New methods for solid phase peptide synthesis of transitionstate analog inhibitors of HIV-1 protease and DPP-IV

Summary A new and stereoselective method to synthesize hydroxyethylamine and hydroxymethylamide peptide bond isosteres is developed. The key step is the addition of 2-trimethylsilylthiazole to -aminoaldehydes, followed by transformation to -hydroxy--aminoaldehydes. The stereochemistry of the addition can be manipulated by the choice of the nitrogen substitution. The isosteres are easily synthesized via Solid Phase Peptide Synthesis, which rapidly gives the desired pseudopeptides.     

Somatostatin analogues containing o-aminomethylphenylacetic acid as a bridge unit

Summary A new cis-peptide bond mimetic, -benzyl-o-aminomethylphenylacetic acid, was synthesized and incorporated in a homodetic somatostatin analogue. Biological binding tests and 2D NMR conformational analysis indicate that the configuration of the bridge-unit asymmetric center and the orientation of the benzyl side chain play a key role in the biological activity of this type of somatostatin analogues.     

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

 Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its K_M was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn²⁺ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities. Accepted: 28 August 1995     

Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes

Rat hepatocytes were incubated in the presence of a high concentrationof the hepatopathogenic agent D-galactosamine (GalN), and theeffect on the cellular concentrations of pyrimidine nucleotidesand nucleotide sugars was determined. The UTP pool became depleted.The pools of UMP and CMP in RNA decreased to 72%, indicativefor an inhibition of RNA synthesis. UDP-HexNAc (where HexNAcis GlcNAc + GalNAc) and UDP-HexN (where HexN is GlcN + GalN)levels increased, and those of UDP-hexose and UDP-GlcA (whereGlcA is glucuronic acid) decreased. The cellular concentrationof CTP did not change, whereas that of CMP-NeuAc (where NeuAcis N-acetylneuraminic add) showed a 2-fold increase. Labellingwith [¹⁴C]orotic acid and [³H]cytidine showed that the metabolicflow via the de novo pathway was not changed. The depletionof the so-called overflow pool of UTP [Pels Rijcken et al, Biochem.J., 293, 207–213, 1993] caused a release of the feedbackinhibition by UTP and thus an increased flow through the salvagepathway. Finally, it appeared that GalN, when added to hepatocytes,gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglueosamine)that is separate from the pool of UDP-GlcNAc that is derivedfrom GlcN. D-galactosamine glycosylation sugar nucleotide biosynthesis     

Identification of cellular prosomatostatin and nonsomatostatin peptides derived from its amino terminus

Rat prosomatostatin was isolated from a somatostatin-producing cell line and was partially microsequenced. This indicated the amino terminal structure of cellular prosomatostatin and implied a 92-amino acid sequence for the somatostatin precursor. Based on the structure for cellular prosomatostatin, a peptide was synthesized and used to develop a radioimmunoassay directed toward the amino terminal portion of prosomatostatin. This assay has revealed two peptides containing the amino-terminal portion of prosomatostatin in a somatostatin-secreting CA-77 rat medullary thyroid carcinoma cell line. These two peptides - MW 4000 and 8000 daltons - lack somatostatin immunoreactivity. Thus, processing of prosomatostatin occurs both at the amino and carboxyl regions. These results open the way for elucidation of the structure, function and metabolism of non-somatostatin peptides derived from the amino terminus of prosomatostatin.     

Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.     

Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.     

Catecholamines in fetal pig plasma and the response to acute hypoxia and chronic fetal decapitation

Summary Dopamine, norepinephrine and epinephrine were measured by radioenzymatic assay in blood plasma samples drawn from the umbilical arteries of 30 anaesthetised Landrace pig fetuses. Just prior to term, the concentrations of dopamine (0.46±0.14 ng·ml^–1) and norepinephrine (1.74±0.60 ng·mg^–1) were lower than earlier in gestation, whereas epinephrine concentrations at term (0.80±0.31 ng·ml^–1) were similar to those at mid-gestation, intervening stages of gestation having higher levels of plasma epinephrine. Fetal hypoxia was induced by clamping the umbilical cord for 2 min and the catecholamines determined in arterial blood samples immediately thereafter, then again 3 min after removal of the clamp. Inconsistent effects of cord clamping on catecholamine levels were seen at 55 days, but thereafter, in all but one instance, the hormone levels were increased. Fetuses near term tended to respond less than fetuses at 75 and 96 days gestation (term=114±1 day). Catecholamines were also present in the circulation of fetuses decapitated at 42 days gestation and studied at 109±1 days. The average concentrations of dopamine (1.12±0.27 ng·ml^–1) and norepinephrine (8.23±3.04 ng·ml^–1) were greater than in intact fetuses, the plasma epinephrine levels being comparable to, or slightly higher than, those in intact fetuses. The results demonstrate that catecholamines are present in the circulation of the intact and decapitated pig fetus and that the actual concentrations and the type of response to umbilical cord clamping are dependent on gestation age.     

Human pituitary thyrotropin. Characterization of five glycoproteins with thyrotropin activity.

Human pituitary thyrotropin prepared by chromatography on hydroxyapatite or on SP-Sephadex was fractionated into five active components by preparative poly-acrylamide gel electrophoresis. The potency of the five components was 4-9 units human Research Standard A/mg. Examination of the components by analytical electrophoresis and by immunological methods revealed no heterogeneity. Ultracentrifugaiton of the three major components showed homogeneity with sedimentaiton coefficinets in the range of 2.4-3.0 S and a value for the molecular weight of about 33 000. Amino acid and carbohydrate analyses indicated close similarites between the five components.     

Nearest-neighbor and next-nearest-neighbor effects in the proton NMR spectra of the oligoribonucleotides ApGpX and CpApX

The variable-temperature proton nmr spectra of the oligoribonucleotides in the series CpApX and the series ApGpX, X = A, G, C, U, together with the parent dimers CpA and ApG have been measured. A complete analysis of all the nonexchangeable base proton resonances and ribose H-1′ proton resonances was made. The presence of trends in the shielding abilities of the various bases at both the nearest-neighbor and next-nearest-neighbor positions were identified. The observed shieldings could be used to predict the chemical shifts of protons in related systems. Based on the empirical results from ribodinucleoside monophosphates, the temperature-dependent behavior of the J_1′2′ coupling constants of the triribonucleotides suggested that the compounds in the CpApX series stacked from the 5′-end to the 3′-end, while those in the ApGpX series stacked from the 3′-end to the 5′-end.