Isolation of a proteolytically derived domain of the insulin receptor containing the major site of cross-linking/binding

Radiolabeled insulin was affinity cross-linked to purified insulin receptor with six separate bifunctional N-hydroxysuccinimide esters of different lengths. Results were qualitatively identical for each cross-linker in that insulin was predominantly cross-linked through its B chain to the receptor's alpha subunit. The maximum efficiencies of cross-linking were 10-15% for the most effective reagents, and this value was dependent upon the concentration and length of the cross-linker. In an effort to locate the cross-linking site, monoiodoinsulin was cross-linked to affinity-purified insulin receptor with disuccinimidyl suberate. Limited proteolysis of the hormone/receptor adduct with Staphylococcus aureus V8 protease, chymotrypsin, or thermolysin in an SDS-containing buffer rapidly generated a 55-kDa, insulin-labeled fragment as shown by SDS-polyacrylamide gel electrophoresis. We reported earlier that the 55-kDa chymotryptic fragment contained multiple internal disulfide bonds as evidenced by its shifting mobility on an SDS gel after dithiothreitol treatment [Boni-Schnetzler et al. (1987) J. Biol. Chem. 262, 8395-8401]. Here we show that the 55-kDa fragment is also formed by proteolysis of the receptor in the absence of prior insulin cross-linking. This fragment was prepared in amounts sufficient for sequence analysis and was purified by passage successively over gel permeation and reverse-phase HPLC columns. The sequence of the fragment's amino terminus corresponds to that of the amino terminus of the receptor's alpha subunit. This fragment also reacts with an antibody raised against a synthetic peptide corresponding to residues 242-253 of the receptor's alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enterococcal and streptococcal resistance to PC190723 and related compounds: Molecular insights from a FtsZ mutational analysis

New antibiotics with novel mechanisms of action are urgently needed to overcome the growing bacterial resistance problem faced by clinicians today. PC190723 and related compounds represent a promising new class of antibacterial compounds that target the essential bacterial cell division protein FtsZ. While this family of compounds exhibits potent antistaphylococcal activity, they have poor activity against enterococci and streptococci. The studies described herein are aimed at investigating the molecular basis of the enterococcal and streptococcal resistance to this family of compounds. We show that the poor activity of the compounds against enterococci and streptococci correlates with a correspondingly weak impact of the compounds on the self-polymerization of the FtsZ proteins from those bacteria. In addition, computational and mutational studies identify two key FtsZ residues (E34 and R308) as being important determinants of enterococcal and streptococcal resistance to the PC190723-type class of compounds.     

Immune cytolysis of human tumor cells mediated by xenogeneic "immune" RNA

Normal, nonimmune, human peripheral blood lymphocytes, when incubated with RNA extracted from lymphoid organs of guinea pigs or sheep immunized with human tumor cells, mediated the immune cytolysis of those tumor cells in vitro. Lymphocytes incubated without RNA, or with control RNA preparations, failed to evidence cytotoxic activity. Treatment of the active RNA preparations with ribonuclease abrogated the cytotoxic activity, but treatment with deoxyribonuclease or pronase did not effect activity.     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

Cavin-3 Knockout Mice Show that Cavin-3 Is Not Essential for Caveolae Formation,for Maintenance of Body Composition,or for Glucose Tolerance

The cavins are a family of proteins associated with caveolae, cavin-1, -2 and -3 being widely expressed while cavin-4 is restricted to striated muscle. Deletion of cavin-1 results in phenotypes including metabolic changes consistent with adipocyte dysfunction, and caveolae are completely absent. Deletion of cavin-2 causes tissue-specific loss of caveolae. The consequences of cavin-3 deletion are less clear, as there are divergent data on the abundance of caveolae in cavin-3 null mice. Here we examine the consequences of cavin-3 deficiency in vivo by making cavin-3 knockout mice. We find that loss of cavin-3 has minimal or no effects on the levels of other caveolar proteins, does not appear to play a major role in formation of protein complexes important for caveolar morphogenesis, and has no significant effect on caveolae abundance. Cavin-3 null mice have the same body weight and fat mass as wild type animals at ages 8 through 30 weeks on both normal chow and high fat diets. Likewise, the two mouse strains exhibit identical glucose tolerance tests on both diets. Microarray analysis from adipose tissue shows that the changes in mRNA expression between cavin-3 null and wild type mouse are minimal. We conclude that cavin-3 is not absolutely required for making caveolae, and suggest that the mechanistic link between cavin-3 and metabolic regulation remains uncertain.     

Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Defining the mode, energetics and specificity with which a macrocyclic hexaoxazole binds to human telomeric G-quadruplex DNA

Oxazole-containing macrocycles represent a promising class of anticancer agents that target G-quadruplex DNA. We report the results of spectroscopic studies aimed at defining the mode, energetics and specificity with which a hexaoxazole-containing macrocycle (HXDV) binds to the intramolecular quadruplex formed by the human telomeric DNA model oligonucleotide d(T2AG3)4 in the presence of potassium ions. HXDV binds solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. HXDV binds d(T2AG3)4 with a stoichiometry of two drug molecules per quadruplex, with these binding reactions being coupled to the destacking of adenine residues from the terminal G-tetrads. HXDV binding to d(T2AG3)4 does not alter the length of the quadruplex. These collective observations are indicative of a nonintercalative 'terminal capping' mode of interaction in which one HXDV molecule binds to each end of the quadruplex. The binding of HXDV to d(T2AG3)4 is entropy driven, with this entropic driving force reflecting contributions from favorable drug-induced alterations in the configurational entropy of the host quadruplex as well as in net hydration. The 'terminal capping' mode of binding revealed by our studies may prove to be a general feature of the interactions between oxazole-containing macrocyclic ligands (including telomestatin) and intramolecular DNA quadruplexes.     

Lysinyl macrocyclic hexaoxazoles: synthesis and selective G-quadruplex stabilizing properties

Macrocyclic hexaoxazoles having one or two lysinyl side chains in which the terminal nitrogen is either a primary amine, N,N-dimethylamine, or an acetamide have been synthesized. Sodium ion has been found to be beneficial to the macrocyclization step by acting as a template around which the linear polyoxazole can organize. Each of the targeted compounds selectivity stabilizes G-quadruplex versus duplex DNA. Compounds with one valine and one lysine residue display the best combination of G-quadruplex stabilizing ability with no detectable stabilization of duplex DNA.