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Animal models of osteoarthritis in an era of molecular biology.

Animal models of osteoarthritis (OA) are used to study the pathogenesis of cartilage degeneration and to evaluate potential anti-arthritic drugs for clinical use. In general, these models fall into 2 categories, spontaneous and induced (surgical instability or genetic manipulation). Animal models of naturally occurring OA occur in knee joints of guinea pigs, mice and Syrian hamsters. Commonly utilized surgical instability models include medial meniscal tear in guinea pigs and rats, medial or lateral partial meniscectomy in rabbits, medial partial or total meniscectomy or anterior cruciate transection in dogs. Transgenic models have been developed in mice. These models all have potential use in the study of molecular mechanisms associated with OA development via use of immunohistochemistry, biochemistry and molecular probes to identify altered matrix molecules at different stages in disease progression. Testing of specific types of inhibitors developed through evaluation of matrix changes in the disease process will ultimately help identify key processes which initiate and perpetuate the disease and will lead to discovery of new disease modifying pharmaceutical agents for OA patients. This paper will focus on the discussion of several models which are likely to be useful in the molecular dissection of processes involved in cartilage degeneration.

Journal Article↗

Structural basis of the gp120 superantigen-binding site on human immunoglobulins.

B cell superantigens (SAg) interact with normal human nonimmune Igs (Igs), independently of the light chain isotype, and activate a large proportion of the B cell repertoire. Recently, the major envelope protein of HIV-1, gp120, was found to exhibit SAg-like properties for B cells with potential pathologic consequences for the infected host. This unconventional mode of interaction contrasts with its binding to immunization-induced Abs, which requires the tertiary structure of the heavy and light chain variable regions. In this report, we have examined the structural basis of the interaction between human Igs and gp120. We found that gp120 binding is restricted to Igs from the V(H)3 gene family and that the two V(H) genes 3-23 and 3-30, known to be overutilized during all stages of B cell development, frequently impart gp120 binding. We also provide evidence that the viral gp120 SAg can interact with only a subset of the human V(H)3+ Igs that can convey binding to the prototypic bacterial B cell SAg protein A from Staphylococcus aureus. Finally, we have identified amino acid positions present primarily in the first and third framework regions of the Ig heavy chain variable region, outside the conventional hypervariable loops, which correlate with gp120 binding. In a three-dimensional sequence-homology model, these residues partially overlap with the predicted SAg protein A binding site for V(H)3+ Igs.

Amino Acid Sequence↗

Male Chinese drinking behavior in Los Angeles.

The purpose of this research was to identify the characteristics that distinguish male Chinese drinkers from abstainers and to clarify differences between Chinese men who drink limited amounts of alcohol and those who drink more heavily. A random sample of 218 adult Chinese men was interviewed using a schedule based on a national study of drinking behavior. The stereotype of Chinese as limited drinkers was partially supported by the data. Approximately 22% of the sample were abstainers whereas only 14% were heavy drinkers. Light drinkers were the modal category. A logistic regression model was used to evaluate the relative importance of the significant characteristics of drinkers and abstainers. Parents' drinking behavior influenced respondent's drinking behavior, and drinkers tended to like to go to parties. Chinese men with higher education were more apt to be drinkers. A second logistic regression model was used to identify the predictive variables related to the level of drinking. Chinese men who go to bars and who have friends who drink were the most likely to be heavy drinkers.

Adult↗

Simulation studies of high-field EPR spectra of spin-labeled lipids in membranes.

