Application of L-threonine aldolase-catalyzed reaction to the preparation of protected 3R,5R-dihydroxy-L-homoproline as a mimetic of idulonic acid

A facile synthesis of 3R,5R-dihydroxy-L-homoproline as idulonic acid mimic, of which the carboxyl and 3-hydroxyl groups were protected, was attained using L-threonine aldolase-catalyzed reaction. Idulonic acid is a key acidic sugar of the b-FGF binding domain in heparin and heparan sulfate. Moreover, the synthetic precursor of N-acetyl-4-deoxy-D-mannosamine, which is a potent inhibitor of NeuAc synthase, was prepared from the side product of the enzymatic aldol condensation.     

Heparin binding by the HIV-1 tat protein transduction domain

The protein transduction domain from the HIV-1 tat protein (termed PTD-tat) has been fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G. We demonstrate that PG-Ctat (PTD-tat fused to the C-terminus of protein G) binds to a heparin affinity column. PG-Ctat binds with relatively high affinity, as shown by its elution at 1.6 M NaCl. The heparin binding properties of PTD-tat are consistent with the idea that heparan sulfate, an analog of heparin found at the cell surface, plays a role in the translocation of PTD-tat fusions. We suggest that the heparin-binding properties of PTD-tat can be exploited for purification of PTD-tat fusions in the absence of affinity tags.     

Calcium oscillations in Xenopus egg cycling extracts

Cell cycle in various types of cells and in early embryos is often accompanied by transient changes in the concentration of free cytosolic calcium. In the present study, using fluorescent indicator fura-2, we demonstrate that Ca(2+) oscillates cyclically with an amplitude of about 100 nM and a period of mitotic cycle in cell-free Xenopus egg cycling extracts. It peaks in early metaphase just preceding mitotic reactivation of Cdc2 kinase and MAPK and reaches a minimum in interphase. The source of Ca(2+) in the extracts is a particulate fraction containing egg intracellular Ca(2+) stores, since the addition of a calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), induced a transient increase in Ca(2+). The inclusion of heparin, an IP3 receptor antagonist, or ultrafiltration of the extracts prevented Ca(2+)-releasing activity of IP3. The depletion of Ca(2+) in the extracts by the calcium chelator BAPTA resulted in the blockade of cell cycle at different stages, depending on the time of drug administration. The addition of BAPTA late in interphase blocked cell cycle at mitotic entry in prophase, whereas its application in anaphase or telophase blocked the extracts in early interphase. BAPTA administration in metaphase before transition to anaphase brought about a metaphase-like arrest in the cycling extracts. Inhibition of IP3-induced calcium release by heparin also arrested cell cycle progression in the cycling extracts.     

Bloodmeal digestion and Leishmania major infections in Phlebotomus duboscqi: effect of carbohydrates inhibiting midgut lectin activity

The carbohydrates galactosamine and heparin, previously shown to inhibit phlebotomine lectin activity in vitro, were fed to the sandfly Phlebotomus duboscqi Neveu-Lemaire (Diptera: Psychodidae) with blood, and the effects on mortality, fecundity, protease activity and susceptibility to Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae) were studied. Previous study revealed that galactosamine considerably enhanced the establishment of L. major infection in P. duboscqi and significantly increased parasite loads in late infections. This work demonstrates a similar but less pronounced effect of heparin. Heparin increased infection rates and parasite loads 3 and 9 days post-feeding but did not affect the location of Leishmania promastigotes and their anterior migration. Galactosamine supplement caused pronounced changes in bloodmeal digestion. It abolished the activity of alkaline proteases and trypsin, caused premature defecation of bloodmeal, increased mortality of female sandflies in days 1-4 post-feeding and decreased their fecundity. Heparin had a less pronounced effect on sandfly physiology. It lowered trypsin activity 12 and 72 h post-bloodmeal but did not alter defecation, mortality and oviposition. The data suggest that the enhancing effect of these carbohydrates on Leishmania infections in sandfly midgut could be explained by their interference with midgut proteases. The study supports the hypothesis that proteolytic activities of midgut proteases strongly influence the vector competence of sandflies.     

