Measurement of structural anisotropy in femoral trabecular bone using clinical-resolution CT images

Discrepancies in finite-element model predictions of bone strength may be attributed to the simplified modeling of bone as an isotropic structure due to the resolution limitations of clinical-level Computed Tomography (CT) data. The aim of this study is to calculate the preferential orientations of bone (the principal directions) and the extent to which bone is deposited more in one direction compared to another (degree of anisotropy). Using 100 femoral trabecular samples, the principal directions and degree of anisotropy were calculated with a Gradient Structure Tensor (GST) and a Sobel Structure Tensor (SST) using clinical-level CT. The results were compared against those calculated with the gold standard Mean-Intercept-Length (MIL) fabric tensor using micro-CT. There was no significant difference between the GST and SST in the calculation of the main principal direction (median error=28°), and the error was inversely correlated to the degree of transverse isotropy (r=−0.34, p<0.01). The degree of anisotropy measured using the structure tensors was weakly correlated with the MIL-based measurements (r=0.2, p<0.001). Combining the principal directions with the degree of anisotropy resulted in a significant increase in the correlation of the tensor distributions (r=0.79, p<0.001). Both structure tensors were robust against simulated noise, kernel sizes, and bone volume fraction. We recommend the use of the GST because of its computational efficiency and ease of implementation. This methodology has the promise to predict the structural anisotropy of bone in areas with a high degree of anisotropy, and may improve the in vivo characterization of bone.

Isolation and analysis of regulatory and structural mutations in the {\it recA\/} gene of {\it Escherichia coli}

The recA gene is essential for homologous recombination and for inducible DNA repair in Escherichia coli. The level of recA expression is important for these functions. The growth defect of a lambda phage carrying a recA-lacZ fusion was used to select mutations that reduced recA expression. Nine of these mutations were single base changes in the recA promoter; each reduced both induced and basal (repressed) levels of expression, indicating that only one promoter is used under both circumstances. Deletion analysis of the promoter region and S1 mapping of transcripts confirmed that there is only one promoter responsible for both basal and induced expression. Some of the mutants, however, displayed a ratio of induced to repressed expression that was much lower than wild-type. For one of these mutants (recA1270) LexA binding studies showed that this was not due to a change in the affinity of LexA repressor for the operator site. The extent of binding of RNA polymerase to this mutant promoter, however, was much reduced, and the complexes formed were qualitatively different. Further binding experiments provided some evidence that LexA does not block RNA polymerase binding to the recA promoter, but inhibits a later step in initiation. Behavior of the mutants with altered induction ratios could be explained if LexA binding to the operator actually increases RNA polymerase binding to the promoter in a closed complex compensating for defects in polymerase binding caused by the mutations.^ In a study of mutations in the recA structural gene, site-directed mutagenesis was used to replace cysteine codons at positions 90, 116, and 129 with a number of different codons. In vivo analysis of the replacements showed that none of the cysteines is absolutely essential and that they do not have a direct role as catalysts in ATP hydrolysis. Some amino acid substitutions abolished all RecA functions, while a few resulted in partial or altered function. Amino acids at positions 90 and 129 tended to affect all functions equally, while the amino acid at position 116 appeared to have a particular effect on the protease activity of the protein. ^

The functional and structural interactions between v-{\it mos\/} proteins and cell cycle control elements

The v-mos gene of Moloney murine sarcoma virus (Mo-MuSv) encodes a serine/threonine protein kinase capable of inducing cellular transformation. The c-mos protein is an important cell cycle regulator that functions during meiotic cell division cycles in germ cells. The overall function of c-mos in controlling meiosis is becoming better understood but the role of v-mos in malignant transformation of cells is largely unknown.^ In this study, v-mos protein was shown to be phosphorylated by M phase kinase in vitro and in vivo. The kinase activity and neoplastic transforming ability of v-mos is positively regulated by the phosphorylation. Together with the earlier finding of activation of M phase kinase by c-mos, these results raise the possibility of mutual regulation between M phase kinase and mos kinases.^ In addition to its functional interaction with the M phase kinase, the v-mos protein was shown to be present in the same protein complex with a cyclin-dependent kinase (cdk). In addition, an antibody that recognizes the cdk proteins was shown to co-precipitate the v-mos proteins in the interphase and mitotic cells transformed by p85$\sp{\rm gag-mos}$. Cdk proteins have been shown to be associated with nonmitotic cyclins which are potential oncogenes. The perturbation of cdk kinase or the activation of non-mitotic cyclins as oncogenes by v-mos could contribute directly to v-mos induced cellular transformation. v-mos proteins were also shown to interact with tubulin and vimentin, the essential components of microtubules and type IV intermediate filaments, respectively. The organizations of both microtubules and intermediate filaments are cell cycle-regulated. These results suggest that the v-mos kinase could be directly involved in inducing morphological changes typically seen in transformed cells.^ The interactions between the v-mos protein and these cell cycle control elements in regards to v-mos induced neoplastic transformation are discussed in detail in the text. ^

