off-Axis Loading Fixture for Spine Biomechanics: Combined Compression and Bending.

The spine is a multi-tissue musculoskeletal system that supports large multi-axial loads and motions during physiological activities. The healthy and pathological biomechanical function of the spine and its subtissues are generally studied using cadaveric specimens that often require multi-axis biomechanical test systems to mimic the complex loading environment of the spine. Unfortunately, an off-the-shelf device can easily exceed 200,000 USD, while a custom device requires extensive time and experience in mechatronics. Our goal was to develop a cost-appropriate compression and bending (flexion-extension and lateral bending) spine testing system that requires little time and minimal technical knowledge. Our solution was an off-axis loading fixture (OLaF) that mounts to an existing uni-axial test frame and requires no additional actuators. OLaF requires little machining, with most components purchased off-the-shelf, and costs less than 10,000 USD. The only external transducer required is a six-axis load cell. Furthermore, OLaF is controlled using the existing uni-axial test frame's software, while the load data is collected using the software included with the six-axis load cell. Here we provide the design rationale for how OLaF develops primary motions and loads and minimizes off-axis secondary constraints, verify the primary kinematics using motion capture, and demonstrate that the system is capable of applying physiologically relevant, noninjurious, axial compression and bending. While OLaF is limited to compression and bending studies it produces repeatable physiologically relevant biomechanics, with high quality data, and minimal startup costs.

Ultrastructure of the spermatozoon of Helicometroides atlanticus (Digenea, Monorchiidae), an intestinal parasite of Parapristipoma octolineatum (Pisces, Teleostei) in Senegal.

The mature Helicometroides atlanticus spermatozoon exhibits an anterior electron-dense material, two axonemes of the 9+"1" pattern, a nucleus, two mitochondria, extramembranous ornamentations located at the level of the first mitochondrion and cortical microtubules arranged into two fields: a ventral field corresponding to the mitochondrial side and a dorsal field corresponding to the nuclear side. It lacks of spine-like body and a cytoplasmic expansion as in Monorchis parvus the only species of Monorchioidea which spermatozoon has been described until now. Nevertheless, it is distinguished from M. parvus by the presence of an anterior electron-dense material and two axonemes which appear one after the other.

The regulation of UDP-glucuronosyltransferase genes by tissue-specific and ligand-activated transcription factors.

Elucidation of the mechanisms regulating UGT genes is of prime importance if the adverse effects of interactions between drugs primarily eliminated by glucuronidation are to be minimized, and if UGT expression is to be manipulated for therapeutic effect. The factors controlling UGT gene expression in the liver include the liver-enriched transcription factors, HNF-1alpha and HNF-4alpha, several members of the nuclear-receptor family (CAR, PXR, FXR, LXR, and PPAR), the arylhydrocarbon receptor, and transcription factors involved in stress responses (Nrf2, Maf). HNF-1alpha, in concert with the intestine-specific transcription factor, Cdx2, and Sp1 regulate UGT gene expression in the gastrointestinal tract, whereas the genes for the major androgen-glucuronidating enzymes, UGT2B15 and UGT2B17, are upregulated by estrogens in breast cell lines and downregulated by androgens in prostate-derived cells. Despite this knowledge, the complex interactions between these transcription factors and their coregulators has not been determined, and the mechanisms regulating UGT gene expression in organs and tissues, other than the liver, gastrointestinal tract, breast, and prostate, remain to be elucidated.

Hepatocyte nuclear factor1 transcription factors are essential for the UDP-glucuronosyltransferase 1A9 promoter response to hepatocyte nuclear factor 4alpha.

In humans, UDP-glucuronosyltransferase 1A9 is known to glucuronidate numerous lipophilic substances of pharmacological and toxicological importance. Although it has been established that individuals vary in their capacity to express this detoxification enzyme, little is known about the mechanisms that dictate the regulation of UGT1A9. In particular, it is not understood why, while the proximal regulatory regions of the UGT1A7-10 gene cluster are highly similar, UGT1A9 is the sole hepatic isoform of the four. Recent data have suggested that the human UGT1A9 promoter is controlled by hepatocyte nuclear factor 4alpha. In this work, we confirm that the human UGT1A9 promoter can indeed be upregulated by human hepatocyte nuclear factor 4alpha in vitro. Our results, however, show that the previously-reported hepatocyte nuclear factor 4alpha-binding site only plays a minor role in this response. Instead, upregulation was found to require a more proximal response element, which was not preserved in the UGT1A7, UGT1A8 or UGT1A10 promoters. Furthermore, hepatocyte nuclear factor 4alpha-mediated transcription from the human UGT1A9 promoter was discovered to be entirely dependent on hepatocyte nuclear factor 1. We have established that two hepatocyte nuclear factor 1-binding elements are involved in this phenomenon, the more distal of which is unique to the UGT1A9 promoter. Interestingly, this second site had no significant role in hepatocyte nuclear factor 1alpha-mediated induction of the UGT1A9 promoter in vitro, yet was critical for upregulation by human hepatocyte nuclear factor 4alpha. The discovery of two unique and cooperative liver-enriched transcription factor binding sites in the UGT1A9 promoter is a significant step towards understanding the unique hepatic expression of UGT1A9 amongst the UGT1A7-10 gene cluster.

