A novel chemically modified analogue of xenin-25 exhibits improved glucose-lowering and insulin-releasing properties

Background: Xenin-25 is a K-cell derived gut peptide with insulin-releasing activity which is rapidly degraded following release into the circulation. We hypothesized that substitution of all naturally-occurring Lys and Arg residues with Gln would lead to prolonged enzyme resistance and enhanced biological efficacy.Methods: Peptide stability was assessed using murine plasma, in vitro insulin-releasing actions evaluated in BRIN-BD11 cells and acute glucose-lowering and insulin-releasing actions examined in high fat fed mice. For sub-chronic studies, a range of metabolic parameters and pancreatic histology were assessed in high fat fed mice which had received saline vehicle or xenin-25(gln) twice-daily for 21 days.Results: In contrast to native xenin-25, xenin-25(gln) was resistant to plasma-mediated degradation and significantly stimulated insulin secretion in BRIN-BD11 cells. Acute administration of xenin-25(gln) in high fat fed mice significantly reduced blood glucose and increased plasma insulin concentrations. Twice-daily administration of xenin-25(gln) in high fat fed mice did not affect food intake, body weight or circulating insulin concentrations but significantly decreased blood glucose from day 9 onwards. Furthermore, glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity and GIP-stimulated insulin-release were significantly enhanced in xenin-25(gln)-treated mice. Pancreatic immunohistochemistry revealed decreased alpha cell area with increased beta cell area and beta-to-alpha cell ratio in xenin-25(gln)-treated mice. In addition, xenin-25(gln) exerted similar beneficial actions in ob/ob mice as demonstrated by reduced blood glucose, superior glycaemic response and glucose-mediated insulin release.Conclusions: Xenin-25(gln) is resistant to plasma-mediated degradation and exerts sustained and beneficial metabolic actions in high fat fed and ob/ob mice.General significance: Glutamine (gln)-modified analogues of xenin may represent an attractive therapeutic approach for type 2 diabetes.     

Chemical and biological properties of a fluorescence derivative of gentamycin

By reaction with 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione a fluorescing gentamycin derivative was produced which is characterized by the following properties: High fluorescence intensity in UV light, which enables simple and sensitive determination up to 10(-9) M. In this way it is possible to determine the localization of this substance in tissues, cells and body fluids. When favorably stored, the derivative is chemically stable. At pH 8 and storage at +5 degrees C in the dark for 27 days the substance loses only 5% of its original fluorescence. In comparison with the nonsubstituted gentamyzin the derivative has a twofold higher binding capacity to serum albumin. The antimicrobial properties are retained; the range of antimicrobial effects is similar to that of gentamycin.     

A novel surface-active peptide derivative exhibits in vitro inhibition of platelet aggregation

A tetrapeptide corresponding to a region of the N-terminal portion of lactotransferrin with hydrophobic alkyl groups at the terminal ends was synthesized and its physicochemical properties as well as its effect on thrombin-stimulated platelet aggregation were examined. The tetrapeptide derivative, in the aggregated state, produced inhibitory effect on platelet aggregation. The concentration dependent activity of the peptide was analyzed in the light of micelle formation, with the micellar aggregate comprising four tetrapeptide units. The unique action of this peptide derivative on the inhibition of platelet aggregation might be useful in the development of potent antithrombotic drugs.     

An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties.

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/microgram; 76 IU/pmol) was significantly greater than that of Epo (352 IU/microgram; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.     

Glucuronoxylomannan exhibits potent immunosuppressive properties

Cryptococcus neoformans is an opportunistic fungus responsible for life-threatening infections in immunocompromised and occasionally in immunocompetent hosts. The fungus is endowed with several virulence factors such as its capsular polysaccharide, which plays a key role in virulence. The major component of capsular material of C. neoformans is glucuronoxylomannan, a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. Here we describe a new aspect relative to glucuronoxylomannan-induced immunosuppression. In this review we analyze the suppressive effects of glucuronoxylomannan and the mechanisms leading to induction of apoptosis in T cells.     

