Normal modes of vibration in bovine pancreatic trypsin inhibitor and its mechanical property

The normal mode analysis of conformational fluctuation is carried out for a small globular protein, bovine pancreatic trypsin inhibitor. Results are analyzed mainly to reveal the mechanical construction of the protein molecule. We take dihedral angles, including peptide omega angles, as independent variables for the normal mode analysis. There are 306 such angles in this molecule. Motions in modes with frequencies lower than 120 cm-1 are shown to involve atoms in the whole protein molecule, and spatial change of displacement vectors is continuous, i.e., those of atoms near in space are similar. To quantitate the observation of the continuity, a correlation function of direction vectors of atomic displacements is calculated. From this function we define a quantity that is interpreted as the wave length of an equivalent elastic plane wave. From this quantity we deduce effective Young's modulus for each mode. For the mode with the lowest frequency 4.4 cm-1, it turned out to be 0.8 x 10(9) dyn cm-2, the value two orders of magnitude softer than, for instance, alpha-helices. Prompted by this observation, the four lowest frequency modes and also the harmonic motions in the thermal equilibrium are analyzed further mainly to detect relatively rigid structural elements in the molecule. From this analysis emerges a mechanical picture of the protein molecule that is made up of relatively rigid elements held together by very soft parts.     

The role of the epidermis as a stiffening agent in Tulipa (Liliaceae) stems

We investigated the hypothesis that the epidermis is a tension-stressed "skin' whose contribution to stem stiffness depends on the turgor pressure exerted on it by an hydrostatically inflated inner "core' of tissues. This hypothesis was tested by relying on the intensities of bending stresses due to stem flexure, which must reach their maximum levels at the outer surface of epidermis such that damage to the surface of the stem should produce the most significant decrease in overall flexural stiffness. We discerned whether the principal tension supporting members at the stem surface (cellulosic microfibrils) were oriented parallel or normal to stem length by comparing the bending stiffness of stems before and after their surface cells first received three parallel longitudinal incisions followed by one helical incision, and by comparing the bending stiffness of stems for which the sequence of cuts was reversed. The same protocol was also applied to stems with various water potentials to determine the effect of hydrostatic pressure on stem stiffness contributed by the surface. Based on the behavior of 82 turgid Tulipa stems, parallel cuts reduced, on average, stem stiffness by 8%, whereas a subsequent helical incision further reduced stiffness by 42%. In contrast, an initial helical incision reduced stem stiffness by 50%, while three subsequent parallel cuts through the same stems did not significantly further reduce stiffness. These results suggested that the net orientation of cellulose microfibrils in the outer epidermal walls was parallel to stem length. This was confirmed by microscopic observations of cells with dichroic staining and polarized light. The responses to surgical damage were directly proportional to stem water potential. We thus conclude that the epidermis, probably in conjunction with a single layer of subepidermal collenchyma cells, acts as a tension-stiffening agent that can contribute as much as 50% to overall stem stiffness We present a simple mechanical model that can account for all our observations.     

Implementation and validation of thoracic side impact injury prediction metrics in a human body model

This study's purpose was to implement injury metrics into the Total Human Model for Safety (THUMS) mirroring the spinal accelerometers, rib accelerometers and chest band instrumentation from two lateral post-mortem human subject sled test configurations. In both sled configurations, THUMS contacted a flat rigid surface (either a wall or beam) at 6.7 m/s. Sled A maximum simulated wall forces for the thorax, abdomen and pelvis were 7.1, 5.0 and 10.0 kN versus 5.7 ± 0.8, 3.4 ± 1.2 and 6.2 ± 2.7 kN experimentally. Sled B maximum simulated beam forces for the torso and pelvis were 8.0 and 7.6 kN versus 8.5 ± 0.2 and 7.9 ± 2.5 kN experimentally. Quantitatively, force magnitude contributed more to variation between simulated and experimental forces than phase shift. Acceleration-based injury metrics were within one standard deviation of experimental means except for the lower spine in the rigid wall sled test. These validated metrics will be useful for quantifying occupant loading conditions and calculating injury risks in various loading configurations.     

Quantitative comparison of ligament formulation and pre-strain in finite element analysis of the human lumbar spine

Data has been published that quantifies the nonlinear, anisotropic material behaviour and pre-strain behaviour of the anterior longitudinal, supraspinous (SSL), and interspinous ligaments of the human lumbar spine. Additionally, data has been published on localized material properties of the SSL. These results have been incrementally incorporated into a previously validated finite element model of the human lumbar spine. Results suggest that the effects of increased ligament model fidelity on bone strain energy were moderate and the effects on disc pressure were slight, and do not justify a change in modelling strategy for most clinical applications. There were significant effects on the ligament stresses of the ligaments that were directly modified, suggesting that these phenomena should be included in FE models where ligament stresses are the desired metric.     

An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored.     

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.     

Molecular Analysis of Two Novel Missense Mutations in the GDF5 Proregion That Reduce Protein Activity and Are Associated with Brachydactyly Type C

Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5^T201P and proGDF5^L263P revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFβ1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.     

Genetic analysis of candidate SNPs for metabolic syndrome in obstructive sleep apnea (OSA)

Obstructive sleep apnea (OSA) is a common disorder characterized by the reduction or complete cessation in airflow resulting from an obstruction of the upper airway. Several studies have observed an increased risk for cardiovascular morbidity and mortality among OSA patients. Metabolic syndrome (MetS), a cluster of cardiovascular risk factors characterized by the presence of insulin resistance, is often found in patients with OSA, but the complex interplay between these two syndromes is not well understood. In this study, we present the results of a genetic association analysis of 373 candidate SNPs for MetS selected in a previous genome wide association analysis (GWAS). The 384 selected SNPs were genotyped using the Illumina VeraCode Technology in 387 subjects retrospectively assessed at the Internal Medicine Unit of the “Virgen de Valme” University Hospital (Seville, Spain). In order to increase the power of this study and to validate our findings in an independent population, we used data from the Framingham Sleep Study which comprises 368 individuals. Only the rs11211631 polymorphism was associated with OSA in both populations, with an estimated OR = 0.57 (0.42–0.79) in the joint analysis (p = 7.21 × 10^− 4). This SNP was selected in the previous GWAS for MetS components using a digenic approach, but was not significant in the monogenic study. We have also identified two SNPs (rs2687855 and rs4299396) with a protective effect from OSA only in the subpopulation with abdominal obesity. As a whole, our study does not support the idea that OSA and MetS share major genetic determinants, although both syndromes share common epidemiological and clinical features.     

Potential roles and targeted therapy of the CXCLs/CXCR2 axis in cancer and inflammatory diseases

The chemokine receptor CXCR2 and its ligands are implicated in the progression of tumours and various inflammatory diseases. Activation of the CXCLs/CXCR2 axis activates multiple signalling pathways, including the PI3K, p38/ERK, and JAK pathways, and regulates cell survival and migration. The CXCLs/CXCR2 axis plays a vital role in the tumour microenvironment and in recruiting neutrophils to inflammatory sites. Extensive infiltration of neutrophils during chronic inflammation is one of the most important pathogenic factors in various inflammatory diseases. Chronic inflammation is considered to be closely correlated with initiation of cancer. In addition, immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) against T cells attenuate the anti-tumour effects of T cells and promote tumour invasion and metastasis. Over the last several decades, many therapeutic strategies targeting CXCR2 have shown promising results and entered clinical trials. In this review, we focus on the features and functions of the CXCLs/CXCR2 axis and highlight its role in cancer and inflammatory diseases. We also discuss its potential use in targeted therapies.