A comparative analysis of heterogeneity in commercially available recombinant factor VIII products.

INTRODUCTION: Advances in analytical technologies enable investigation of possible correlations between molecular structure, aggregation and subvisible particle content. Regulatory agencies place increasing attention on potential risks associated with protein aggregates in the micron range in biological therapeutics. AIM: Assess the heterogeneity, high-molecular-weight protein (HMWP) species, subvisible particle content and posttranslational modifications in six commercially available recombinant FVIII (rFVIII) products. METHODS: Three B-domain-deleted (BDD) or B-domain truncated rFVIII products (turoctocog alfa, simoctocog alfa and moroctocog alfa) and three full-length rFVIII products (octocog alfa FS and two octocog alfa) were analysed. HMWP content, amount of micron range subvisible particles, tyrosine-1680 sulphation and N-glycan analysis were investigated. RESULTS: The B-domain-modified products had more protein size homogeneity vs the full-length products. Size exclusion-high-performance liquid chromatography data indicated no association between B-domain structure and aggregate content or size of the products tested. The rFVIII products showed large variation in subvisible particle concentration, with turoctocog alfa and simoctocog alfa having the lowest numbers (1000-1600 and 1800-2400 particles/100 IU, respectively). Turoctocog alfa and simoctocog alfa displayed the most complete tyrosine sulphation (>99.5%). CONCLUSION: Overall, there was no association between molecular structure (full-length B-domain, BDD or truncated) and subvisible particle or HMWP content. Dissimilarities may be related to production and product handling differences. In this study, turoctocog alfa, such as simoctocog alfa, had one of the lowest levels of subvisible particles and HMWP content, and high protein size homogeneity.

Fast and direct measurements of the electrical properties of graphene using micro four-point probes.

We present measurements of the electronic properties of graphene using a repositionable micro four-point probe system, which we show here to have unique advantages over measurements made on lithographically defined devices; namely speed, simplicity and lack of a need to pattern graphene. Measurements are performed in ambient, vacuum and controlled environmental conditions using an environmental scanning electron microscope (SEM). The results are comparable to previous results for microcleaved graphene on silicon dioxide (SiO(2)). We observe a pronounced hysteresis of the charge neutrality point, dependent on the sweep rate of the gate voltage; and environmental measurements provide insight into the sensor application prospects of graphene. The method offers a fast, local and non-destructive technique for electronic measurements on graphene, which can be positioned freely on a graphene flake.

Is the ACT two minutes after heparin injection reliable?

BACKGROUND: Blood for activated clotting time (ACT) measurement to verify the effect of the initial dose of heparin before cannulation in heart surgery has traditionally been drawn 5 minutes (min) after injection of the heparin. However, there has been an increasing demand to reduce the waiting time. The aim of this study was to investigate if ACT measured 1, 2, 3 and 4 min after heparin injection is as reliable as ACT measured 5 min after heparin injection. MATERIALS AND METHODS: Fifty adult patients undergoing routine cardiac surgery with a heart-lung machine. Heparinization was obtained with unfractioned porcine heparin. The ACT was measured with 5 Hemochron® Jr. machines 1, 2, 3, 4 and 5 min after the heparin injection. Full heparinization was defined as an ACT >400 seconds. RESULTS: At 1 and 2 min, 94% (n=47) of the ACTs were > 400. All ACTs >400 seconds after 2 min remained >400 seconds at 3, 4 and 5 min. Mean values declined from 533 to 498. ANOVA analysis showed statistically significantly higher values at 1, 2 and 3 min, compared to 5 min, but not at 4 min. However, the estimated differences were small: 3.7-36 seconds. There was no significant difference between variances for the five sample times. Standard deviation declined from 123 to 100. Values at 2 min correlated as well as those at 5 min with mean 1-5 min values. CONCLUSION: The range of the ACT values tends to diminish over time and, consequently, the reliability of the results increases. However, the difference is small and has little or no clinical relevance. Giving time for the circulation to distribute the heparin in the bloodstream, we recommend measuring the ACT two min after heparin administration.

