Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin‐dependent kinase II

Calmodulin (CaM) functions depend on interactions with CaM‐binding proteins, regulated by . Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM‐binding region of /calmodulin‐dependent kinase II (CaMKII_290−309) have been studied using biophysical methods. These proteins have opposite dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that and CaM interact very rapidly, and with moderate affinity ( ). Calmodulin‐CaMKII_290−309 interactions were only detected in the presence of , exhibiting fast kinetics and nanomolar affinity ( ). The CaM–Ng interaction had higher affinity under ‐depleted ( and k ₋₁ = 1.6 × 10⁻¹s⁻¹) than ‐saturated conditions ( ). The IQ motif of Ng (Ng₂₇₋₅₀) had similar affinity for CaM as Ng under ‐saturated conditions ( ), but no interaction was seen under ‐depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( ) and CaMKII_290−309( ) interactions. Although CaMKII_290−309 showed expected interaction characteristics, they may be different for full‐length CaMKII. The data for full‐length Ng, but not Ng₂₇₋₅₀, agree with the current model on Ng regulation of /CaM signaling.     

Pituitary Secretory Granule Topography: Influence of Different Electron Microscopic Preparation Procedures on the Surface of Epon Sections

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO₄. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Structural and functional studies of differentially O‐glycosylated analogs of a thrombin inhibitory peptide – variegin

Variegin is a 32‐amino acid long thrombin inhibitory peptide isolated from the salivary gland extract of tropical bont tick Amblyomma variegatum. It was identified to be O‐glycosylated on its Thr‐14 side chain, and this glycosylated form was 14‐fold more potent than that of its non‐glycosylated form. However, as the identity of this glycosylation remained elusive, the mechanistic details underlying its functional impact are not yet known. In this report, we synthesized four different O‐glycosylated analogs of variegin bearing physiologically relevant sugars on its Thr‐14. Functional characterization of these analogs by enzyme inhibitory kinetics and surface plasmon resonance methods showed that all the synthesized glycopeptides are strong thrombin inhibitors. Structural studies by macromolecular docking identified that the sugar moiety of these peptides can potentially mediate favorable interactions with amino acids at the base of thrombin's autolysis loop. This report, for the first time, describes the impact of differential glycosylation on the function of a thrombin inhibitory peptide and tries to provide structural insights into the relevance of peptide glycosylation in thrombin inhibition. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

CELL SURFACE ALTERATIONS BY TAXOL ASSOCIATED WITH ABNORMAL MORPHOGENESIS IN THE CHICK EMBRYO

The anticancer drug taxol brings about its biological effects by altering the stability of microtubules. We have examined the effects of taxol on early morphogenesis in chick embryos culturedin vitro. Taxol induced various abnormalities in the developing nervous system, heart and somites as well as general retardation of development. SEM studies revealed that taxol treatment leads to dramatic alterations in the embryonic cell surfaces. Time-course experiments demonstrated that the action of taxol is very rapid and becomes evident within a few minutes at the ultrastructural level. Taxol thus throws embryonic cell adhesion and motility out of balance. This appears to be the major cause of abnormal morphogenesis in taxol-treated embryos.     

Zosteric acid and salicylic acid bound to a low density polyethylene surface successfully control bacterial biofilm formation

The active moieties of the anti-biofilm natural compounds zosteric (ZA) and salicylic (SA) acids have been covalently immobilized on a low density polyethylene (LDPE) surface. The grafting procedure provided new non-toxic eco-friendly materials (LDPE-CA and LDPE-SA) with anti-biofilm properties superior to the conventional biocide-based approaches and with features suitable for applications in challenging fields where the use of antimicrobial agents is limited. Microbiological investigation proved that LDPE-CA and LDPE-SA: (1) reduced Escherichia coli biofilm biomass by up to 61% with a mechanism that did not affect bacterial viability; (2) significantly affected biofilm morphology, decreasing biofilm thickness, roughness, substratum coverage, cell and matrix polysaccharide bio-volumes by >80% and increasing the surface to bio-volume ratio; (3) made the biofilm more susceptible to ampicillin and ethanol. Since no molecules were leached from the surface, they remained constantly effective and below the lethal level; therefore, the risk of inducing resistance was minimized.     

Evidence for the functions of surface‐active behaviors in humpback whales (Megaptera novaeangliae)

As part of their social sound repertoire, migrating humpback whales (Megaptera novaeangliae) perform a large variety of surface‐active behaviors, such as breaching and repetitive slapping of the pectoral fins and tail flukes; however, little is known about what factors influence these behaviors and what their functions might be. We investigated the potential functions of surface‐active behaviors in humpback whale groups by examining the social and environmental contexts in which they occurred. Focal observations on 94 different groups of whales were collected in conjunction with continuous acoustic monitoring, and data on the social and environmental context of each group. We propose that breaching may play a role in communication between distant groups as the probability of observing this behavior decreased significantly when the nearest whale group was within 4,000 m compared to beyond 4,000 m. Involvement in group interactions, such as the splitting of a group or a group joining with other whales, was an important factor in predicting the occurrence of pectoral, fluke, and peduncle slapping, and we suggest that they play a role in close‐range or within‐group communication. This study highlights the potentially important and diverse roles of surface‐active behaviors in the communication of migrating humpback whales.     

Meloidogyne incognita Surface Antigen Epitopes in Infected Arabidopsis Roots

Surface-coat epitopes of Meloidogyne incognita were detected in root tissues of Arabidopsis thaliana during migration and feeding site formation. A whole-mount root technique was used for immunolocalization of surface coat epitopes in A. thaliana, with the aid of a monoclonal antibody raised specifically against the outer surface of infective juveniles of M. incognita. The antibody, which was Meloidogyne-specific, recognized a fucosyl-bearing glycoprotein in the surface coat. During migration in host tissues the surface coat was shed, initially accumulating in the intercellular spaces next to the juvenile and later at cell junctions farther from the nematode. Upon induction of giant cell formation, the antibody bound to proximally located companion cells and sieve elements of the phloem.     

Tuning cellular responses to BMP-2 with material surfaces

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications.