Adrenomedullin in mammalian embryogenesis

Here are summarized data supporting that adrenomedullin (AM) is a multifunctional factor involved in the complex regulatory mechanisms of mammalian development. During rodent embryogenesis, AM is first expressed in the heart, followed by a broader but also defined spatio-temporal pattern of expression in vascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs. AM pattern of expression is suggestive of its involvement in the control of embryonic invasion, proliferation, and differentiation processes, probably through autocrine or paracrine modes of action. AM levels in fetoplacental tissues, uterus, maternal and umbilical plasma are highly increased during normal gestation. These findings in addition to other physiological and gene targeting studies support the importance of AM as a vasorelaxant factor implicated in the regulation of maternal vascular adaptation to pregnancy, as well as of fetal and fetoplacental circulations. AM is also present in amniotic fluid and milk, which is suggestive of additional functions in the maturation and immunological protection of the fetus. Altered expression of AM has been found in some gestational pathologies, although it is not yet clear whether this corresponds to causative or compensatory mechanisms. Future studies in regard to the distribution and expression levels of the molecules known to function as AM receptors, together with data on the action of complement factor H (an AM binding protein), may help to better define the roles of AM during embryonic development.     

Immune response by host after allogeneic chondrocyte transplant to the cartilage

We discovered adrenomedullin (AM) from human pheochromocytoma tissue by monitoring the elevating activity of intracellular cyclic AMP (cAMP) in rat platelets in 1993. Since the discovery of AM, it has attracted intense interest from cardiovascular researchers because AM elicits multiple biological activities, including a potent and powerful hypotensive activity caused by dilatation of resistance vessels. AM is biosynthesized and secreted from tissues, including cardiovascular organs. In addition to AM, "proadrenomedullin N-terminal 20 peptide (PAMP)," another biologically active peptide, was found to be processed from the AM precursor. Plasma AM levels are increased in various cardiovascular and renal diseases. AM, therefore, seems to function as a novel system that controls circulation and body fluid, and may be involved in pathophysiological changes in cardiovascular diseases. Therefore, in this review we will focus on the structure of AM and its gene, distribution, receptor, and the physiological and pathological roles of AM in cardiovascular disease.     

Adrenomedullin as a pancreatic hormone

Adrenomedullin (AM) is a multiregulatory peptide which is expressed in a wide range of tissues. In the pancreas, AM was first found in mammals, including man, and its colocalization with the pancreatic polypeptide (PP) was established in islet F cells. In addition, three different AM receptors have been characterized in B-cells. AM has been also located in the pancreatic cells of other vertebrate classes. The frequency and distribution of AM cells vary between different animals; they can be found scattered among the exocrine tissue, in the islets, or in ductal epithelia. The colocalization of AM with other hormones presents different patterns, although in birds, as in mammals, it seems to colocalize only with PP. The best-determined pancreatic AM function is the inhibition of insulin secretion in B-cells, which seems to be linked to a recently discovered binding protein, factor H. In relation to this physiological role, clinical data show that AM is raised in some groups of both types I and II diabetic patients and AM might have triggered the disease in a subset of them. On the other hand, AM pancreatic cells are also involved in the response to septic shock by increasing AM circulating levels. A third putative function is the inhibition of amylase secretion by the exocrine pancreatic cells. AM has been found in embryonic mammalian pancreas from the earliest stages of the development, colocalizing with all pancreatic hormones, although in adults only coexpression with PP is kept. AM may play a role in the growth and morphogenesis of the pancreas.     

Adrenomedullin functions as an important tumor survival factor in human carcinogenesis

Adrenomedullin (AM) is a pluripotent regulatory peptide initially isolated from a human pheochromocytoma (adrenal tumor) and subsequently shown to play a critical role in cancer cell division, tumor neovascularization, and circumvention of programmed cell death, thus it is an important tumor cell survival factor underlying human carcinogenesis. A variety of neural and epithelial cancers have been shown to produce abundant amounts of AM. Recent findings have implicated elevation of serum AM with the onset of malignant expression. In addition, patients with tumors producing high levels of this peptide have a poor prognostic clinical outcome. Given that most human epithelial cancers display a microenvironment of reduced oxygen tension, it is interesting to note that AM and several of its receptors are upregulated during hypoxic insult. The existence of such a regulatory pathway has been implicated as the basis for the overexpression of AM/AM-R in human malignancies, thereby generating a subsequent autocrine/paracrine growth advantage for the tumor cell. Furthermore, AM has been implicated as a potential immune suppressor substance, inhibiting macrophage function and acting as a newly identified negative regulator of the complement cascade, protective properties which may help cancer cells to circumvent immune surveillance. Hence, AM's traditional participation in normal physiology (cited elsewhere in this issue) can be extended to a primary player in human carcinogenesis and may have clinical relevance as a biological target for the intervention of tumor progression.     

