SHOX at a glance: from gene to protein

The Short Stature Homeobox-containing Gene SHOX was identified as the genetic cause of the short stature phenotype in patients with Turner Syndrome and in certain patients with idiopathic short stature. Shortly after, SHOX mutations were also associated with the growth failure and skeletal deformities seen in patients with Léri - Weill dyschondrosteosis and Langer mesomelic dysplasia. Today it is estimated that SHOX mutations occur with an incidence of roughly 1:1,000 in newborns, making mutations of this gene one of the most common genetic defects leading to growth failure in humans. This review summarises the involvement of SHOX in several short stature syndromes and describes recent advances in our understanding of SHOX functions and regulation. We also discuss the current evidence in the literature that points to a role of this protein in growth and bone development. These studies have improved our knowledge of the SHOX gene and protein functions, and have given insight into the etiopathogenesis of short stature. However, the exact role of SHOX in bone development still remains elusive and poses the next major challenge for researchers in this field.     

Information relay from gene to protein: the mRNP connection

Eukaryotic messenger RNAs and their binding proteins are organized into structural units called ribonucleoprotein particles (mRNPs). Some mRNP proteins are ubiquitous, and might bind all mRNAs to ensure efficient translation. Other mRNA proteins, however, are cell-specific and bind only certain mRNAs that display regulated translation. This is particularly evident in early development, where some mRNP particles can be sequestered from the translational apparatus for months before they enter polysomes. Recent investigations suggest that these and other mRNP proteins bind specific sequences and regulate translation.     

Fibronectin and cell attachment to cell and protein resistant polyelectrolyte surfaces

Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces.     

Fibronectin gene expression during limb cartilage differentiation

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo. In high-density micromass cultures of limb mesenchymal cells, FN mRNA levels increase about 5-fold coincident with the crucial condensation process, and remain relatively high during the initial deposition of cartilage matrix by the cells. Thereafter, FN mRNA levels progressively decline to relatively low levels as the cultures form a virtually uniform mass of cartilage. The changes in FN mRNA levels in vitro are paralleled closely by changes in the relative rate of FN synthesis as determined by pulse-labeling and immunoprecipitation analysis. The relative rate of FN synthesis increases 4- to 5-fold at condensation and the onset of chondrogenesis, after which it progressively declines to low levels as cartilage matrix accumulates. High levels of FN gene expression also occur at the onset of chondrogenesis in vivo. In the proximal central core regions of the limb bud in which condensation and cartilage matrix deposition are being initiated, FN mRNA levels and the relative rates of FN synthesis become progressively about 4-fold higher than in the distal subridge region, which consists of undifferentiated mesenchymal cells that have not yet initiated condensation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Advances in Na+,K+-ATPase studies: from protein to gene and back to protein

Complete primary structures of both subunits of Na+,K+-ATPase from various sources have been established by a combination of the methods for molecular cloning and protein chemistry. The gene family homologous to the alpha-subunit cDNA of animal Na+,K+-ATPases has been found in the human genome. Some genes of this family encode the known isoforms (alpha I and alpha II) of the Na+,K+-ATPase catalytic subunit. The proteins coded by other genes can be either new isoforms of the Na+,K+-ATPase catalytic subunit or other ion-transporting ATPases. Expression of the genes of this family proceeds in a tissue-specific manner and changes during the postnatal development and neoplastic transformation. The complete exon-intron structure of one of the genes of this family has been established. This gene codes for the form of the catalytic subunit, the existence of which has been unknown. Apparently, all the genes of the discovered family have a similar intron-exon structure. There is certain correlation between the gene structure and the proposed domain arrangement of the alpha-subunit. The results obtained have become the basis for the experiments which prove the existence of the earlier unknown alpha III isoform of the Na+,K+-ATPase catalytic subunit and have made possible the study of its function.     

