Massachusetts Institute of Technology

Commissariat a L'energie Atomique

Dartmouth College

Beth Israel Hospital

Robert D Rosenberg

Zhengliang L Wu

Nicholas W Shworak

Lijuan Zhang

David L Beeler

Kuberan Balagurunathan

Robert Samuel Langer

Elazer Reuven Edelman

Jian Liu

Michael Simons

Marcel Jozefowicz

Jay M Edelberg

Patricia G Spear

John J Schwartz

Benjamin G Neel

Christine Fougnot

David J Kuter

Robert T Palac

Jorge Plutzky

Robert M Freeman, Jr

Jean-Luc DeKeyser

Deepak Shukla

Linda M S Fritze

The invention relates to methods for detecting and characterizing enzymatic modifications of oligosaccharides, such as heparan sulfate, and their interaction with binding partners, such as proteins, using an oligosaccharide-binding partner binding assay, such as a gel mobility shift assay. The instant invention relates to a rapid, convenient, sensitive and inexpensive method for identifying or studying oligosaccharide-binding partner interactions, identifying and characterizing structural features on oligosaccharides, identifying and characterizing binding partners, identifying agents capable of interfering with, enhancing, or facilitating the binding of an oligosaccharide to its binding partner, diagnosing conditions associated with altered oligosaccharide-binding partner binding, and generating oligosaccharide libraries and kits therefor.

Methods for determining oligosaccharide binding

The present invention provides methods for the production of N-deacetylate N-sulfate derivatives of non-sulfated N-acetyl heparosan (HS) polysaccharides, compounds thus obtained and compositions comprising same. This invention also provides applications of N-deacetylate N-sulfate derivatives of non-sulfated N-acetyl heparosan (HS) polysaccharides, and compositions comprising same, for use in controlling coagulation and treating thrombosis.

Rapid two-step synthesis of anti-coagulants

Disclosed are methods of 6-O-sulfating glucosaminyl N-acetylglucosamine residues (GlcNAc) in a polysaccharide preparation and methods of converting anticoagulant-inactive heparan sulfate to anticoagulant-active heparan sulfate and substantially pure polysaccharide preparations may by such methods. Also disclosed is a mutant CHO cell which hyper-produces anticoagulant-active heparan sulfate. Methods for elucidating the sequence of activity of enzymes in a biosynthetic pathway are provided.

6-O sulfated polysaccharides and methods of preparation thereof

The present invention provides methods, processes and reaction mixtures, which produce sulfated heparosan polysaccharides. This invention also provides methods and reaction mixtures for the synthesis of N-deacetylate N-sulfate derivatives of non-sulfated N-acetyl heparosan (HS) polysaccharides.

Methods for synthesis of sulfated saccharides

An animal selected for lacking heparan sulfate 3-O-sulfotransferase-1 activity is provided. This animal exhibits characteristics associated with myxomatous valvular disease and is useful for identifying agents which prevent, delay or treat myxomatous valvular disease. Methods of diagnosing myxomatous valvular disease are also provided.

Mouse model of myxomatous valvular disease

Disclosed herein are polysaccharide preparations enriched in 3-OST-3 modified heparan sulfate. Also disclosed are methods of treating herpes simplex viral type-1 infection using the pharmaceutical preparations of the invention.

Pharmaceutical preparations for the inhibition of herpes simplex virus 1 entry

Disclosed are novel isolated nucleic acids and substantially pure protein preparations for naturally occurring and synthetic or chimeric heparan sulfate D-glicosaminyl 3-O-sulfo-transferases (3-OSTs). Also disclosed are uses for these genes and proteins, including uses for the modification and sequencing of glycosaminoglycans.

Heparan sulfate D-glucosaminyl 3-O-sulfotransferases, and uses therefor

Molecularly-mediated and cellular-based &bgr;-adrenergic receptor-dependent biological pacemakers are disclosed. Methods of using these compositions to improve cardiac chrontropic responsiveness by upregulating heart rate and altering cardiac rhythm are also disclosed.

