Circular dichroism studies of low molecular weight hydrogelators: The use of SRCD and addressing practical issues

Circular dichroism (CD) spectroscopy has been used extensively for the investigation of the conformation and configuration of chiral molecules, but its use for evaluating the mode of self‐assembly in soft materials has been limited. Herein, we report a protocol for the study of such materials by electronic CD spectroscopy using commercial/benchtop instruments and synchrotron radiation (SR) using the B23 beamline available at Diamond Light Source. The use of the B23 beamtime for SRCD was advantageous because of the unique enhanced spatial resolution achieved because of its highly collimated and small beamlight cross section (ca. 250 μm) and higher photon flux in the far UV region (175‐250 nm) enhancing the signal‐to‐noise ratio relative to benchtop CD instruments. A set of low molecular weight (LMW) hydrogelators, comprising two Fmoc‐protected enantiomeric monosaccharides and one Fmoc dipeptide (Fmoc‐FF), were studied. The research focused on the optimization of sample preparation and handling, which then enabled the characterization of sample conformational homogeneity and thermal stability. CD spectroscopy, in combination with other spectroscopic techniques and microscopy, will allow a better insight into the self‐assembly of chiral building blocks into higher order structural architectures.     

Discovery of isatin and 1H-indazol-3-ol derivatives as d-amino acid oxidase (DAAO) inhibitors

d-Amino acid oxidase (DAAO) is a potential target in the treatment of schizophrenia as its inhibition increases brain d-serine level and thus contributes to NMDA receptor activation. Inhibitors of DAAO were sought testing [6+5] type heterocycles and identified isatin derivatives as micromolar DAAO inhibitors. A pharmacophore and structure-activity relationship analysis of isatins and reported DAAO inhibitors led us to investigate 1H-indazol-3-ol derivatives and nanomolar inhibitors were identified. The series was further characterized by pK_a and isothermal titration calorimetry measurements. Representative compounds exhibited beneficial properties in in vitro metabolic stability and PAMPA assays. 6-fluoro-1H-indazol-3-ol (37) significantly increased plasma d-serine level in an in vivo study on mice. These results show that the 1H-indazol-3-ol series represents a novel class of DAAO inhibitors with the potential to develop drug candidates.     

Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family.     

Crystallization of recombinant rat cathepsin B

A glycosylation-minus mutant of rat cathepsin B expressed in yeast has been purified and crystallized. X-ray diffraction data have been collected and molecular replacement for solving the structure is in progress. The space group for the recombinant rat cathepsin B was determined to be P2(1) with unit cell dimensions alpha = 62.2 A, b = 90.19 A, c = 47.07 A, and beta = 97.43 degrees. A unit cell contains 4 molecules and 2 molecules per asymmetric unit.     

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family

We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the version reported by Moore et al. [Moore, R. E., Davies, M. S., O'Connell, K. M., Harding, E. I., Wiegand, R. C., and Tiemeier, D. C. (1986) Nucleic Acids Res. 14:7227-7235] in eight reading frame shifts which result in partial amino acid sequence conservation with the rat GSH S-transferase sequences. The GST I and GST III sequences share approximately 45% amino acid sequence homology. Both the GST I and the GST III mRNAs contain different repeating motifs in front of the initiation codon ATG. Multiple poly(A) addition sites have been identified for these two classes of maize GSH S-transferase messages. Genomic Southern blotting results suggest that both GST I and GST III are present in single or low copies in the maize (GT112 RfRf) genome.     

Effect of 5-isosorbide mononitrate on isosorbide dinitrate-induced relaxation of rabbit aortic rings

The ability of isosorbide dinitrate (ISDN) and its two metabolites, 5-isosorbide mononitrate (5-ISMN) and 2-isosorbide mononitrate (2-ISMN), to relax phenylephrine-contracted rabbit aortic rings was compared. The three organic nitrates demonstrated similar efficacy. ISDN was found to be the most potent (median effective dose (ED50); 1.5 X 10(-7) +/- 1.1 X 10(-7) M), followed by 2-ISMN (ED50, 1.8 X 10(-6) +/- 9 X 10(-7) M) and 5-ISMN (ED50, 8.2 X 10(-6) +/- 3.6 X 10(-6) M). The log dose-response curve of ISDN in rabbit aortic rings was constructed in the absence and presence of three fixed concentrations of 5-ISMN (5 X 10(-6), 10(-5), and 3 X 10(-5) M). No shift in the ISDN dose-response curve at high ISDN concentrations was noted in the presence of 5-ISMN. Using the isobolographic method with fixed ISDN/5-ISMN ratio mixtures, no evidence for an antagonistic effect of 5-ISMN on ISDN-induced vasodilation was obtained. Analysis of the fixed ISDN/5-ISMN ratio mixture responses by the median-effect plot showed no antagonistic effect. It is concluded that in rabbit aortic rings 5-ISMN, the major metabolite of ISDN, is not an antagonist of ISDN at a "nitrate receptor," and no support is provided for the hypothesis that the accumulation in plasma of metabolites (e.g., 5-ISMN) with longer half-lives than the parent drug explains tolerance to organic nitrates.