Mapping T-cell epitopes of the rubella virus structural proteins

Rubella virus (RV) typically causes a mild childhood illness, but complications can result from both viral and immune-mediated pathogenesis. RV can persist in the presence of neutralizing antibodies, suggesting that cell-mediated immune responses may be necessary for viral clearance. However, the molecular determinants recognized by RV-specific T-cells have not been identified. Using recombinant proteins which express the entire RV structural open reading frame in proliferation assays with lymphocytes of RV-immune individuals, domains which elicit major histocompatibility complex class II-restricted helper T-cells were identified. Synthetic peptides representing these domains were used to define specific epitopes. Two immunodominant domains were mapped to the capsid protein sequence C$\sb1$-C$\sb{29}$ and the E1 glycoprotein sequence E1$\sb{202}$-E1$\sb{283}.$ RV-specific MHC class I-restricted cytotoxic T lymphocytes (CTLs) were identified using a chromium-release assay with infected fibroblasts as target cells. An infectious Sindbis virus vector expressing each of the RV structural proteins identified the capsid, E2 and E1 proteins as targets of CTLs. Specific CTL epitopes were mapped within the previously identified immunodominant domains. This study identified domains of the RV structural proteins that may be beneficial for development of a synthetic vaccine, and provides normative data on RV-specific T-cell responses that should enhance our ability to understand RV persistence and associated complications. ^

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Structural equation modeling of the medical outcome study: Short Form 36 (SF-36)

The factorial validity of the SF-36 was evaluated using confirmatory factor analysis (CFA) methods, structural equation modeling (SEM), and multigroup structural equation modeling (MSEM). First, the measurement and structural model of the hypothesized SF-36 was explicated. Second, the model was tested for the validity of a second-order factorial structure, upon evidence of model misfit, determined the best-fitting model, and tested the validity of the best-fitting model on a second random sample from the same population. Third, the best-fitting model was tested for invariance of the factorial structure across race, age, and educational subgroups using MSEM.^ The findings support the second-order factorial structure of the SF-36 as proposed by Ware and Sherbourne (1992). However, the results suggest that: (a) Mental Health and Physical Health covary; (b) general mental health cross-loads onto Physical Health; (c) general health perception loads onto Mental Health instead of Physical Health; (d) many of the error terms are correlated; and (e) the physical function scale is not reliable across these two samples. This hierarchical factor pattern was replicated across both samples of health care workers, suggesting that the post hoc model fitting was not data specific. Subgroup analysis suggests that the physical function scale is not reliable across the "age" or "education" subgroups and that the general mental health scale path from Mental Health is not reliable across the "white/nonwhite" or "education" subgroups.^ The importance of this study is in the use of SEM and MSEM in evaluating sample data from the use of the SF-36. These methods are uniquely suited to the analysis of latent variable structures and are widely used in other fields. The use of latent variable models for self reported outcome measures has become widespread, and should now be applied to medical outcomes research. Invariance testing is superior to mean scores or summary scores when evaluating differences between groups. From a practical, as well as, psychometric perspective, it seems imperative that construct validity research related to the SF-36 establish whether this same hierarchical structure and invariance holds for other populations.^ This project is presented as three articles to be submitted for publication. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

How best to preserve and reveal the structural intricacies of cartilaginous tissue.

No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABA(A) receptors.

Biphenylic compounds related to the natural products magnolol and 4'-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA(A) receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5'-hexylbiphenyl-2,2'-diol (45) and the honokiol analogs 4'-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4'-methoxybiphenyl-2-ol (62) and 5-hexyl-4'-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA(A) receptor agonists.