Enhancement of cardiac chronotropy

Disclosed is a method for localized application of antisense oligonucleotides, which has been found to be effective in inhibiting expression and translation of a variety of genes. The method utilizes antisense oligonucleotides which are specific for the mRNA transcribed from the gene of interest. The antisense oligonucleotides are applied directly to the desired locus of the cells to be treated, where they hybridize with the mRNA and inhibit expression of the gene. Devices for localized antisense application and methods for making them also are described.

Localized oligonucleotide therapy

New therapies for treating blood platelet disorders based on the megapoietin gene and protein are described. Additional utilities for megapoietin such as increasing the storage life of platelet and whole blood preparations, a means for selectively targeting therapeutic or imaging agents to arterial clots, and a means for selectively stimulating platelet production from megakaryocytes in vivo and in vitro (e.g., as a source of platelets for transplantation) and to stimulate stem cell growth are also described.

Method of using megapoietin for prolonging the survival & viability of

The present invention relates to the isolation of genes encoding novel protein tyrosine phosphatases (PTPs) having SH2 domains, the nucleic acid sequences isolated, and the encoded phosphatases. The invention further relates to methods of altering tyrosine phosphatase activities encoded by the novel phosphatases. By altering (i.e., increasing or decreasing) tyrosine phosphatase activity, one can alter megakaryocyte cell function, and thereby alter platelet production. Alteration of the genes is associated with neoplastic disease.

Methods for identifying a tyrosine phosphatase abnormality associated

A megakaryocyte stimulatory factor (MSF), purified to homogeneity, is an acidic protein (pI=5.1) with an Mr=15,000 which stimulates PF4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF exhibits no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage. In the given examples, MSF was purified to homogeneity (as judged by SDS-PAGE and isoelectric focusing in the presence of 9.2 M urea) from serum-free conditioned medium obtained from cultured human embryonic kidney (HEK) cells, and to near homogeneity from thrombocytopenic plasma. The MSF is isolated by precipitating the MSF with ammonium sulfate at 80% saturation, removing insoluble, non-MSF material and applying the soluble protein to a WGA-Sepharose column, eluting the MSF protein with chitin oligosaccharides, applying the concentrated eluant containing MSF activity to a Biogel P200 column, and chromatographing the eluted MSF fractions on a TSK-G3000 HPLC size exclusion column.

Megakaryocyte stimulatory factor

The present invention relates to a process for separating and purifying proteases and antiproteases. This process is characterized in that there are placed in contact an insoluble cross-linked polymer including in its chain the group --SO.sub.3 R.sub.1 -- and/or the group --SO.sub.2 R.sub.2 -- in which R.sub.1 denotes a hydrogen atom or metal and R.sub.2 denotes the remainder of an amino acid linked to the --SO.sub.2 -- bridge through its amine --NH--, function, with the solution containing proteases and antiproteases or their complex; separating the polymer which has absorbed the desired protein, washing it carefully with the buffer, desorbing the absorbed protein by a solution of a polycationic compound in the case of T or by an albumin solution in the case of AT or of the complex T-AT, and isolating the protein, if desired, by known means, such as, especially, freeze drying. The invention is useful for studying the mechanism of blood coagulation.

Process for the separation and purification of proteases and

A megakarytocyte stimulatory factor (MSF), purified to homogeneity, is an acidic protein (pI=5.1) with an Mr=15,000 which stimulates PF4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF exhibits no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage. In the given examples, MSF was purified to homogeneity (as judged by SDS-PAGE and isoelectric focusing in the presence of 9.2 M urea) from serum-free conditioned medium obtained from cultured human embryonic kidney (HEK) cells, and to near homogeneity from thrombocytopenic plasma. The MSF is isolated by precipitating the MSF with ammonium sulfate at 80% saturation, removing insoluble, non-MSF material and applying the soluble protein to a WGA-Sepharose column, eluting the MSF protein with chitin oligosaccharides, applying the concentrated eluant containing MSF activity to a Biogel P200 column, and chromatographing the eluted MSF fractions on a TSK-G3000 HPLC size exclusion column.

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Boston, MA, United States of America

Robert D Rosenberg