A method for visualizing CellML models.

MOTIVATION: The Physiome Project was established in 1997 to develop tools to facilitate international collaboration in the physiological sciences and the sharing of biological models and experimental data. The CellML language was developed to represent and exchange mathematical models of biological processes. CellML models can be very complicated, making it difficult to interpret the underlying physical and biological concepts and relationships captured/described in the mathematical model. RESULTS: To address this issue a set of ontologies was developed to explicitly annotate the biophysical concepts represented in the CellML models. This article presents a framework that combines a visual language, together with CellML ontologies, to support the visualization of the underlying physical and biological concepts described by the mathematical model and also their relationships with the CellML model. Automated CellML model visualization assists in the interpretation of model concepts and facilitates model communication and exchange between different communities.

Biophysical annotation and representation of CellML models.

MOTIVATION: CellML is an implementation-independent model description language for specifying and exchanging biological processes. The focus of CellML is the representation of mathematical formulations of biological processes. The language captures the mathematical and model building constructs well, but does not lend itself to capturing the biology these models represent. RESULTS: This article describes the development of an ontological framework for annotating CellML models with biophysical concepts. We demonstrate that, by using these ontological mappings, in combination with a set of graph reduction rules, it is possible to represent the underlying biological process described in a CellML model.

Facilitating modularity and reuse: guidelines for structuring CellML 1.1 models by isolating common biophysical concepts.

The CellML language was developed in response to the need for a high-level language to represent and exchange mathematical models of biological processes. The flexible structure of CellML allows modellers to construct mathematical models of the same biological system in many different ways. However, some modelling styles do not naturally lead to clear abstractions of the biophysical concepts and produce CellML models that are hard to understand and from which it is difficult to isolate parts that may be useful for constructing other models. In this article, we advocate building CellML models which isolate common biophysical concepts and, using these, to build mathematical models of biological processes that provide a close correspondence between the CellML model and the underlying biological process. Subsequently, models of higher complexity can be constructed by reusing these modularized CellML models in part or in whole. Development of CellML models that best describe the underlying biophysical concepts thus avoids the need to code models from scratch and enhances the extensibility, reusability, consistency and interpretation of the models.

Influence of venous reservoir level on microbubbles in cardiopulmonary bypass.

Patients undergoing open-heart surgery may, post-operatively, suffer from neurological disorders due to microbubbles created during extracorporeal circulation. Venous air is not completely removed in open hard-shell venous reservoirs. We, therefore, investigated the relationship between venous reservoir blood level and the amount of microbubbles in different commercially available reservoirs for comparison and determination of safe level. An in vitro flow loop with a heart-lung machine was used to compare three different reservoirs (Maquet, Sorin and Medtronic) at different levels. Microbubbles were measured after the reservoir and after the arterial filter with a GAMPT BCC200 detector. Microbubble count and volume were significantly higher with decreasing reservoir level (p = 0.014), but not as much as earlier studies have shown. Reducing the level from 1000 ml to 250 ml resulted in a 12.4% increase in bubble volume after the reservoir and 40.2% after the arterial filter. There was an almost linear trend towards more bubble volume with decreasing reservoir level (R2 = 0.98-0.83). There was a significant difference in microbubbles between the 3 tested reservoirs, up to 32.6%, p < 0.001 measured after the reservoir. Bubble volume from the Sorin reservoir was markedly lower after the arterial filter than from the Medtronic and Maquet reservoirs (up to 60 times p < 0.001). A lower reservoir level results in a moderate rise in microbubbles passing the reservoir. The minimum levels recommended by the manufacturers are safe. There was a significant difference in bubbles between the different reservoirs, especially after the arterial filter.

The evolution of CellML.

CellML is an open XML-based markup language for describing and exchanging mathematical models of biological processes. While CellML was originally designed to describe and exchange models of cellular and subcellular processes, the design principles that were applied to the construction of a language with this relatively narrow focus are equally applicable to the specification of a language with a much wider scope. In this paper we describe the structure of CellML, how the language is evolving to broaden its scope, and what tools are being developed to facilitate its use.

