Flexibility of the pyrrolidine ring in proline peptides

A survey of over 50 crystal structures indicates that both imino acid and peptide derivatives of proline populate ring conformers consistent with the torsional potentials about single bonds. In both cases, lower barriers for rotation about C? N bonds relative to those about C? C bonds favor smaller values for dihedral angles about the former bonds. In peptides a minimum in the torsional potential about C? N bonds occurs at zero dihedral angle, further favoring small angles. The pyrrolidine-ring dihedral angles of the proline compounds in the solid state obey a cyclopentane-type pseudorotation function. Thus the puckering of the five-membered ring can be quantitatively described by two parameters. Consistent with small dihedral angles about C? N bonds, C^β and/or C^γ are puckered out of the mean plane of the ring in nearly all of the nonstrained compounds. Utilizing the consistent force-field method of Lifson and coworkers [see A. Warshel, M. Levitt, and S. Lifson (1970) J. Mol. Spectrosc. 33 , 84] the intramolecular energy of five proline peptides was minimized with respect to all internal coordinates. In addition, the energy surface near minima was explored by constraining a particular dihedral angle and reminimizing the energy with respect to all remaining variables. In linear peptides two types of pyrrolidine-ring conformers have identical predicted energies. In the cyclic dipeptide cyclo (Pro-Gly) one of the ring conformers is favored by about 3 kcal/mol, while the cyclic tripeptide cyclo(Pro-Gly-Gly) favors the other conformer by a comparable margin. In agreement with observations in the solid state and in solution, C^β and/or C^γ are puckered in the predicted conformers. A correlation between proline Φ and the details of the puckered conformation was predicted and found to match precisely conformers observed in crystals. For the diamides N-acetyl-L -proline-N′-methyl-amide and N-acetyl-L -proline-N′,N′-dimethylamide (AcProMe₂A) 30% and 60% cis acetyl peptide bonds were predicted in good agreement with observations in nonpolar solvents for the respective compounds. The conformational distributions with respect to proline Ψ are also in accord with experimental observations. For AcProMe₂A, a model for a -Pro-Pro-sequence in a peptide chain, this study is the first to predict stable conformers for proline Ψ either ca. ?50° or 140° for both cis and trans peptides.     

Quinine-induced agranulocytosis: toxic effect of quinine bisulphate on bone marrow cultures in vitro.

In a case of quinine-induced agranulocytosis marrow culture studies confirmed the inhibitory effect on the patient''s cells of equivalent therapeutic plasma concentrations of quinine. Similar concentrations had no effect on normal marrow cells. Quinidine, the stereoisomer of quinine, had no effect on either cells from the patient or normal cells. The results encourage the use of in-vitro bone marrow cultures for identifying drugs responsible for agranulocytosis.     

Treatment of intact striatal neurones with cholera toxin or 8-bromoadenosine 3',5'-(cyclic)phosphate decreases the ability of pertussis toxin to ADP-ribosylate the alpha-subunits of inhibitory and other guanine-nucleotide-binding regulatory proteins, Gi and Go. Evidence for two distinct mechanisms.

Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 micrograms/ml, 22 h) decreases the subsequent ADP-ribosylation of the alpha subunit of the guanine-nucleotide-binding regulatory protein Go (Go alpha) and the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory protein (Gi alpha) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin. The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones. Treatment of cells with 0.1 microM 8-bromoadenosine 3',5'-(cyclic)phosphate (BrcAMP) for 16 h, or with 0.1 mM BrcAMP for 5 min, mimicked the effect of cholera toxin on the ADP-ribosylation of Go alpha and Gi alpha in vitro. However, the two agents seem to act through distinct mechanisms. The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin. In addition, measurements of the pI of the Go alpha deduced from immunoblots of two-dimensional gels performed using a specific antibody directed against Go alpha suggest that treatment of neurones with cholera toxin induces ADP-ribosylation of Go alpha in intact cells, while BrcAMP does not.     

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.     

Interleukin-6 administration has no acute hemodynamic or hematologic effect in the dog

To investigate the possible hemodynamic effects of interleukin-6 (IL-6), a single dose of 15 mcg/kg of recombinant IL-6 isolated from Escherichia coli was injected intravenously in six pentobarbital-anesthetized dogs. After 30 min, saline infusion was performed to maintain the pulmonary artery balloon-occluded pressure at baseline level. The animals were observed for up to 5 hours. No other hemodynamic alteration was observed than a gradual decline in cardiac output attributed to anesthesia. Hematologic variables, blood glucose, and total serum proteins were also constant. IL-6 levels were markedly elevated in the blood, but no tumor necrosis factor activity was detected. Thus a primary role for IL-6 in the early cardiovascular alterations associated with septic shock seems unlikely.     

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

In vitro propagation and cytology of wild yams,Dioscorea abyssinica Hoch. and D. mangenotiana Miège

Two wild yams of West Africa, Dioscorea abyssinica Hoch, and D. mangenotiana Miège were micropropagated from nodal cultures. Both species produced 4–5 nodes per each node cultured. The size of nodal cuttings was critical, segments shorter than 0.5 cm being less suitable for micropropagation. The number of nodes produced was constant even after 5 cycles of subculture; however, D. abyssinica continuous subculture decreased propagation efficiency, resulting in a reduced number of reculturable nodes at each cycle. In D. mangenotiana, the decrease in multiplication efficiency affected both the number of total and reculturable nodes. Large-sized microtubers were induced on nodal segments maintained under 8-h daylength in both species. In D. abyssinica, however, microtubers were induced on media containing 20, 40, 60 and 80 g l^-1 sucrose, whereas in D. mangenotiana only 40 and 60 g l^-1 sucrose favoured tuberization. Cytological studies confirmed that the chromosome number of D. abyssinica was 2n=40, although a high incidence of cytochimerism and cells with 2n=38 were observed in root meristems. In D. mangenotiana clones, the chromosome number was 2n=40, as against 2n=72 and 2n=80 reported in literature. This species also displayed karyological stability.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - PPF photosynthetic photon flux