The high-field (i.e., 94 GHz) membrane EPR spectra of lipids spin labeled in their fatty acid chains have been simulated by using two limiting motional models. The aim was to identify the dynamic origin of the residual (g(xx) - g(yy)) anisotropy observed in the nonaxial EPR spectra of cholesterol-containing membranes. It is concluded that the residual spectral anisotropy arises from in-plane ordering of the lipid chains by cholesterol. The partial averaging of the (g(xx) - g(yy)) anisotropy was best described by restricted axial rotation with a frequency in the region of tau(-1)(R||) approximately 0.5-1 x 10(9) s(-1). Simulations for slower axial rotation of unrestricted amplitude produced less satisfactory fits. In phospholipid membranes not containing cholesterol, the nonaxial anisotropy is completely averaged in the fluid phase and substantially reduced even in the gel phase. The unrestricted axial rotation in the gel phase is of comparable frequency to that of the limited axial rotation in the liquid-ordered phase of membranes containing cholesterol. These results on in-plane ordering by cholesterol in the liquid-ordered phase could be significant for current proposals regarding domain formation in cellular membranes.

Algorithms↗

Reversed-phase liquid chromatography column testing: robustness study of the test.

Choosing the right RPLC column for an actual separation among the more than 600 commercially available ones still represents a real challenge for the analyst particularly when basic solutes are involved. Many tests dedicated to the characterization and the classification of stationary phases have been proposed in the literature and some of them highlighted the need of a better understanding of retention properties to lead to a rational choice of columns. However, unlike classical chromatographic methods, the problem of their robustness evaluation has often been left unaddressed. In the present study, we present a robustness study that was applied to the chromatographic testing procedure we had developed and optimized previously. A design of experiment (DoE) approach was implemented. Four factors, previously identified as potentially influent, were selected and subjected to small controlled variations: solvent fraction, temperature, pH and buffer concentration. As our model comprised quadratic terms instead of a simple linear model, we chose a D-optimal design in order to minimize the experiment number. As a previous batch-to-batch study [K. Le Mapihan, Caractérisation et classification des phases stationnaires utilisées pour l'analyse CPL de produits pharmaceutiques, Ph.D. Thesis, Pierre and Marie Curie University, 2004] had shown a low variability on the selected stationary phase, it was then possible to split the design into two parts, according to the solvent nature, each using one column. Actually, our testing procedure involving assays both with methanol and with acetonitrile as organic modifier, such an approach enabled to avoid a possible bias due to the column ageing considering the number of experiments required (16 + 6 center points). Experimental results were computed thanks to a Partial Least Squares regression procedure, more adapted than the classical regression to handle factors and responses not completely independent. The results showed the behavior of the solutes in relation to their physico-chemical properties and the relevance of the second term degree of our model. Finally, the robust domain of the test has been fairly identified, so that any potential user precisely knows to which extend each experimental parameter must be controlled when our testing procedure is to be implemented.

Chromatography, Liquid↗

Automated 3-D extraction of inner and outer surfaces of cerebral cortex from MRI.

Automatic computer processing of large multidimensional images such as those produced by magnetic resonance imaging (MRI) is greatly aided by deformable models, which are used to extract, identify, and quantify specific neuroanatomic structures. A general method of deforming polyhedra is presented here, with two novel features. First, explicit prevention of self-intersecting surface geometries is provided, unlike conventional deformable models, which use regularization constraints to discourage but not necessarily prevent such behavior. Second, deformation of multiple surfaces with intersurface proximity constraints allows each surface to help guide other surfaces into place using model-based constraints such as expected thickness of an anatomic surface. These two features are used advantageously to identify automatically the total surface of the outer and inner boundaries of cerebral cortical gray matter from normal human MR images, accurately locating the depths of the sulci, even where noise and partial volume artifacts in the image obscure the visibility of sulci. The extracted surfaces are enforced to be simple two-dimensional manifolds (having the topology of a sphere), even though the data may have topological holes. This automatic 3-D cortex segmentation technique has been applied to 150 normal subjects, simultaneously extracting both the gray/white and gray/cerebrospinal fluid interface from each individual. The collection of surfaces has been used to create a spatial map of the mean and standard deviation for the location and the thickness of cortical gray matter. Three alternative criteria for defining cortical thickness at each cortical location were developed and compared. These results are shown to corroborate published postmortem and in vivo measurements of cortical thickness.