Multiple roles of adrenomedullin revealed by animal models

The newly identified adrenomedullin (AM) gene codes for a potent, highly conserved vasodilator that is expressed in many tissues. Many biological functions have been ascribed to AM based on its broad expression pattern and numerous in vitro studies, and it is currently viewed as a multifunctional peptide hormone. Recent advances in gene manipulation have permitted the development of experimental animal systems to help distinguish between gene causes and effects in the context of otherwise normal physiology, and so the normal biological function of the AM gene can be studied within the intact physiological milieu of a whole animal. In this review article, we summarize the recent findings from three different types of genetic experiments involving the AM gene.     

A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: in vivo characterization and structure/function relationships

Mass spectrometry has evolved in recent years to a well-accepted and increasingly important complementary technique in molecular and structural biology. Here we review the many contributions mass spectrometry based studies have made in recent years in our understanding of the important cyclic nucleotide activated protein kinase A (PKA) and protein kinase G (PKG). We both describe the characterization of kinase isozymes, substrate phosphorylation, binding partners and post-translational modifications by proteomics based methodologies as well as their structural and functional properties as revealed by native mass spectrometry, H/D exchange MS and ion mobility. Combining all these mass spectrometry based data with other biophysical and biochemical data has been of great help to unravel the intricate regulation of kinase function in the cell in all its magnificent complexity.     

Additive manufacturing technology: the status,applications, and prospects

Additive manufacturing (AM) has first emerged in 1987 with the invention of stereolithography. The AM is an important, rapidly emerging, manufacturing technology that takes the information from a computer-aided design (CAD) and builds parts in a layer-by-layer style. As this technology offers many advantages such as manufacturing of complex geometries, reducing manufacturing cost and energy consumption, it has transformed manufacturing from the mass production to the mass customization. Also, it has found wide applications in several fields although some drawbacks. This paper presents the state of the art of the different AM processes, the material processing issues, and the post-processing operations. A comparison between AM and conventional processes is presented as well. We finish by presenting some prospects of this technology such as hybrid manufacturing and 4D printing.     

Dephosphorylated mutations affect the protein-protein interactions of ERF in <i>Populus simonii x P. nigra</i>

Phosphorylation is a common type of post-translational modification (PTM). It plays a vital role in many cellular processes. The reversible phosphorylation and dephosphorylation affect protein structures and proteinprotein interactions. Previously, we obtained five proteins that interact with ethylene-responsive factor (ERF) from the cDNA library of Populus simonii x Populus nigra. To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability, we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay (Y2H). The secondary structures and 3D structures of PsnERF, ERFm, TrunERF, and psnerf^197/198/202a were predicted by homology modeling. The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions, while dephosphorylated mutations blocked the interactions. Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation, and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.     

Adrenomedullin has multiple roles in disease stress: development and remission of the inflammatory response

The upregulation of adrenomedullin (AM) gene expression and increases in systemic circulatory as well as localized tissue AM concentrations is well coordinated with the onset and progression of trauma, infection, and sepsis. As such, the coordinated change in AM suggests a key role for this peptide in the inflammatory response. By clinical definition, the process of inflammation constitutes an orchestrated cascade of localized tissue and systemic responses to immunological challenges. Classical responses to the onset of disease stresses are manifested in the timely elaboration of humoral, blood-borne signal effectors (such as adrenocortical and locally produced tissue hormones, immune cytokines, and inorganic signals such as nitric oxide) as well as patterned migration and infiltration of circulating bone marrow-derived cells (mononuclear cells such as monocyte-macrophages and polymorphonuclear cells like neutrophils) largely associated with or delivered through the vascular system. The body's attempts to combat acute infection to restore homeostatic equilibrium are further compromised by underlying disease situations. Atherosclerosis, diabetes, and cardiovascular disease, as well as nutritional metabolic derangements and persistent subclinical infection perturb the regulatory feedback loops necessary for proper control of response effectors like hormones and cytokines. When imbalances occur, tissue necrosis can ensue as driven by free radical damage to cell components. A true appreciation of the inflammatory response can only be grasped through an integrative approach in which the relationship between the different physiological systems is viewed in terms of a changing, dynamic interaction. In essence, the inflammatory response can be thought of in three phases: a period of severity assessment, a period of remediation, and a period of homeostatic restoration. Indeed, AM has differential effects on cellular metabolism, immune function, endocrine function, and cardiovascular function. This peptide appears to play a pivotal role in both reprioritizing the biological needs of tissues and organs during the three phases of inflammatory response as well as a role in restoring homeostatic equilibrium to the body.     