Immunobiological properties of a concanavalin A derivative

A monovalent subunit of concanavalin A (Con A) was tested for mitogenic effects on murine splenic lymphocytes in vitro. In contrast to the effects of intact Con A, the monovalent derivative was not mitogenic at any concentration tested. Furthermore, prior exposure of splenic lymphocytes to monovalent Con A rendered the cells refractory to stimulation by the unmodified lectin.     

Lipase-mediated production of specific lipids with improved biological and physicochemical properties

The use of lipases in the modification of lipids has grown significantly in recent years. This increased interest is mainly due to the ability of these enzymes to catalyze the production of lipids with specific distributions of fatty acids that better fit the current needs of consumers, who are looking for healthier foods that are manufactured with the highest quality. The successful use of lipases to obtain modified lipids with low caloric content, high concentrations of n?3 fatty acids or high amounts of phenolic compounds demonstrate the great potential of these enzymes. The lipase-catalyzed production of lipids with reduced caloric content is made possible by the addition of a medium or a very long chain fatty acid to the triacylglyceride. Diacylglicerols with low caloric content can also be produced using lipases. Due to the deficiency of n?3 fatty acids in the current diet, strategies for the lipase-mediated incorporation of these acids in the TAG have shown promising results. Finally, studies have successfully used lipases for the incorporation of phenolic compounds in the lipid structure, which produce compounds with improved oxidative stability and more beneficial health effects.     

Hormonal regulation of hair follicles exhibits a biological paradox

Hair's importance for insulation and camouflage or human communication means that hairs need to change with season, age or sexual development. Regular, regenerating hair follicle growth cycles produce new hairs which may differ in colour and/or size, e.g., beard development. Hormones of the pineal-hypothalamus-pituitary axis coordinate seasonal changes, while androgens regulate most sexual aspects with paradoxically different effects depending on body site; compare beard growth and balding! Hormones affect follicular mesenchymal-epithelial interactions altering growing time, dermal papilla size and dermal papilla cell, keratinocyte and melanocyte activity. Greater understanding of these mechanisms should improve treatments for poorly controlled hair disorders, alopecia and hirsutism.     

A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC₅₀’s <5 μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold’s history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.     

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.     

A novel MaxiK splice variant exhibits dominant-negative properties for surface expression

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.     

A new psoralen derivative with enlarged antiproliferative properties

Following our results with benzopsoralens as potent photochemotherapeutic agents, we report the antiproliferative evaluation of nitrogenated isoster upon and without UVA irradiation. The evaluated pyridazinopsoralen showed a higher photochemotherapeutic activity with respect to the well-known drug, 8-MOP, and a significant cytotoxicity, also in the dark. This result enlarges the interest in this tetracyclic psoralen derivative skeleton in the search of new anticancer agents.     

Threshold secretory mechanism: A model of derivative element in biological control

A secretory system can be considered a collection of packets of a stored substance, each pecket characterized by a threshold to a stimulatory releasing agent. In terms of macroscopic release of the substance, the rate equation contains explicitly and naturally the time derivative of the stimulus intensity. Elaboration of this general model for threshold secretory mechanisms was motivated by insulin secretion data discussed here. Experimental data on the same type of secretory system (pancreas), the hormone secretion as well as electrical characteristics of the secretory cells, lead to a conjecture that the packets might be identified with the whole secretory cells, rather than the granules or vessicles of the stored hormone.     

A physico-chemical and biological study of novel chitosan-chloroquinoline derivative for biomedical applications

This paper describes an elegant cross-linking technique for the preparation of chitosan-chloroquinoline derivative by using a greener technique. Chitosan solution in aqueous acetic acid was treated with 2-chloroquinoline-3-carbaldehyde solution to form hydrogel; the resulting hydrogel was subjected to solvent exchange. Combining the results of FTIR and XRD confirmed that 2-chloroquinoline-3-carbaldehyde have been reacted to chitosan. The morphology of the derivative was investigated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The thermal stability of the derivative was examined by thermogravimetric analysis (TGA). The photoluminescence (PL) spectra of chitosan-chloroquinoline derivative show red-shifted emission maximum. The microbiological screening has demonstrated the antimicrobial activity of the derivative against bacteria viz. Staphylococcus aureus, Escherichia coli and Candida albicans. The obtained results showed that the chitosan-chloroquinoline derivative might be a promising candidate for novel antimicrobial agents for biomedical applications.     