Algorithms↗

Polypeptide components of oligomeric legumin-like thiamin-binding protein from buckwheat seeds characterized by partial amino acid sequencing and photoaffinity labeling.

Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for "basic subunits" of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.

Binding Sites↗

A partial catalog of proteins secreted by epidermal keratinocytes in culture.

Proteins secreted by epidermal keratinocytes are known to engage in functions other than those directly associated with barrier formation. We have used a previously published culture model to collect proteins secreted by adult human epidermal keratinocytes. Electrophoresis and microsequencing allowed us to identify 20 proteins. The list of proteins includes those known to be produced by keratinocytes (beta-2 microglobulin, betaIG-H3, calgranulin A, cathepsin B and D, E-cadherin, gelatinase B, gelsolin, interstitial collagenase, laminin B2t, plasminogen activator inhibitor-1, protein 14-3-3epsilon, SCC antigen, stratifin, and translationally controlled tumor protein) as well as those not previously known to be secreted by keratinocytes (epididymis secretory protein, maspin, and anti-neoplastic urinary protein). In addition, two proteins were identified that are not known to be secreted (glutathione-S-transferase and heat shock protein 27/28 kDa). The varied nature of the proteins identified suggests that epidermal keratinocytes have physiologic functions that have yet to be identified.

3T3 Cells↗

Kernel Cox regression models for linking gene expression profiles to censored survival data.

In functional genomics, one important problem is to relate the microarray gene expression profiles to various clinical phenotypes from patients. The success has been demonstrated in molecular classification of cancer in which gene expression data serve as predictors and different types of cancer are the binary or multi-categorical outcome variable. However, there has been less research in linking gene expression profiles to other types of phenotypes, in particular, the censored survival data such as patients' overall survival or cancer relapse times. In the paper, we develop a kernel Cox regression model for relating gene expression profiles to censored phenotypes in the framework the penalization method in terms of function estimation in reproducing kernel Hilbert spaces. To circumvent the problem of censoring, we use the negative partial likelihood as a loss function in the estimation procedure. The functional combinations of the original gene expression data identified by the method are highly correlated with the patients' survival times and at the same time account for the variability in the gene expression levels. We apply our method to data sets from diffuse large B-cell lymphoma, lung adenocarcinoma and breast carcinoma studies to verify its effectiveness. The results from these analyses indicate that the proposed method works very well in identifying subgroups of patients with different risks of death or relapse and in predicting the risk of relapse or death based on the gene expression profiles measured from the tumor samples taken from the patients.

Artificial Intelligence↗

Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice.

Nerve-evoked contractions were studied in vitro in phrenic nerve-hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE-/-) mice and their wild-type littermates (AChE+/+). The AChE-/- mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE-/- mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals. In AChE-/- preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW 284C51). The ability of diaphragm muscles from AChE-/- mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms--including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region--were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors.

Acetylcholine↗

"To enrol or not to enrol?": A qualitative investigation of demand for health insurance in rural West Africa.

In spite of the fact that feeble levels of participation have long been identified as a major constraint to the successful long-term implementation of community-based health insurance (CBI) in low-income countries, evidence on determinants of enrolment in CBI is still lacking. The application of econometric modelling has provided a partial answer to the question, but on its own it has proved to be insufficient to guide policy making. This paper aims to fill this gap in knowledge using qualitative research methods. In-depth interviews with 32 household heads were conducted in the Nouna Health District, Burkina Faso, West Africa to assess determinants of enrolment in a newly established CBI scheme. The findings highlight that factors previously neglected in the literature, such as institutional rigidities and socio-cultural practices, play an important role in shaping the decision to enrol. The discussion of the findings focuses on the policy implications, offering concrete recommendations to maximise enrolment, within and beyond Burkina Faso.

