Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition

Thiol-functionalized dextrans (dex-SH) (M(n,dextran) = 14K or 31K) with degrees of substitution (DS) ranging from 12 to 25 were synthesized and investigated for in situ hydrogel formation via Michael type addition using poly(ethylene glycol) tetra-acrylate (PEG-4-Acr) or a dextran vinyl sulfone conjugate with DS 10 (dex-VS DS 10). Dex-SH was prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl chloroformate and subsequent reaction with cysteamine. Hydrogels were rapidly formed in situ under physiological conditions upon mixing aqueous solutions of dex-SH and either PEG-4-Acr or dex-VS DS 10 at polymer concentrations of 10 to 20 w/v%. Rheological studies showed that these hydrogels are highly elastic. By varying the DS, concentration, dextran molecular weight, and type of cross-linker, hydrogels with a broad range of storage moduli of 9 to 100 kPa could be obtained. Varying the ratio of thiol to vinyl sulfone groups from 0.9 to 1.1 did not alter the storage modulus of the hydrogels, whereas larger deviations from equimolarity (thiol to vinyl sulfone ratios of 0.75 and 1.5) considerably decreased the storage modulus. The plateau value of hydrogel storage modulus was reached much faster at pH 7.4 compared to pH 7, due to a higher concentration of the thiolate anion at higher pH. These hydrogels were degradable under physiological conditions. Degradation times were 3 to 7 weeks for dex-SH/dex-VS DS 10 hydrogels and 7 to over 21 weeks for dex-SH/PEG-4-Acr hydrogels, depending on the DS, concentration, and dextran molecular weight.     

Hox genes from the earthworm Perionyx excavatus

The Hox genes of the oligochaete, Perionyx excavatus, were surveyed using PCR and phylogenetic analysis. We were able to identify 11 different Hox gene fragments. Comparative and phylogenetic analyses revealed that this oligochaete would have at least five Hox genes of the anterior group, including three copies of labial-type, five of the central group and one of the posterior group. This is the first report regarding sequence information and phylogenetic analysis of Hox genes in the earthworm.     

Distribution of catecholamine-containing nerve cells in the walls of the major vessels of the lamprey, Lampetra fluviatilis

Using histochemical fluorescence technique, two types of adrenergic nervous cells, i.e. small with intense fluorescence and large with weak fluorescence, have been detected in tissues and walls of the main blood vessels in the lamprey Lampetra fluviatilis. The first type of cells is distributed along the whole vascular net concentrating mainly in branching nodes. The second type of cells was found only in the adventitial layer of the dorsal aorta and intestinal artery. Adrenergic nervous cells are also present in the walls of the main veins. Possibility of presence of catecholamines of different nature in the bodies of the nerve cells is discussed.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Joint axes of rotation and body segment parameters of pig limbs

To enable a quantification of net joint moments and joint reaction forces, indicators of joint loading, this study aimed to locate the mediolateral joint axes of rotation and establish the body segment parameters of the limbs of pigs (Sus scrofa). To locate the joint axes of rotation the scapulohumeral, humeroradial, carpal complex, metacarpophalangeal, coxofemoral, femorotibial, tarsal, and metatarsophalangeal joints from 12 carcasses were studied. The joints were photographed in three positions, bisecting lines drawn at fixed landmarks with their intersection marking the joint axes of rotation. The body segment parameters, i.e. the segment mass, center of mass and moment of inertia were measured on the humerus, radius/ulna, metacarpus, forepastern, foretoe, femur, tibia, metatarsus, hindpastern, and hindtoe segments from five carcasses. The segments were weighed, and their center of mass was found by balancing them. The moments of inertia of the humerus, radius/ulna, femur and tibia were found by rotating the segments. The moments of inertia of the remaining segments were calculated. Generally, the joint axes of rotation were near the attachment site of the lateral collateral ligaments. The forelimb, with segments taken as one, was significantly lighter and shorter than the hindlimb (P < 0.001). In all segments the center of mass was located 31 to 50% distal to the proximal segment end. The segment mass decreased with distance from the trunk, as did the segment moment of inertia. The results may serve as reference on the location of the joint axes of rotation and on the body segment parameters for inverse dynamic modeling of pigs.     

HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H(2)N-(His)(6)-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO(2)H, which consists of four functional elements: i) an affinity ligand (His(6)-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d(4)) or no (d(0)) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d(4)) or (d(0)) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d(4) co-elute with their d(0)-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni(2+) affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of (13)C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.