‘Heparin’ – from anticoagulant drug into the new biology

This overview attempts to cover, from a personal viewpoint, the development of the heparin field during the last four decades. In particular, it emphasizes the metamorphosis of heparan sulfate (HS), from a disturbing contaminant in heparin production to the present-day key player in cell and developmental biology. Our understanding of the structural properties of the polysaccharides has been greatly promoted by studies of their biosynthesis. We now have a fairly detailed view of the various enzymatic reactions, that convert the initial [4GlcA1-4GlcNAc1-]_n polymer into sulfated products with highly variable proportions of GlcA/IdoA and of N-acetyl, N-sulfate and O-sulfate substituents. It is also recognized that the variously substituted domains of the polysaccharide serve to interact, in more or less specific fashion, with a multitude of proteins, and that these interactions are essential to the biological functions of the proteins. Molecular genetics has unravelled the gene structures for almost all of the enzymes required to synthesize a heparin or HS chain, and has shown that several of these proteins exhibit genetic polymorphism. While differences in substrate specificity between enzyme isoforms may help to explain the structural variability of, in particular, HS chains, we still only partly understand the key features of heparin/HS biosynthesis and its regulation.     

How to analyze the anticoagulant and antithrombotic mechanisms of action in fucanome and galactanome?

Through the perspective of the current glycomics age, fucanomics and galactanomics denote the international projects concerned with the studies of the biomedically active marine sulfated fucose- or galactose-composed polysaccharides, named sulfated fucans (SFs), and sulfated galactans (SGs), respectively. SFs and SGs are isolated from algae or marine invertebrates. The range of therapeutic actions of SFs and SGs is impressively broad. When certain structural requirements are found, some SFs and SGs may exhibit beneficial properties in inflammation, nociception, hemostasis (coagulation and thrombosis), vascular biology (angiogenesis), oncology, oxidative-stress, and virus infections. Although many biomedical applications for SFs and SGs have been pointed out over the past two decades, only inflammation, hemostasis, cancer, and vascular biology have their mechanisms of action satisfactorily elucidated. In addition, advanced structure-function relationships have been achieved only for the anticoagulant and antithrombotic activities, in which glycans of well-defined structures have been assayed. Because of this, the activities of SFs and SGs in stopping the clot and thrombus formation represent the closest therapeutic areas of having these glycans truly explored for drug development. Here, through an analytical viewpoint, we present the common methods and protocols employed to achieve such advanced structure-function relationships of SFs and SGs in anticoagulation and antithrombosis.     

Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?

We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.     

Is the shoot a root with a view?

Recently, it has been shown that the same sets of genes act in both root and shoot to regulate cell fate and patterning. One gene cassette regulates epidermal cell fate, another cassette regulates ground tissue derived cell fate and organization. Ectopic expression and laser ablation have been used to probe the mechanisms by which these genes perform their tissue and organ-specific functions.     

The “therapeutic range” of the one-stage prothrombin time in the control of anticoagulant therapy: The effect of different thromboplastin preparations

Commercially available thromboplastin reagents and two human brain preparations have been compared using the one-stage prothrombin time and plasma samples from patients receiving long-term oral anticoagulant therapy. Considerable variation is noted between various thromboplastins using the same plasma sample. The commercially available thromboplastins give shorter prothrombin times than do human brain preparations. With the latter, the “therapeutic range” is represented by a prothrombin time of about 1.8 to 3.0 times the normal control value, whereas with commercial preparations the “therapeutic range” is about 1.25 to 1.75 times normal. The implications of these observations are discussed; the desirability of standardization of the one-stage prothrombin time is emphasized.     

Antigenic similarity between the β-subunits of the ATPases of a bacterium,a yeast and a higher plant

Antiserum raised against the β-subunit of wheat (Triticum aestivum) chloroplast ATPase cross-reacts with a 51000 protein located in the membrane fraction of Escherichia coli. The differential solubility of this polypeptide after chloroform treatment of unc⁺ and uncD409 strains indicates that this cross-reacting polypeptide is the bacterial β-subunit of ATPase. Thus a high degree of conservation of antigenic determinant sites exists between a bacterial β-subunit and the β-subunit of a monocot. This conservation also seems to extend to the β-subunit of mitochondrial ATPase of yeast (Saccharomyces cerevisiae).     