Adult↗

Topomimetics of amphipathic beta-sheet and helix-forming bactericidal peptides neutralize lipopolysaccharide endotoxins.

Release of lipopolysaccharide (LPS) endotoxin from Gram negative bacterial membranes triggers macrophages to produce large quantities of cytokines that can lead to septic shock and eventual death. Agents that bind to and neutralize LPS may provide a means to clinically prevent septic shock upon bacterial infection. Previously, we reported the design of antibacterial helix peptide SC4 and beta-sheet-forming betapep peptides that neutralize LPS in vitro. We hypothesized that the ability of these and other such peptides to neutralize LPS rested in the common denominator of positively charged amphipathic structure. Here, we describe the design and synthesis of nonpeptide, calixarene-based helix/sheet topomimetics that mimic the folded conformations of these peptides in their molecular dimensions, amphipathic surface topology, and compositional properties. From a small library of topomimetics, we identified several compounds that neutralize LPS in the 10-8 M range, making them as effective as bactericidal/permeability increasing protein and polymyxin B. In an endotoxemia mouse model, three of the most in vitro effective topomimetics are shown to be at least partially protective against challenges of LPS from different bacterial species. NMR studies provide mechanistic insight by suggesting the site of molecular interaction between topomimetics and the lipid A component of LPS, with binding being mediated by electrostatic and hydrophobic interactions. This research contributes to the development of pharmaceutical agents against endotoxemia and septic shock.

Animals↗

Sensitivity of Caenorhabditis elegans clk-1 mutants to ubiquinone side-chain length reveals multiple ubiquinone-dependent processes.

Ubiquinone (coenzyme Q, or Q) is a membrane constituent, whose head group is capable of accepting and donating electrons and whose lipidic side chain is composed of a variable number of isoprene subunits. A possible role for Q as a dietary antioxidant for treating conditions that involve altered cellular redox states is being intensely studied. Mutations in the clk-1 gene of the nematode Caenorhabditis elegans affect numerous physiological rates including behavioral rates, developmental rates, reproduction, and life span. clk-1 encodes a protein associated with the inner mitochondrial membrane that is necessary for Q biosynthesis in C. elegans. clk-1 mutants do not synthesize Q but accumulate demethoxyubiquinone, a Q synthesis intermediate that is able to partially sustain mitochondrial respiration in worms as well as in mammals. Recently, we and others have found that exogenous Q is necessary for the fertility and development of clk-1 mutants. Here, we take advantage of the clk-1 genetic model to identify structural features of Q that are functionally important in vivo. We show that clk-1 mutants are exquisitely sensitive to the length of the side chain of the Q they consume. We also identified differential sensitivity to Q side-chain length between null alleles of clk-1 (qm30 and qm51) and the weaker allele e2519. This allows us to propose a model where we distinguish several types of Q-dependent processes in vivo: processes that are very sensitive to Q side-chain length and processes that are permissive to Q with shorter chains.

Alleles↗

A Drosophila clathrin light-chain gene: sequence, mapping, and absence of neuronal specialization.

Because mammalian light chains have been implicated in the regulation of clathrin coat assembly and neuronal specialization of clathrin-mediated vesicle trafficking, a clathrin light-chain gene of Drosophila has been sought as a genetically tractable model for these developmental membrane-trafficking systems. A light-chain gene has been identified and its expression examined in various developmental stages and tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). A cDNA clone, originally identified from a partial sequence in the Berkeley Drosophila Genome Project EST database, has been sequenced completely and shown to encode a polypeptide with extensive sequence similarity to vertebrate and invertebrate clathrin light chains. Secondary structure algorithms predict an extensive coiled-coil over a region extending from amino acid residues 100 to 170, in excellent agreement with previous analyses of mammalian light chains. By in situ hybridization to larval polytene chromosomes, the gene has been mapped to cytologic position 77A on the left arm of chromosome 3. An RT-PCR analysis, coupled with PCR analysis of genomic DNA, showed that there is no neural specialization of the Drosophila clathrin light chain corresponding to that observed in mammalian neuronal light chains. The neuron-specific alternative splicing of clathrin light chains thus appears to be restricted to vertebrates, where it may contribute to the more complex information-processing capacity of higher nervous systems.

