Design and optimization of selective serotonin re-uptake inhibitors with high synthetic accessibility Part 1

The reported selective serotonin Re-uptake Inhibitor Litoxetine was used as the starting point in the design of a range of potential SSRIs with high ease of synthetic accessibility. Preparation and subsequent optimization yielded a range of potent and highly selective SSRIs.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Electron cytochemistry of mycobacteria: Evidence that strongly acidic sulfate groups are present on the surface of H37Rv (virulent) strain ofMycobacterium tuberculosis

Cytochemical characterization of mycobacterial surfaces was carried out on virulent (H37Rv) and avirulent (H37Ra) strains ofMycobacterium tuberculosis. The results were quantified and compared with those obtained with three colony types of the opportunistic pathogenMycobacterium avium. Mycobacterium aurum, a rapidly growing, nonpathogenic species, served as a model for the cytochemical methods. Concanavalin A (ConA) reacted with -d-mannose and -d-glucose residues, whereas negative charged residues were detected with either the ionized ferritin (CF) or the colloidal ferric hydroxide (CIH) method. Strongly acidic sulfate groups were detected by their selective blockage with alcian blue (AB) at pH 1 prior to the CIH labeling at pH 1.8. Weakly acidic groups were demonstrated by AB blockage at pH 2.5 prior to staining with CF stain. Except forM. aurum, all other strains showed a marked heterogeneity in regard to the abundance of their surface labeling. Accessible sulfate groups were present on the cell surface of the virulent H37Rv strain ofM. tuberculosis, but not on the avirulent strain H37Ra. Distribution of ConA receptors, on the other hand, was unrelated to the virulence or pathogenicity of the bacterial strain.     

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family

Summary The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)_n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.Professor Dr. Otto Prokop (Humboldt-Universität Berlin) on the occasion of his 70th birthday     

Computation of relaxation matrix elements from incomplete NOESY data sets

Summary The structural determination of biological molecules in solution by NMR relies on the determination of a set of interatomic distances obtained by measurement of intramolecular nuclear Overhauser effects (NOE). It is shown in this paper that it is possible to obtain the accurate relaxation rate (and hence the interatomic distance) from the direct measurement of a single NOE signal. The precise analysis of a NOESY peak evolution with respect to the mixing time allows the evaluation of the relaxation parameters for the pair of spins under consideration. This is done without any assumption on the relaxation of unmeasured spins, or on the movement of the molecule. The theoretical basis of this method is presented. In order to evaluate the proposed method, a simulated case on the protein BPTI is studied, which shows that the method performs very well even in the case of noisy data sets.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.