Is the Pope a Catholic?

The whole-part relationship is generally considered transitive, but there are some apparent exceptions. Componential sortals create some apparent problems. Homo sapiens, the Pope, and his heart are all individuals. A human being, such as the Pope, is an organism-level component of Homo sapiens. The Pope’s heart is an organ-level component of both Homo sapiens and the Pope. Although the Pope is a part, and not an instance, of the Roman Catholic Church, it seems odd to say that his heart is a part of that church. This is largely because the Pope’s heart does not have a place in the ecclesiastical government. However, it does contribute to the functioning of the organization. One popular alternative to the view that Homo sapiens is an individual is the notion that it is a natural kind. This has been done by redefining ‘natural kind’ in such a manner that not just the Roman Catholic Church, but the Pope and every other human being is a natural kind as well.     

Is the meniscus of the knee joint a fibrocartilage?

A histological analysis of the structure of intact knee joint menisci was carried out in adult dogs. By means of specific histochemical methods for the connective tissue and cartilage, it was found that the meniscus as a whole does not have a unique structure. The anterior and posterior horns are populated by round chondroid cells encircled by abundant interstitial substance and branched wavy connective fibers; blood vessels are present. The outer third of the meniscus is constituted of cross bundles of connective fibers, fibrocytes and spindle-like areas of loose connective tissue with blood vessels. The inner avascular two thirds of the meniscus are filled with parallel circumferentially oriented fascicles of connective fibers, ovally elongated chondroid cells, and a small quantity of chondroid interstitial substance. In some menisci, in the inner two thirds of the body, there are isles of typical cartilage, which show metachromasia of the beta type and rarely of the gamma type. The occurrence and way of the manifestation of cartilage are of an individual character. The structural duality of the knee meniscus is accounted for by its functional duality manifested in offering resistance to the forces of traction and pressure, the latter ones favoring the process of evolution of tissue from connective, through chondroid, to cartilaginous.     

Does the immune system of a mouse age faster than the immune system of a human?

One of the characteristics of all somatic cells is a finite life span. Cells may proliferate until they reach a point after which, although they are metabolically active, they can no longer produce daughter cells. This observation is central to the clonal exhaustion hypothesis, a mechanism cited to explain age-associated immune dysfunction. In this hypothesis, repeated division of lymphocytes leads to a replicative limit, after which they enter the senescent phase but are not lost from the pool of T cells. Advancing age would then be associated with an increase in the number of T cells that are unable to proliferate to a stimulus which induces a proliferative response in T cells from younger individuals. This hypothesis seems both logical and reasonable and is supported by data from both humans and mice with the demonstration of an age-related accumulation of senescent T cells in both species. However, there is an apparent paradox. The paradox arises because the onset of immunosenescence appears to be more closely linked to the life span of the animal rather than the life span of the lymphocyte. BioEssays 21:519–524, 1999. © 1999 John Wiley & Sons, Inc.     

Is the dynein motor a winch?

Dyneins are the largest and most complex of the three classes of linear motor proteins in eukaryotic cells. The mass of the dynein motor domain is about ten times that of the other microtubule motor, kinesin. Dynein's homology with the AAA+ superfamily of mechanoenzymes distinguishes it from both kinesin and myosin, which share a common fold and ancestry as members of the G-protein superfamily. In contrast to the other motor proteins, little is known about the mechanism of dynein; its three-dimensional structure is unknown even at low resolution. Recent two-dimensional images from electron microscopy have revealed new details of its structure and how this changes to produce movement. These and the recently solved crystal structure of another AAA+ protein, ClpB, offer tantalising hints about dynein's mechanism, suggesting it may act like a molecular winch.     

Was the ASN a Myelin Society?