ICP‐MS‐Based strategies for protein quantification

In the post‐genomics era, proteomics has become a central branch in life sciences. An understanding of biological functions will not only rely on protein identification, but also on protein quantification in a living organism. Most of the existing methods for quantitative proteomics are based on isotope labeling combined with molecular mass spectrometry. Recently, a remarkable progress that utilizes inductively coupled plasma‐mass spectrometry (ICP‐MS) as an attractive complement to electrospray MS and MALDI MS for protein quantification, especially for absolute quantification, has been achieved. This review will selectively discuss the recent advances of ICP‐MS‐based technique, which will be expected to further mature and to become one of the key methods in quantitative proteomics. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:326–348, 2010     

The substructure of immunoglobulin G resolved to 25 kDa using amplitude modulation AFM in air

Amplitude modulation (or tapping-mode) atomic force microscopy (AM AFM or TM AFM) in air can reveal sub-molecular details of isolated multi-subunit proteins, such as immunoglobulin G (IgG) antibodies, on atomically flat support surfaces such as mica [A. San Paulo, R. Garcia, Biophys. J. 78(3) (2000) 1599]. This is achieved by controlling the microscope imaging parameters (e.g. cantilever drive frequency and set-point amplitude) to keep the AFM tip predominantly in the attractive force regime. Under these conditions, the 50 kDa F(c) and F(ab) subunits can be resolved when the molecule has the appropriate orientation on the surface. The presence of a water layer on hydrophilic mica is an important factor affecting imaging contrast, a consequence of capillary neck formation between tip and surface [L. Zitzler, S. Herminghaus, F. Mugele, Phys. Rev. B 66(15) (2002) 155436]. Desiccation of samples to remove surface bound water layers can yield reproducible imaging of the IgG substructure [N.H. Thomson, J. Microsc. (Oxford) 217(3) (2004) 193]. This approach has also given higher resolution than previously achieved, down to about 25 kDa, and these data are detailed here. These subdomains are formed as two immunoglobulin folds from the light and heavy peptide chains of the IgG crossover. This result has been validated by comparing the AFM images with X-ray crystallography data from the protein data bank. These data show that the AFM can obtain 25 kDa resolution on isolated protein molecules with commercially available silicon tips, but, as expected for a local probe technique, resolution is highly dependent on the macromolecular orientation on the support surface.     

Adrenomedullin in the central nervous system

Adrenomedullin (AM) is a novel vasodilator peptide first purified from human pheochromocytoma by tracing its capacity to stimulate cAMP production in platelets. AM immunoreactivity is widely distributed in the central nervous system (CNS) and in the rat has been demonstrated by immunohistochemical techniques to be present in many neurons throughout the brain and spinal cord, as well as in some vascular endothelial cells and perivascular glial cells. Electron microscopy shows that the immunoreactivity is located mainly in the neuronal cytoplasm, but also occurs in the cell nucleus in some cells of the caudate putamen and olfactory tubercle. Biochemical analyses suggest that higher molecular forms, presumably precursor forms, may predominate over fully processed AM in some brain areas. The expression of AM immunoreactivity is increased in cortical neurons, endothelial cells, and perivascular processes after a simulation of ischemia by oxygen and glucose deprivation. Immunohistochemical, electrophysiological, and pharmacological studies suggest that AM in the CNS can act as a neurotransmitter, neuromodulator, or neurohormone, or as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role.     

Additive manufacturing: technology,applications and research needs

Additive manufacturing (AM) technology has been researched and developed for more than 20 years. Rather than removing materials, AM processes make three-dimensional parts directly from CAD models by adding materials layer by layer, offering the beneficial ability to build parts with geometric and material complexities that could not be produced by subtractive manufacturing processes. Through intensive research over the past two decades, significant progress has been made in the development and commercialization of new and innovative AM processes, as well as numerous practical applications in aerospace, automotive, biomedical, energy and other fields. This paper reviews the main processes, materials and applications of the current AM technology and presents future research needs for this technology.     