Isolation of peptides of the brevinin-1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii.

The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions from the foothill yellow-legged frog Rana boylii. Brevinin-1BYa (FLPILASLAA10KFGPKLF CLV20TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 microm] and also active against Escherichia coli (MIC = 17 microm) and Staphylococcus aureus (MIC = 2 microm), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC50 = 4 microm). The single amino acid substitution (Phe12 --> Leu) in brevinin-1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys11 --> Thr, Phe17 --> Leu and Val20 --> Ile) in brevinin-1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin-2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.

The systems biology markup language (SBML): a medium for representation and exchange of biochemical network models.

MOTIVATION: Molecular biotechnology now makes it possible to build elaborate systems models, but the systems biology community needs information standards if models are to be shared, evaluated and developed cooperatively. RESULTS: We summarize the Systems Biology Markup Language (SBML) Level 1, a free, open, XML-based format for representing biochemical reaction networks. SBML is a software-independent language for describing models common to research in many areas of computational biology, including cell signaling pathways, metabolic pathways, gene regulation, and others. AVAILABILITY: The specification of SBML Level 1 is freely available from http://www.sbml.org/

Location of crosslinks in chemically stabilized horseradish peroxidase: implications for design of crosslinks.

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker.

Human pS2/trefoil factor 1: production and characterization in Pichia pastoris.

The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris. hTFF1 was secreted in large amounts in the extracellular medium of P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography. The resulting hTFF1 was found to be intact by Western blot analysis. Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide. Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells.

Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration.

A series of very potent derivatives of the 30-amino acid peptide hormone glucagon-like peptide-1 (GLP-1) is described. The compounds were all derivatized with fatty acids in order to protract their action by facilitating binding to serum albumin. GLP-1 had a potency (EC(50)) of 55 pM for the cloned human GLP-1 receptor. Many of the compounds had similar or even higher potencies, despite quite large substituents. All compounds derivatized with fatty acids equal to or longer than 12 carbon atoms were very protracted compared to GLP-1 and thus seem suitable for once daily administration to type 2 diabetic patients. A structure-activity relationship was obtained. GLP-1 could be derivatized with linear fatty acids up to the length of 16 carbon atoms, sometimes longer, almost anywhere in the C-terminal part without considerable loss of potency. Derivatization with two fatty acid substituents led to a considerable loss of potency. A structure-activity relationship on derivatization of specific amino acids generally was obtained. It was found that the longer the fatty acid, the more potency was lost. Simultaneous modification of the N-terminus (in order to obtain better metabolic stability) interfered with fatty acid derivatization and led to loss of potency.

Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26.

A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC ), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26(-/-) mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26(-/-), mice. This inhibitor also improved glucose tolerance in GLP-1 receptor(-/-) mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.

Tachykinins (substance P and neuropeptide gamma) from the brains of the pallid sturgeon, Scaphirhynchus albus and the paddlefish, Polyodon spathula (Acipenseriformes).

A peptide with substance P-like immunoreactivity was isolated from extracts of the brains of the pallid sturgeon, Scaphirhynchus albus and the North American paddlefish, Polyodon spathula. The primary structure of the peptide (Lys-Pro-Lys-Pro-His-Gln-Phe-Phe-Gly-Leu-Met.NH(2)) is the same in both species and contains 2 amino acid substitutions (Arg(1) --> Lys and Gln(5) --> His) compared with human substance P and 1 substitution (Arg(3) --> Lys) compared with substance P from the trout (Teleostei). Scyliorhinin I, a tachykinin previously isolated from an extract of sturgeon intestine, was not detected in either brain extract. A peptide with neurokinin A-like immunoreactivity (Ser-Ser-Ala-Asn-Arg-Gln-Ile-Thr-Gly-Lys(10)Arg-Gln-Lys-Ile-Asn-Ser-P he-Val-Gly-Leu(20)Met.NH(2)) was isolated from sturgeon brain and contains 10 amino acid substitutions compared with human neuropeptide gamma (a specific product of the posttranslational processing of gamma-preprotachykinin A) but only 4 substitutions compared with trout neuropeptide gamma. It was not possible to obtain the paddlefish neurokinin A-related peptide in pure form. The structural similarity between the sturgeon and the trout tachykinins supports the hypothesis that the Acipenseriformes (sturgeons and paddlefish) represent the sister group of the Neopterygii (gars, bowfin, and teleosts).