Neurochemical Research -     

“Timing” of arrival and in-hospital mortality in a cohort of patients under anticoagulant therapy presenting to the emergency departments with cerebral hemorrhage: A multicenter chronobiological study in Italy

Therapy with oral anticoagulants (OACs) is a risk factor for cerebral hemorrhage (CH). Although different studies have been undertaken to investigate the timing of the onset of major cardiovascular events, no data exist on temporal patterns of the onset of CH in subjects treated with OACs. The aim of this study is to evaluate the timing of CH in patients treated with OACs. All patients who developed CH under OACs therapy and admitted to 28 Italian Emergency Departments (EDs) between September 2011 and July 2013 were enrolled. Age, sex, time and location of the hemorrhagic lesion, type of the bleeding events (idiopathic or post-traumatic), anticoagulant therapy (warfarin or new oral anticoagulants - NOAs) and time of ED admission (i.e., hour, day, month and season) were recorded. Five hundred and seventeen patients (63.2% male aged 80 ± 7.9 yrs) with CH were involved. Warfarin was taken by 494 patients (95.6%), and NOAs by 23 (4.4%). In-hospital mortality (IHM) was recorded in 208 cases (40.2%). Cosinor analysis showed a peak of CH arrival between 12:00 and 14:00 h both in the whole population (PR 73.9%, p = 0.002) and the male subgroup (PR 65.2%, p = 0.009), whereas females showed an anticipated morning peak between 08:00 and 10:00 h (PR 65.7%, p = 0.008). A further analysis between idiopathic and post-traumatic CH confirmed the presence of a 24 h pattern with a peak between 12:00 and 14:00 h (PR 58.5%, p = 0.019) and between 08:00 and 10:00 h (PR80.1%, p < 0.001) for idiopathic events and post-traumatic hemorrhages, respectively. Moreover, a seasonal winter peak was identified for idiopathic forms (PR 74%, p = 0.035), and a summer peak for post-traumatic forms (PR 77%, p = 0.025). The present study suggests the presence of a temporal pattern of ED arrivals in CH patients treated with OACs.     

Making a Mechanical Organism Being the fourth in a series of essays on the materials of nature

Most multicellular organisms can be categorised by two words： hierarchy and composite. The underlying fractal geometry of nature - at least in terms of provision of infrastructure - provides much of the hierarchy, although many materials for which infrastructure is not an integral factor are also strongly hierarchical. Plants can therefore be modelled using recursive computer programs which add structures as the size increases. However, problems with mechanical stability also increase as the structure grows, so the plant changes from deriving stiffness from intevaal pressure to cross-linking the cell wall components permanently. However, this compromises the ability of the plant to grow and repair itself.     

Is the multidrug transporter a flippase?

The phenomenon of multidrug resistance is correlated with the presence of a membrane protein, P-glycoprotein, which pumps a wide variety of drugs out of cells thus reducing their toxicity. However, the mechanism of this pumping action remains unclear. In this article, we suggest that several properties of the multidrug transporter may be explained if it acts as a 'flippase' to transport drugs from the inner leaflet of the lipid bilayer to the outer or to the external medium.     

Mannitol in the rhodophyceae—a reappraisal

Experiments were performed to determine whether mannitol occurs as a native constituent of marine and freshwater Rhodophyceae. Those red algae which had previously been reported to contain mannitol were tested. In none of these species could mannitol or any other hexitol be detected, either as ¹⁴C-assimilate after photosynthetic assimilation of ¹⁴C from H¹⁴CO₃^– or in trace amounts of the EtOH-soluble fraction. Attempts to qualify the action of a specific mannitol synthesizing enzyme (mannitol-1-phosphate dehydrogenase, EC 1.1.1.17) also failed. Though mannitol [¹⁴C] is taken up when exogenously supplied by a seawater medium, less than 5% of ¹⁴C from mannitol [¹⁴C] taken up after 3 hr is recovered in other compounds. Mannitol is therefore not regarded as a natural metabolite (assimilate) of Rhodophyceae.     

Is the enzyme a powerful reactant of the biochemical reaction?

The mainstream explanation of enzyme catalysis relies on the assumption that enzymes can utilize the binding energy. The author suggest that (i) an enzyme with excess free energy first gives a group from its active site into the final place of the bound reactant (substrate) in order to break the first initial chemical bond; (ii) this enzyme accepts a similar group from the second bound reactant (or second group in the case of the single-substrate) into active site and finish the substrate conversion and enzyme regeneration. The detailed mechanisms of the well-studied reactions of peptide bond hydrolysis catalyzed by α-chymotrypsin and the glyceraldehyde-3-phosphate interconversion steps in glycolysis are in accordance with the proposed theoretical conclusions.