Exposure to Biomass Smoke Extract Enhances Fibronectin Release from Fibroblasts

COPD induced following biomass smoke exposure has been reported to be associated with a more fibrotic phenotype than cigarette smoke induced COPD. This study aimed to investigate if biomass smoke induced extracellular matrix (ECM) protein production from primary human lung fibroblasts in vitro. Primary human lung fibroblasts (n = 5–10) were stimulated in vitro for up to 72 hours with increasing concentrations of biomass smoke extract (BME) or cigarette smoke extract (CSE) prior to being assessed for deposition of ECM proteins, cytokine release, and activation of intracellular signalling molecules. Deposition of the ECM proteins perlecan and fibronectin was upregulated by both CSE (p<0.05) and BME (p<0.05). The release of the neutrophilic chemokine IL-8 was also enhanced by BME. ERK1/2 phosphorylation was significantly upregulated by BME (p<0.05). Chemical inhibition of ERK signalling molecules partially attenuated these effects (p<0.05). Stimulation with endotoxin had no effect. This study demonstrated that BME had similar effects to CSE in vitro and had the capacity to directly induce fibrosis by upregulating production of ECM proteins. The mechanisms by which both biomass and cigarette smoke exposure cause lung damage may be similar.     

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.     

Fibronectin binding to Salmonella strains

Abstract Recent investigations by many workers have elucidated the mechanisms which may explain the clinical observation that an excessive intake of dietary carbohydrates can aggravate oral candidosis. These include the enhancement of growth, multiplication and adherence of Candida species to oral surfaces and the production of excessive quantities of short-chain carboxylic acids as byproducts of sugar metabolism. The resultant acidic milieu, in addition to provoking a mucosal inflammatory reaction, could also activate the highly potent phospholipases and acid proteases of Candida and suppress the growth of normal commensal flora. A unifying hypothesis encompassing the above is proposed to explain the exacerbation of mucosal candidoses observed in environments replete with carbohydrates such as glucose and sucrose.     

纤维连接蛋白上调兔支气管上皮细胞过氧化氢酶表达

为从基因转录水平阐明纤维连接蛋白(fibronectin,Fn)与整合素(integrins)结合反应对支气管上皮细胞(bronchial epithelialCells,BECs)的抗氧化保护机制,本文在先前的工作基础上用臭氧(ozone,O_3)攻击原代培养的免BEC,RT-PCR扩增过氧化氢酶(catalase,CAT)的cDNA,PCR产物经琼脂糖凝胶电泳后用凝胶成像系统进行灰度分析,反映CAT mRNA的原始表达丰度,观察Fn处理的影响及蛋白酪氨酸激酶抑制剂genistein和钙调素抑制剂W_7的作用。同时,将电泳展开的PCR产物电转移至尼龙膜上,用CAT特异性寡核苷酸探针杂交,证实PCR扩增产物为特异性目的基因的转录产物。结果证实:Fn(10μg/ml)处理可提高CAT表达(P<0.01),蛋白酪氨酸激酶抑制剂genistein可阻断Fn对CAT mRNA表达的增强效应(P<0.01);钙调素抑制剂W_7对Fn处理后CAT mRNA表达增强也有抑制作用。提示:Fn可提高BEC细胞内CAT编码基因的转录水平,其上游信号途径与整合素介导的酪氨酸磷酸化或Ca~(2+)-钙调素通路有关。     

Fibronectin binding to the Treponema pallidum adhesin protein fragment rTp0483 on functionalized self-assembled monolayers

Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface. This research seeks to prepare a surface capable of mimicking the FN binding ability of T. pallidum in order to investigate the impact of FN binding with adsorbed Tp0483 on the host response to the surface. By understanding this interaction, it may be possible to develop more effective treatments for infection and possibly mimic the stealth properties of the bacteria. Functionalized self-assembled monolayers (SAMs) on gold were used to investigate rTp0483 and FN adsorption. Using a quartz crystal microbalance (QCM), rTp0483 adsorption and subsequent FN adsorption onto rTp0483 were determined to be higher on negatively charged carboxylate-terminated self-assembled monolayers (-COO(-) SAMs) compared to the other surfaces analyzed. Kinetic analysis of rTp0483 adsorption using surface plasmon resonance (SPR) supported this finding. Kinetic analysis of FN adsorption using SPR revealed a multistep event, where the concentration of immobilized rTp0483 plays a role in FN binding. An examination of relative QCM dissipation energy compared to the shift in frequency showed a correlation between the physical properties of adsorbed rTp0483 and SAM surface chemistry. In addition, AFM images of rTp0483 on selected SAMs illustrated a preference of rTp0483 to bind as aggregates. Adsorption on -COO(-) SAMs was more uniform across the surface, which may help further explain why FN bound more strongly. rTp0483 antibody studies suggested the involvement of amino acids 274-289 and 316-333 in binding between rTp0483 to FN, while a peptide blocking study only showed inhibition of binding with amino acids 316-333. Finally, surface adsorbed rTp0483 with FN bound significantly less anti-RGD and gelatin compared to FN adsorbed directly to -COO(-) SAMs, indicating that one or both binding regions may play a role in binding between rTp0483 and FN.     

Fibronectin in evolution: presence in invertebrates and isolation from Microciona prolifera

Fibronectin is found in the tissues of a series of vertebrates and invertebrates which suggests its appearance with the simplest multicellular organisms. Fibronectin is specifically localized on the surface and on the substrate in the immediate vicinity of some, but not all, dissociated Microciona prolifera cells, suggesting that the expression of fibronectin in this organism might be dependent on cell type and/or developmental stage. Fibronectin has been partially purified and characterized from intact Microciona prolifera tissue on the basis of its immunological and biochemical properties.     

Designing gene libraries from protein profiles for combinatorial protein experiments

Protein combinatorial libraries provide new ways to probe the determinants of folding and to discover novel proteins. Such libraries are often constructed by expressing an ensemble of partially random gene sequences. Given the intractably large number of possible sequences, some limitation on diversity must be imposed. A non-uniform distribution of nucleotides can be used to reduce the number of possible sequences and encode peptide sequences having a predetermined set of amino acid probabilities at each residue position, i.e., the amino acid sequence profile. Such profiles can be determined by inspection, multiple sequence alignment or physically-based computational methods. Here we present a computational method that takes as input a desired sequence profile and calculates the individual nucleotide probabilities among partially random genes. The calculated gene library can be readily used in the context of standard DNA synthesis to generate a protein library with essentially the desired profile. The fidelity between the desired profile and the calculated one coded by these partially random genes is quantitatively evaluated using the linear correlation coefficient and a relative entropy, each of which provides a measure of profile agreement at each position of the sequence. On average, this method of identifying such codon frequencies performs as well or better than other methods with regard to fidelity to the original profile. Importantly, the method presented here provides much better yields of complete sequences that do not contain stop codons, a feature that is particularly important when all or large fractions of a gene are subject to combinatorial mutation.     

Fibers with Integrated Mechanochemical Switches: Minimalistic Design Principles Derived from Fibronectin

Inspired by molecular mechanisms that cells exploit to sense mechanical forces and convert them into biochemical signals, chemists dream of designing mechanochemical switches integrated into materials. Using the adhesion protein fibronectin, whose multiple repeats essentially display distinct molecular recognition motifs, we derived a computational model to explain how minimalistic designs of repeats translate into the mechanical characteristics of their fibrillar assemblies. The hierarchy of repeat-unfolding within fibrils is controlled not only by their relative mechanical stabilities, as found for single molecules, but also by the strength of cryptic interactions between adjacent molecules that become activated by stretching. The force-induced exposure of cryptic sites furthermore regulates the nonlinearity of stress-strain curves, the strain at which such fibers break, and the refolding kinetics and fraction of misfolded repeats. Gaining such computational insights at the mesoscale is important because translating protein-based concepts into novel polymer designs has proven difficult.