Neuroendocrine peptides (insulin, pancreatic polypeptide, neuropeptide Y, galanin, somatostatin, substance P, and neuropeptide gamma) from the desert tortoise, Gopherus agassizii.

The traditional view that Testudines (tortoises and turtles) should be regarded as the surviving clade of the anapsid reptiles rather than classified with the diapsid reptiles (snakes, lizards, and crocodiles) has recently been challenged. Neuropeptide Y, neuropeptide gamma, and somatostatin-14 were isolated from an extract of the brain, substance P and galanin from an extract of the intestine, and insulin and pancreatic polypeptide from an extract of the pancreas of the desert tortoise, Gopherus agassizii. Despite that crocodilians did not appear until the late Triassic, the amino acid sequences of the tortoise peptides resemble those of the American alligator quite closely. The primary structures of neuropeptide Y, somatostatin-14, and neuropeptide gamma are the same in tortoise and alligator. The primary structures of substance P, insulin, galanin, and pancreatic polypeptide in the two species differ by 1, 3, 5, and 8 amino acid residues, respectively. Although fewer neurohormonal peptides from squamates (lizards and snakes) have been characterized, the primary structures of neuropeptide gamma, insulin, and pancreatic polypeptide from the Burmese python and the desert tortoise differ by 3, 8, and 18 residues, respectively. The data suggest, therefore, a closer phylogenetic relationship between Testudines and Crocodilians than that derived from 'classical' analyses based on morphological criteria and the fossil record.

Molecular evolution of peptide tyrosine--tyrosine: primary structure of PYY from the lampreys Geotria australis and Lampetra fluviatilis, bichir, python and desert tortoise.

Peptide tyrosine-tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10/DAPPEELAKY20/YSALR HYINL30/ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10-->Gln and Leu22-->Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (Söderberg et al. J. Neurosci. Res. 1994;37:633-640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10-->Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.

Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.

Multiple forms of glucagon and somatostatin isolated from the intestine of the southern-hemisphere lamprey Geotria australis.

Current views on Agnathan phylogeny favor the hypothesis that the genera of holarctic lampreys belong to a single family (Petromyzontidae) and form an interrelated progression in which Petromyzon is near to Ichthomyzon at the base of the phylogenetic tree and Lampetra is the most derived. A stock similar to that of contemporary Ichthomyzon is considered to have given rise to the southern hemisphere lamprey Geotria australis, the sole member of the Geotriidae. In the present study, two molecular forms of glucagon were isolated from an extract of G. australis intestine that differed in structure by six amino acid residues. One form shows two amino acid substitutions (Leu14 --> Met and Ala29 --> Ser) compared with the single molecular form of glucagon isolated from the sea lamprey Petromyzon marinus and the second form shows three substitutions (Asp15 --> Glu, Ser16 --> Ala, Ile24 --> Thr) compared with the single glucagon isolated from the river lamprey Lampetra fluviatilis. As Petromyzon and Lampetra glucagons differ by six amino acid residues, the data suggest that a duplication of the glucagon gene occurred prior to or early in lamprey evolution. Although both genes are strongly expressed in G. australis, the expression of one gene predominates in P. marinus while that of the other gene predominates in L. fluviatilis. Previous work has shown that, in the islet organ of G. australis, preprosomatostatin is processed almost exclusively to somatostatin-33. However, the present study demonstrates that somatostatin-14 is the major molecular form in G. australis intestine with somatostatin-33 present only as a minor component. This result demonstrates a tissue-dependent pathway of posttranslational processing of preprosomatostatin in the Geotria enteropancreatic system.