Analysis of sulfates on low molecular weight heparin using mass spectrometry: structural characterization of enoxaparin

Introduction: Structural characterization of low molecular weight heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly enoxaparin.

Areas covered: This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Furthermore, popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as ‘enoxaparin’, ‘mass spectrometry’, ‘low molecular weight heparin’, ‘structural characterization’, etc.

Expert commentary: This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-α and interleukin-1β contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Enzymatic Improvement of Food Flavor I. Purification and Characterization of Bovine Liver Mitochondrial Aldehyde Dehydrogenase

Bovine liver mitochondrial aldehyde dehydrogenase (aldehyde: NAD⁺ oxidoreductase, EC 1.2.1.3) has been purified to homogeneity by conventional purification procedures. The enzyme was found to have a molecular weight of 215,000 based on gel filtration. The protein is composed of polypeptides having the same molecular weight, 54,000 and thus it appears to consist of four subunits of equal size. The enzyme exhibited a broad aldehyde specificity, oxidizing irreversibly a wide variety of aliphatic and aromatic aldehydes to corresponding carboxylic acids. Km values for straight-chain saturated aldehydes were below 0.1 µm, and relatively constant independent of the carbon chain lengths of the aldehydes. The maximum velocities for saturated aldehydes also did not vary appreciably with their carbon chain lengths. Maximum activity was observed at pH 9.3 and 50°C. The enzyme activity was affected by some divalent cations. Ca²⁺ enhanced the activity, while Mg²⁺ inhibited it. The enzyme was quite stable at neutral pH, but was unstable above pH 9 or below pH 6. Bovine liver has three isozymes of aldehyde dehydrogenase which are located in the mitochondrial, cytosolic, and microsomal fractions. Comparison of enzymic properties among these isozymes and yeast enzyme indicates that the mitochondrial enzyme is very suitable for improving the objectionable flavor due to aldehydes in foods.     

A Systems View of the Heparan Sulfate Interactome

Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate–binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called “heparan sulfate interactome,” with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate–binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.     

Structural insight into the evolution of a new chemokine family from zebrafish

The mammalian chemokine family is segregated into four families – CC, CXC, CX3C, and XC—based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N‐terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur‐SAD phasing to determine the three‐dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin‐binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family. Proteins 2014; 82:708–716. © 2013 Wiley Periodicals, Inc.     

A simple venous thromboembolism prophylaxis protocol for patients undergoing bariatric surgery

Objective: Pulmonary embolism is a leading cause of death for bariatric patients. Numerous regimens have been proposed, but a comprehensive, simple approach is lacking. This study provides a simple, easily implemented prophylaxis regimen. Research Methods and Procedures: One hundred fifty bariatric surgery patients were evaluated. Patients considered at high risk for venous thromboembolism had heart failure, a BMI of ≥50 kg/m², or a history of venous thromboembolism or pelvic surgery. Preoperatively and postoperatively, all patients received subcutaneous enoxaparin or unfractionated heparin. High‐risk patients received either preoperatively inserted inferior vena cava filters or continuous heparin infusions intraoperatively. All high‐risk patients were anticoagulated with warfarin (Coumadin; Bristol Myers‐Squibb, Princeton, NJ) for at least 3 months postoperatively. Initially, some patients experienced significant hemorrhage; to prevent this, sutures were oversewn into staple lines. Results: No patient experienced venous thromboembolism; a binomial test showed that the regimen reduced the risk of this complication to less than 2% (p < 0.05). Hemorrhage sufficient to require transfusion occurred in 4 of the first 20 patients; of the remaining 130 patients, into whose staple lines sutures were oversewn, none required transfusion (p < 0.05). Discussion: Patients should be divided into those who are at high risk and those who are at low risk for venous thromboembolism. All patients should receive pre‐ and postoperative anticoagulation. High‐risk patients should also receive either an inferior vena cava filter or intraoperative heparin infusions, as well as at least 3 months of Coumadin therapy. Oversewing of staple lines may reduce the risk of hemorrhage.