Nucleophilic substitution at C-2 of S-alkylated 2-thiocytidines by cysteine and lysine : A new method for specific covalent linking of peptides to nucleic acids

S-Alkylated 2-thiocytidine can be substituted at C-2 by nucleophilic agents. This reaction has been investigated with model compounds as well as with tRNA using the amino acids cysteine and lysine in order to develop a new affinity label linking covalently tRNA and a protein. Reaction with N-protected cysteine gives 2-S-alkyl-pyrimidines, while unprotected cysteine yields an N-alkyl-pyrimidine, after intramolecular substitution. With the -amino group of lysine a fast replacement at C-2 is observed, leading to an unstable 2-N-alkyl-pyrimidine. All products have been characterized both chemically and spectroscopically.     

Quantitative analysis of the microtubule system in isolated fish melanophores

Isolated melanophores of the angelfish, Pterophyllum scalare, have been used in a morphometric analysis and a quantitative study of their microtubule system. Using transverse sections spaced at regular intervals, the changes associated with the process of pigment aggregation have been determined. Upon the concentration of pigment granules in the central cell region, almost half of the cytoplasmic portion is also withdrawn from the peripheral cell regions. Counts of microtubules within a cell sector in cells with pigment aggregated and dispersed, respectively, reveal (a) a constancy of the number of microtubules in this sector regardless of the distance from the cell center, and (b) a reduction of microtubule number in cells with pigment aggregated by about 58%. On the basis of these counts, the total number of microtubules has been calculated. In the dispersed state, about 2,400 microtubules extend between the center and the periphery of the cell, while their number is about 1,000 in the aggregated state. Using a 13-protofilament model of a microtubule and relevant data on size and molecular weight of microtubule subunits, the amount of tubulin present as microtubules is calculated. In the average, the cells contain 1.95·10⁸ monomers corresponding to 1.78·10^?8 mg tubulin. A tentative estimation of the concentration of tubulin inside a melanophore yields values of 6.1 mg/ml for the whole cell and 16.5 mg/ml for the cytoplasm alone (excluding membrane-bound organelles). Based on this estimation, a comparison, with microtubule assembly in vitro is made.     

Fusion of neurohypophyseal membranes in vitro

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca²⁺ in micromolar concentrations. Mg²⁺ and Sr²⁺ were ineffective. Mg²⁺ inhibited Ca²⁺-induced fusion.In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca²⁺. Mg²⁺ and Sr²⁺ were ineffective in replacing Ca²⁺ as trigger for fusion or vasopressin release.Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosome were exposed to cold.     

1H-nmr studies of polyoxyethylene-bound homo-oligo-L-methionines

The use of ¹H-nmr spectroscopy is demonstrated to be a useful analytical method to characterize the structure of synthetic peptides attached to soluble, macromolecular polyoxyethylene (POE) supports in the liquid-phase method (LPM) of peptide synthesis. We report an extensive 360-MHz ¹H-nmr study of POE-bound homo-oligo-L -methionine peptides. A combination of high field and selective saturation or Redfield pulse methods allows resolution of individual backbone NH and α-CH resonances of dilute peptides in the presence of strong resonances from macromolecular POE and/or protonated solvents. The nmr spectra for the POE-bound peptides in CDCl₃ are qualitatively similar to those of the low-molecular-weight Boc-L -Met_n-OMe peptide esters. This corroborates other observations that POE has little effect on peptide stucture. The backbone α-CH region of peptides is overlapped by signals from the terminal oxyethylene group of POE, but the peptide side-chain and low-field backbone NH resonances are well resolved. In trifluoroethanol the Boc-(L -Met)_n-NH-POE heptamer and octamer adopt the right-handed α-helical structure, and the present nmr studies provide evidence for two strong intramolecular hydrogen bonds to stabilize the helices. In water, the N-deblocked derivatives, (L -Met)_n-NH-POE oligomers adopt β-sheet structure and manifest well-resolved nonequivalent NH resonances with 6–7 Hz ³J_NH-CH coupling constants.     

Monoclonal antibodies directed against the sugar sequence of lacto-N-fucopentaose III are obtained from mice immunized with human tumors

Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III,

but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues.     

Different concentrations of thyroid peroxidase and thyroglobulin in the nuclear envelope and the endoplasmic reticulum throughout the cytoplasm.

Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Br?kelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some properties of an extracellular l-amino acid oxidase from Cellulomonas cellulans AM8 isolated from soil

Summary The soil isolate Cellulomonas cellulans AM8 produces an extracellular l-amino acid oxidase (L-AAO) with broad substrate specificity. The strain produced up to 0.35 unit (U)/ml of the extracellular L-AAO in a simple medium containing glycerol and yeast extract. The enzyme was easily purified up to 30 U/mg protein using Phenyl-Sepharose fast flow. The purified enzyme migrated as single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 55 kDa. On native PAGE the molecular mass was approx. 300 000 kDa, which may be due to aggregation. With the exception of glycine, proline, and threonine, all the amino acids normally constituting proteins were oxidized. The V _max values from 0.7 to 35.2 U/mg for aspartic acid and lysine, respectively, and the K _m values from 0.007 to 7.1 mm for cysteine and valine, respectively, were obtained at 25° C and pH 7.0 in oxygen-saturated solutions. The L-AAO had a pH optimum of 6.5–7.5. It was stable for several months at — 30° C and for some days at 35° C. Ferricyanide served as an electron acceptor with a V _max of 50 U/mg and K _m for 0.3 mm with phenylalanine as the substrate. Correspondence to: R. D. Schmid     

Conformation and formation tendency of the cyclotetrapeptide cyclo (D-Pro-D-Pro-L-Pro-L-Pro): experimental results and molecular modeling studies

The title compound represents the smallest member of cyclic proline peptides corresponding to the general formula c(DDLL-Pro4)n with a strictly D,D,L,L double-alternating sequence of the chiral amino acid residues. The cyclopeptides with n greater than or equal to 2 could be synthesized from both DDLL-Pro4 (1) and DLLD-Pro4 (2). The cyclic monomer (n = 1) resulted only from 2, whereas not even a trace could be found by cyclization of 1. The peptide exists in a strongly strained Ci symmetrical conformation (x-ray analysis) with alternating cis and trans peptide bonds (ctct form I). The cis peptide bonds deviate from planarity (omega = 22 degrees); two of the pyrrolidine rings show a "South" conformation (phi = -94 degrees), whereas the other residues exhibit C alpha-endo puckering (phi = -124 degrees). Two of the psi angles surprisingly occur at +41 degrees (anti-cis'), the others are located in the trans' region. A quantitative ring opening occurs with trifluoroacetic acid at room temperature. In solution the existence of an isomeric ctcc sequence (form Ia) is indicated. Dreiding model studies also suggested a favorable conformation with a tctc sequence (form II). Consequently, we performed molecular mechanics calculations, based on the CHARMM force field and semiempirical quantum mechanical AM1 calculations (MOPAC program). Pronounced differences in the backbone parameters were found using these two methods. However, the theoretical studies evidenced the experimentally obtained differences in the cyclization tendencies of the linear precursors.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.