Alternative Splicing↗

On the equivalence of mathematical models for cell proliferation kinetics.

Various types of mathematical models, such as partial differential equations, ordinary differential equations and difference equations, are available in the literature to describe the kinetics of cell proliferation, and different studies of cell kinetic phenomena have been conducted using these models. This paper discusses the equivalence between the different models identifying the conditions and approximations under which one type of models may be derived from another. Such an equivalence study is highly useful for an integration of the diverse results that have been obtained using different models in order to gain a more complete understanding of cell kinetic phenomena.

Animals↗

Transcriptional changes underlying the secretory activation phase of mammary gland development.

The secretory activation stage of mammary gland development occurs after parturition and converts inactive lobuloalveoli to active milk secretion. This process is triggered by progestin withdrawal and depends upon augmented prolactin (Prl) signaling. Little is known about the Prl-induced transcriptional changes that occur in the mammary gland to drive this process. To examine changes in the mammary transcriptome responsible for secretory activation, we have used transcript profiling of three mouse models that exhibit failure of secretory activation: knockout of galanin (a regulator of pituitary Prl production and a mammary cell autonomous modulator of Prl action); treatment with S179D Prl (a phosphoprolactin mimic); and knockout of a single Prl receptor allele. A significant reduction in expression was observed in genes belonging to 46 gene ontologies including those representing milk proteins, metabolism, lipid, cholesterol and fatty acid biosynthetic enzymes, immune response, and key transcription factors. A set of 35 genes, commonly regulated in all three models, was identified and their role in lactogenesis was validated by examining their expression in response to Prl stimulation or signal transducer and activator of transcription 5 knockdown in the HC11 mouse mammary cell culture model. The transcript profiles provided by these experiments identify 35 key genes (many for the first time) involved in the secretory activation phase of mammary gland development, show that S179D acts as an antagonist of Prl action, and provide insight into the partial penetrance of failed lactation in Prl receptor heterozygous females.

Alleles↗

A probability-based multivariate statistical algorithm for autofluorescence spectroscopic identification of oral carcinogenesis.

A probability-based multivariate statistical algorithm combining partial least-squares (PLS) and logistic regression was developed to identify the development stages of oral cancer through analysis of autofluorescence spectra of oral tissues. Tissues were taken from a 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis model. Analyses were conducted at various excitation wavelengths, ranging from 280 nm to 400 nm in 20 nm increments, to assess classification performance at different excitations. For each excitation the PLS analysis and logistic regression were combined, on the basis of cross validation, to calculate the posterior probabilities of samples belonging to four stages of cancer development: normal tissues, hyperplasia, dysplasia and early cancers and frankly invasive cancers. Results showed that the 320 nm excitation wavelength optimally classified the cancer development stages: the accuracy rates for identifying samples at that excitation were 91.7%, 83.3%, 66.7% and 83.3% for the four respective stages. The average accuracy rate was 81.3%. These results suggest that the algorithm described in this study might be useful for the detection of human oral cancers.

9,10-Dimethyl-1,2-benzanthracene↗

[Characteristics of protein metabolism in labyrinthine tissue].

The synthesis of total protein in organic culture of the internal ear was studied in 16-day embryo of CBA mice exposed to altering factors. The experiments showed feasibility of partial recovery for impaired metabolic processes in the labyrinth following phonophoretic introduction of mitochondrial coenzymes and inhibitors of lysosomal activity. Formation of systemic structural trace by modelling of acoustic stress and verification of protein stress agents was tested making it possible to identify an important component in dysadaption mechanism in mature CBA mice labyrinth.

Animals↗