Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering

Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin and Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2 mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.     

Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol

Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included β-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4-epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2011-31 July 2011

This article documents the addition of 112 microsatellite marker loci and 24 pairs of single nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Agelaius phoeniceus, Austrolittorina cincta, Circus cyaneus, Circus macrourus, Circus pygargus, Cryptocoryne × purpurea Ridl. nothovar. purpurea, Mya arenaria, Patagioenas squamosa, Prochilodus mariae, Scylla serrata and Scytalopus speluncae. These loci were cross-tested on the following species: Cryptocoryne × purpurea nothovar. purpurea, Cryptocoryne affinis, Cryptocoryne ciliata, Cryptocoryne cordata var. cordata, Cryptocoryne elliptica, Cryptocoryne griffithii, Cryptocoryne minima, Cryptocoryne nurii and Cryptocoryne schulzei. This article also documents the addition of 24 sequencing primer pairs and 24 allele-specific primers or probes for Aphis glycines.     

Suppression of soft tissue sarcoma growth by a host defense-like lytic peptide

Background

Soft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K₃H₃L₉ on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum.

Methods

In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K₃H₃L₉, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K₃H₃L₉ was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed.

Results

The peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K₃H₃L₉ in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group.

Conclusion

These findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K₃H₃L₉ in athymic and syngeneic mouse models.     

Train shunting at a workshop area

We consider the problem of planning the shunting of train units at a railway workshop area. Before and after the maintenance check, a train unit is parked at a depository track. The problem is to schedule the trains to workshops and depot tracks in order to complete the repairs as soon as possible, while avoiding train blockings at the tracks. We give a formal definition of the problem and present three heuristic approaches based on, respectively, Guided Local Search (GLS), Guided Fast Local Search (GFLS) and Simulated Annealing (SA). Computational experiments are reported for realistic instances. It turns out, that both GLS and SA find within a few minutes solutions that are a few percent from the best MIP solution found.     

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.Human gp340, also known as DMBT1 (deleted in malignant brain tumors 1), belongs to the innate immune protein family of scavenger receptor cysteine-rich (SRCR)² proteins, all of which contain one or more evolutionarily conserved SRCR domain linked to other conserved protein domains (1, 2). Many of these proteins serve as pattern recognition receptors for innate immunity. gp340 is expressed by epithelial cells and cells of the immune system, and its expression is up-regulated after inflammatory stimuli (3, 4). It inhibits bacterial invasion to epithelial cells and the secretion of proinflammatory cytokines (5–7). Thus, it appears to be an important mediator of host immune responses to various microbes and was recently linked to Crohn disease, a human inflammatory bowel disease (8). gp340 is also found in human secretions like tears and saliva, and the salivary form has long been known as salivary agglutinin, which is an important molecule in oral biofilm formation and is suggested to have a role in dental caries development (9–12). The mechanisms of gp340 action in these different biological contexts are not known.Common to all scavenger receptors, the ligand repertoire of gp340 is wide; it binds many different types of bacteria as well as viruses (10, 13, 14). The wide ligand recognition pattern of scavenger receptors is thought to be based on the recognition of common microbial structures, such as lipopolysaccharides and lipoteichoic acids, but in the case of gp340, specific bacterial surface proteins are reported to be involved in the interactions characterized (15–18). Because the importance of gp340/salivary agglutinin in the oral environment has been evident for a long time, most of our knowledge of gp340-microbial interactions is from studies with oral bacteria. For example, viridans streptococci, such as Streptococcus mutans and Streptococcus gordonii, interact with saliva gp340 via their surface proteins AgI/II and the sialic acid-binding Hsa or GspB adhesin. In these interactions, gp340 shows a peculiar fluid phase versus surface-adsorbed behavior, as evidenced by AgI/II polypeptides primarily mediating aggregation of bacteria by fluid phase gp340, whereas the Hsa adhesin primarily mediates adhesion of S. gordonii to surface-bound gp340 (18).gp340 binds also to many non-oral human Gram-negative and Gram-positive pathogens, such as Helicobacter pylori and S. pyogenes (10), but these interactions are less characterized. There are few studies suggesting that both carbohydrates and the protein core of the gp340 can be involved in these interactions. For example, a VEVLXXXXW peptide derived from the SRCR domain of gp340 is shown to bind different types of bacteria (19), whereas sialic acid residues may mediate binding to, for example, influenza virus (20). However, the molecular basis of the ability of gp340 or peptides derived thereof to bind a large ligand repertoire is not understood.The aim of the present study was to use the common human pathogen S. pyogenes as a model bacterium to identify novel bacterial proteins binding to gp340 and in this way shed light on the ligand recognition capability of gp340. We report a novel S. pyogenes-host interaction mediated by bacterial surface protein Spy0843 and gp340. gp340 binds to conserved leucine-rich repeat (Lrr) motifs in the Spy0843 and recognizes the same motif also in other bacterial proteins, from both Gram-negative and Gram-positive bacteria. Moreover, the inhibition of Lrr binding with SRCR-derived peptides differed from that previously reported, which suggests a novel mode for ligand recognition of human scavenger receptor gp340.     

Impact of the biological control agent Phlebiopsis gigantea on its resident genetic structure in the Baltic Sea area

The basidiomycete Phlebiop sis gigantea (Fr.) Jülich has been used in Swedish forestry as a biocontrol agent against the root and butt-rot pathogen Heterobasidion annosum s.l. on freshly cut Picea abies stumps since the early 1990s. Until 2005, the commercial preparation of this biological stump treatment, Rotstop®, has been based on a single strain of P. gigantea that has been applied on more than 47,000 ha annually in Fennoscandia. This paper reports on the spread of genetic material from the Rotstop® biocontrol strain of P. gigantea to resident populations of P. gigantea. We conclude that the inoculated fungus remained to a large extent restricted to the treated plots and did not spread to the adjacent areas, dominated by the natural spore deposition from resident populations of the fungus. Furthermore, the study demonstrates high genetic diversity and low geographic differentiation in P. gigantea populations in the geographical area around the Baltic Sea.     

Investigation of aminopyridiopyrazinones as PDE5 inhibitors: Evaluation of modifications to the central ring system

Efforts to improve the potency and physical properties of the aminopyridiopyrazinone class of PDE5 inhibitors through modification of the core ring system are described. Five new ring systems are evaluated and features that impart improved potency and improved solubility are delineated.