Starmaker exhibits properties of an intrinsically disordered protein

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.     

A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties

ABSTRACT

Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.     

Synthesis and biological evaluation of alpha-galactosylceramide (KRN7000) and isoglobotrihexosylceramide (iGb3)

Glycoceramides can activate NKT cells by binding with CD1d to produce IFN-gamma, IL-4, and other cytokines. An efficient synthetic pathway for alpha-galactosylceramide (KRN7000) was established by coupling a protected galactose donor to a properly protected ceramide. During the investigation, it was discovered that when the ceramide was protected with benzyl groups, only beta-galactosylceramide was produced from the glycosylation reaction. In contrast, the ceramide with benzoyl protecting groups produced alpha-galactosylceramide. Isoglobotrihexosylceramide (iGb3) was prepared by glycosylation of Galalpha1-3Galbeta1-4Glc donor with 2-azido-sphingosine in high yield. Biological assays on the synthetic KRN7000 and iGb3 were performed using human and murine iNKT cell clones or hybridomas.     

Hydrogen-bond effects on the electronic absorption spectrum and evaluation of nonlinear optical properties of an aminobenzodifuranone derivative that exhibits the largest positive solvatochromism

In this work, for the first time, a theoretical approach to describing the influence of hydrogen-bond formation on the electronic absorption spectrum and nonlinear optical properties of an aminobenzodifuranone derivative (ABF) that exhibits the largest positive solvatochromic shift compared to other known chromophores is given. The solvent effect was included via the supermolecule (SM) method. The calculations were performed for a strong low-lying (^*) transition based on the configuration interaction singles (CIS) and time-dependent DFT (TDDFT) methods. The first-order hyperpolarizabilities () were computed using the finite-field (FF) technique combined with the Hartree–Fock (HF) theory. Reasonable agreement between theory and experiment was obtained for the solvatochromic shifts of the ABF molecule. Moreover, it was found that H-bond formation strongly influences the NLO response of the systems investigated.Figure The interaction difference-density maps of the systems studied: a II - complex ABF with NFTB; b III - complex ABF with HMPA. The red color designates an increase of the electron density caused by the intermolecular interactions, whereas blue indicates a corresponding decrease of the electron density. The isodensity contours were plotted for ±0.01 electron/bohr³ (DFT/B3LYP/6-31G(d,p)).     

Mesothelium of the murine allantois exhibits distinct regional properties

The rodent allantois is thought to be unique amongst mammals in not having an endodermal component. Here, we have investigated the mesothelium, or outer surface, of murine umbilical precursor tissue, the allantois (～7.25–8.5 days postcoitum, dpc) to discover whether it exhibits the properties of an epithelium. A combination of morphology, challenge with biotinylated dextran amines (BDAs), and immunohistochemistry revealed that the mesothelium of the mouse allantois exhibits distinct regional properties. By headfold stages (～7.75–8.0 dpc), distal mesothelium was generally squamous in shape, and highly permeable to BDA challenge, whereas ventral proximal mesothelium, referred to as “ventral cuboidal mesothelium” (VCM) for the characteristic cuboidal shape of its cells, was relatively impermeable. Although “dorsal cuboidal mesothelium” (DCM) resembled the VCM in cell shape, its permeability to BDA was intermediate between the other two regions. Results of immunostaining for Zonula Occludens‐1 (ZO‐1) and Epithelial‐cadherin (E‐cadherin), together with transmission electron microscopy (TEM), suggested that impermeability in the VCM may be due to greater cellular contact area between cells and close packing rather than to maturity of tight junctions, the latter of which, by comparison with the visceral yolk sac, appeared to be rare or absent from the allantoic surface. Both VCM and DCM exhibited an ultrastructure more favorable for protein synthesis than did the distal squamous mesothelium; however, at most stages, VCM exhibited robust afadin (AF‐6), whereas the DCM uniquely contained alpha‐4‐integrin. These observations demonstrate that the allantoic mesothelium is not a conventional epithelium but possesses regional ultrastructural, functional and molecular differences that may play important roles in the correct deployment of the umbilical cord and its associated vascular, hematopoietic, and other cell types. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.