Isolation of the UDP-glucuronosyltransferase 1A3 and 1A4 proximal promoters and characterization of their dependence on the transcription factor hepatocyte nuclear factor 1alpha.

The UGT1A3-1A5 genes are a highly related UDP-glucuronosyltransferase (UGT) cluster exhibiting high levels of coding and regulatory region homology. However, the ensuing proteins have both differing substrate specificities and differing expression patterns. The expression profile of each enzyme also varies considerably from one individual to the next. Differences in UGT expression have been predicted to contribute to an individual's response to pharmaceuticals and to predisposition toward cancer in the event of carcinogen exposure. Therefore, it is desirable to elucidate the mechanisms that drive the transcription of UGT genes and identify the factors responsible for their variable expression. To this end, we have isolated the UGT1A3, UGT1A4, and UGT1A5 proximal promoters and begun to investigate the regulatory elements necessary for activity in vitro. We have established that the nucleotide sequence upstream of the UGT1A5 exon 1 is an ineffective promoter, correlating with the lack of substantial expression of this UGT in human tissues. In contrast, the UGT1A3 and UGT1A4 proximal promoters are both highly active in hepatic and colonic cell lines, with maximal activity being encoded by the proximal 500 base pairs. However, the UGT1A3 and UGT1A4 promoters exhibit low activity in the human embryonic kidney cell line HEK293, unless coexpressed with hepatocyte nuclear factor (HNF) 1alpha. Furthermore, mutation of the consensus-like HNF1-binding site in the UGT1A3 promoter abolishes promoter function in all cell types. This study suggests an important role for HNF1alpha in the transcriptional regulation of the human UGT1A3 and UGT1A4 genes.

The caudal-related homeodomain protein Cdx2 and hepatocyte nuclear factor 1alpha cooperatively regulate the UDP-glucuronosyltransferase 2B7 gene promoter.

The gastrointestinal tract, contains several UDP-glucuronosyltransferases (UGTs) of the UGT1A and UGT2B subfamilies. UGT2B7 is one particular enzyme expressed throughout the gastrointestinal tract that possesses broad substrate specificity towards orally administered drugs. Because the caudal-related homeodomain protein 2 (Cdx2) regulates many gastrointestinal properties, we sought to determine whether it could regulate the UGT2B7 promoter in the colon-derived cell line Caco-2. Levels of Cdx2 and UGT2B7 were measured in differentiated and non-differentiated Caco-2 cells by the quantitative polymerase chain reaction. The capacity of the UGT2B7 gene promoter to drive expression of the luciferase reporter gene was assessed by transfection into Caco-2 cells, with transcription factor expression plasmids. Mutation of putative transcription factor binding sites and electrophoretic mobility shift assays were used to define important regulatory regions of the UGT2B7 gene promoter. The levels of Cdx2 and UGT2B7 mRNAs were co-ordinately increased in differentiated Caco2 cells compared to non-differentiated cells. Cdx2 activates the UGT2B7 proximal promoter by binding to two adjacent sites. Promoter activation requires Cdx2 binding to both sites wherein these proteins interact to form a putative functional dimer. Dimerization was shown to be dependent on redox state using extracts depleted of dithiothreitol. In addition, Cdx2 was shown to cooperatively activate the UGT2B7 promoter in conjunction with hepatocyte nuclear factor 1alpha (HNF1alpha), a mechanism previously observed to regulate other intestine-specific genes. The present study is the first to define transcription factors involved in the control of intestinal UGT2B expression. The demonstration that Cdx2 and HNF1alpha are important regulators of UGT2B7 expression will aid in defining pathways for coordinate control of drug metabolism in the gastrointestinal tract.

Identification and characterization of functional hepatocyte nuclear factor 1-binding sites in UDP-glucuronosyltransferase genes.

The hepatocyte nuclear factor 1 (HNF1) transcription factor family is composed of two closely related homeodomain proteins with similar but distinct expression profiles. Homodimers and heterodimers of these transcription factors, HNF1alpha and HNF1beta, increase transcription from target genes through direct physical interaction with one or more elements of sufficient similarity to a 13 nucleotide-inverted dyad consensus-binding sequence. Potential HNF1-binding sites have been found in the proximal upstream regulatory regions of most known human UDP-glucuronosyltransferase (UGT) genes. As the liver and gastrointestinal tract are both important sites of glucuronidation and express significant levels of one or both HNF1 proteins, it is thought that these homeoproteins may play a role in transcriptional regulation of UGTs. This chapter explores the current evidence that HNF1 transcription factors are explicitly involved in the transcription of mammalian UGT genes. Most data supporting this hypothesis come from in vitro reporter assays, site-directed mutagenesis, and electrophoretic mobility-shift assays, for which methods are detailed. However, as in vitro functionality of transcription factors does not necessarily imply significance in vivo, some of the limitations of these techniques are also examined. In addition, available in vivo data are discussed, with particular attention given to contributions made by HNF1alpha knockout mouse models and microarray studies of human tissue. Finally, possible scenarios in which HNF1-mediated regulation of UGT expression may be clinically relevant are suggested.

Regulation of UDP glucuronosyltransferases in the gastrointestinal tract.

The UDP glucuronosyltransferases (UGT) of the gastrointestinal (GI) tract have a crucial role in protection against the toxic effects of lipophilic chemicals in the environment. UGTs such as UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in gastrointestinal tissues, each with a unique tissue distribution pattern that is subject to interindividual variation. The factors regulating this tissue-specific expression and that contribute to variability are beginning to be elucidated. Studies on the UGT1A7, 1A8, 1A9, and 1A10 gene promoters in Caco-2 cells, an in vitro model of enterocytes of the gastrointestinal tract, have identified the caudal homeodomain transcription factor, Cdx2, as an important regulator of the UGT1A8 and 1A10 gene proximal promoters. This transcription factor is found exclusively in the small intestine and colon: it is absent in the gastric epithelium and the esophagus. Cdx2 regulates the UGT1A8 and 1A10 promoters in cooperation with hepatocyte nuclear factor 1alpha (HNF1alpha). It is noteworthy that UGT1A7 is not expressed in gastrointestinal tissue distal to the gastric mucosa and does not contain a Cdx2 binding site in its proximal promoter. Transcription factors, including Sp1, which differentially bind to the initiator regions of the UGT1A8, 1A9, and 1A10 promoters, also contribute to the differences in expression of these UGTs in Caco-2 cells. The identification of important regulatory regions of UGT genes expressed in the gastrointestinal tract, and the transcription factors that bind to these regions, will aid in the elucidation of factors that contribute to interindividual differences in gastrointestinal UGT expression. In turn, this will lead to further understanding of interindividual variation in the capacity of the GI tract to metabolize lipophilic chemicals and to act as a barrier to dietary toxins and orally administered drugs.

Coordinate regulation of the human UDP-glucuronosyltransferase 1A8, 1A9, and 1A10 genes by hepatocyte nuclear factor 1alpha and the caudal-related homeodomain protein 2.

The human UDP-glucuronosyltransferases (UGT) -1A8 and -1A10 are exclusively expressed in extrahepatic tissues and primarily in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. We have demonstrated previously that the UGT1A8 and UGT1A10 genes, in contrast to the UGT1A9 gene, are regulated via an initiator-like element in their proximal promoters. To determine the elements that contribute to the gastrointestinal expression of UGT1A8 and -1A10, we conducted deletion analysis of the UGT1A8, -1A9, and -1A10 promoters in the colon-derived cell line Caco2. DNA elements contributing significantly to UGT1A8, -1A9, and -1A10 promoter activity were found to reside primarily within 140 base pairs of the transcription start site. Within this region, putative binding sites for the intestine-specific transcription factor, caudal-related homeodomain protein 2 (Cdx2), and hepatocyte nuclear factor 1 (HNF1) were identified. Using gel shift and functional assays, HNF1alpha was demonstrated to bind to and activate the UGT1A8, -1A9, and -1A10 promoters. In contrast, Cdx2 bound to and activated the UGT1A8 and -1A10 promoters but could not activate the UGT1A9 promoter. A single base pair difference between the UGT1A8 and -1A10 promoters, three base pairs downstream of the consensus Cdx2 site, contributed to the observed difference in Cdx2 binding and Cdx2-mediated promoter activation of these two promoters. In addition, Cdx2 was shown to cooperate with HNF1alpha to synergistically activate the UGT1A8, -1A9, and -1A10 promoters. These studies provide insight into the mechanisms controlling the extrahepatic expression of the UGT1A8, -1A9, and -1A10 genes.

Cloning and characterization of the human UDP-glucuronosyltransferase 1A8, 1A9, and 1A10 gene promoters: differential regulation through an interior-like region.

The human UDP-glucuronosyltransferases, UGT1A8, 1A9, and 1A10, are closely related in sequence and have a major role in the elimination of lipophilic chemicals by glucuronidation. UGT1A8 and 1A10 are expressed exclusively in the gastrointestinal tract, whereas UGT1A9 is expressed mainly in the liver and kidneys. To determine the factors contributing to the extrahepatic expression of these UDP-glucuronosyltransferases, we have cloned and characterized the promoters of the UGT1A8, 1A9, and 1A10 genes and studied their regulation in the colon cell line, Caco2. Their transcription start sites were mapped, and a functional overlapping Sp1/initiator-like site was identified which strongly contributed to UGT1A8 and 1A10 promoter activity. The high promoter activity of UGT1A8 and 1A10 correlated with the binding of nuclear proteins (complex B) to this region. Two-bp differences in the corresponding site in the UGT1A9 promoter prevented the binding of complex B and reduced promoter activity. Although Sp1 was able to bind to the Sp1/initiator-like site, its binding was dispensable for promoter activity. However, the binding of Sp1 to a second Sp1 site 30 bp 5' to the Sp1/initiator-like site greatly enhanced the activity of the UGT1A8 and 1A10 promoters. These results provide evidence that the UGT1A8, 1A9, and 1A10 genes are differentially regulated through an initiator element in their 5'-flanking regions.