Angiogenesis of the heart

Despite continued advances in the prevention and treatment of coronary artery disease, there are still a large number of patients who are not candidates for the conventional revascularization techniques of balloon angioplasty and stenting, or coronary artery bypass grafting (CABG). Therapeutic angiogenesis, in the form of the administration of growth factor protein or gene therapy, has emerged as a promising new method of treatment for patients with coronary artery disease. The goal of this strategy is to promote the development of supplemental blood conduits that will act as endogenous bypass vessels. New vessel formation occurs through the processes of angiogenesis, vasculogenesis, and arteriogenesis, under the control of growth factors such as those that belong to the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and angiopoeitin (Ang) families of molecules. Preclinical studies have suggested that such an approach is both feasible and effective; however many questions remain to be answered. This review will address the elements of pharmacologic revascularization, focusing on gene and protein-based therapy. The important growth factors, the vector (for gene therapy), routes of delivery, the desired therapeutic effect, and quantifiable clinical end points for trials of angiogenesis will all be addressed.     

Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy,surface replication,freeze-etching and label fracture

In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno-gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO₄ fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors.     

Biomedical overview of melanin. 2. Updating molecular modeling,synthesis mechanism,and supramolecular properties regarding melanoma therapy

Melanins represent one of the most ancient and important group of natural macromolecular pigments. They have multiple biological roles in almost all organisms across the Phyla, examples being photoprotection, anti-oxidative action, radical scavenger activity, and heavy metal removal. From the biomedical point of view, melanocytes are involved in the origin of melanoma tumors, and the main therapeutic advances for their treatment have been revised in Part 1 of this review. The chemical structure of eumelanin is a biological concern of great importance, and therefore, exploring theoretical molecular models and synthesis mechanisms will be here described, as well as molecular orbital features and supramolecular organization, which are responsible for the key properties that make these biological pigments so important, and so fascinating. Ultimately, this updated overview is devoted to describe present structural models and physico-chemical characteristics of eumelanin, in order to explain and utilize melanin properties on which new photothermal and ultrasonic protocols for melanoma treatment can be devised and applied.

     

Mapping of the receptor-associated protein (RAP) binding proteins on living fibroblast cells using an atomic force microscope

The distribution of the receptor-associated protein (RAP) binding protein and the adhesion forces between RAP and its binding protein on living fibroblast cells were examined using an atomic force microscope (AFM). The distribution of RAP binding protein was obtained on 256 (16x16) locations in 2x2 micro m sections over the surface of living cells. The adhesion forces between RAP and the binding protein were measured with an AFM tip functionalized with RAP. In the presence of RAP in the scanning solution, the number of force curves with large adhesion force decreased. These results indicate that the adhesive forces observed here represent specific binding between RAP and the binding protein. This method will be a useful application of AFM to examine receptors on cell surfaces in high resolution.     

A combined additive manufacturing and micro-syringe deposition technique for realization of bio-ceramic structures with micro-scale channels

This article presents a novel rapid layered manufacturing approach based on a combined additive manufacturing (AM) process and a UV-based micro-syringe deposition (μSD) technique to be used in the fabrication of bio-ceramic structures with controlled micro-sized channels for bone and osteochondral tissue regeneration. In the proposed rapid manufacturing method, micro-scale sacrificial photopolymer networks are integrated within the manufactured part by depositing the photopolymer on selected bio-ceramic powder layers using an injection system. This AM–μSD method along with a post-processing protocol can potentially overcome current limitations of traditional powder-based AM approaches that are restricted in terms of complexity of internal architecture and feature size. For bone or osteochondral repair applications, the material system composed of the bio-ceramic and sacrificial photopolymer, along with the post-processing protocol, must ensure that the final implants are free from manufacturing residuals that could trigger an immune response post-implantation. In this study, calcium polyphosphate bio-ceramic was used as the substrate material based on prior art, polyvinyl alcohol solution was used as the powder binding agent, and ethoxylated (10 bisphenol A diacrylate) photopolymer solution was used as the sacrificial photopolymer element. Material characterization suggests that the proposed material system along with heat treatment protocol is suitable for the targeted applications where micro-scale channels within the implant are produced by AM–μSD.     

电磁轴承系统中的辅助轴承与振动影响

出于安全性考虑,实验或应用中的电磁轴承系统通常需要考虑安装辅助轴承。由于辅助轴承并非具有实质性的支承作用,因此一般情况下,在电磁轴承系统设计时,对辅助轴承的振动情况及其对整个磁悬浮系统的影响考虑是极少的。通过一台磁悬浮铣床电主轴样机的实验结果表明,辅助轴承的振动影响有时不仅不能忽略,甚至它对系统的安全运行的影响是至关重要的。