Stability and motion of a hairpin and the corresponding mismatched duplex: a theoretical exploration using molecular mechanics and normal mode analysis of 2D NMR results on d(GCCGCAGC)

The oligomer d(GCCGCAGC) can adopt two different conformations: i) a duplex with two mismatched A.C base pairs and ii) a hairpin with two C.G base pairs and a single stranded loop. We report molecular mechanics, normal mode analysis, and thermodynamic stability calculations for both structures. We show that the energy-minimized structure and harmonic-dynamics results are in complete agreement with the observed NOE spectrum and imino proton exchange data. We conclude that the high stability of the hairpin structure over the duplex at low salt concentration is due to the higher vibrational entropy contribution to the system free energy by the single stranded loop and to the lack of minor groove phosphate/phosphate electrostatic repulsions that tend to destabilize the duplex.     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

Biological effects of human gastrin I and II chemically modified at the C-terminal tetrapeptide amide.

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C-terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors (Kd approximately 10 nM) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -omega(CH2-NH)- bond resulted in a compound which also bound to gastrin receptors (Kd approximately 10 nM) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -omega(CH2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor (Kd greater than 10(-6) M) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors (Kd approximately 0.3 nM). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

Respiratory electron transport activity in plankton of the Weddell and Scotia Seas during late spring — early summer: relationships with other biological parameters

Summary As a means to estimate potential oxygen consumption, profiles of elctron transport system (ETS) activity were made along three transects across the Weddell-Scotia Confluence zone (WSC) and the marginal ice zone (which overlapped in part) during the EPOS leg 2 cruise of the RV Polarstern. The integrated ETS activity between 0 and 100 m depth (referred to in situ temperatures) ranged from 261 meq (mili-electron equivalents) m^–2 day^–1 in the WSC to 45 meq m^–2 day^–1 in the southernmost stations at 62° S. The temporal changes in the overall distribution of ETS activity were small compared with the spatial variations. The main feature of the ETS activity distribution was the presence of maxima located in the WSC, coinciding with peaks of phytoplankton biomass. Different relationships between ETS and chlorophyll a concentration in these maxima appeared to be related to diatom or flagellate dominance. Vertically integrated ETS activities were significantly correlated with chlorophyll a and paniculate organic carbon concentrations, primary production and bacterial thymidine uptake.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Analysis of the human regulators of complement activation (RCA) gene cluster with yeast artificial chromosomes (YACs).

The human regulators of complement activation gene cluster (RCA cluster) have been partially characterized with yeast artificial chromosomes (YACs). While the data confirm many points previously elucidated, the finer resolution of YAC mapping has allowed the discovery and/or localization of partial gene duplications, the determination of gene orientations, and the measurement of gaps between known genes. Here nine overlapping YACs that encompass a genomic region of 800 kb, encoding four RCA genes and three gene-like elements, are described. The encoded genes and two of the gene-like elements share the same orientation and are ordered (5' to 3') DAF, CR2, CR1, MCP-like, CR1-like, and MCP. A C4bp-like region lies upstream from DAF and is likely to correspond to one recently observed by F. Pardo-Manuel, J. Rey-Campos, A. Hillarp, B. Dahlback, and S. Rodriguez de Cordoba (1990, Proc. Natl. Acad. Sci. USA 87: 4529-4533). MCP-like, a new genetic element, was discovered and found to be homologous to the 5' portion of the MCP gene. Two large gaps of 85 kb (between CR2 and DAF) and 110 kb (between DAF and the C4bp-like element) could carry additional RCA genes. The arrangement of CR1, MCP-like, CR1-like, and MCP, in that order, strongly suggests that this region was generated by a single duplication of neighboring CR1/CR1-like and MCP/MCP-like forerunners. The RCA YACs will now serve as convenient DNA sources for the subcloning and further characterization of this region.     

Localization of the amphotropic murine leukemia virus receptor gene to the pericentromeric region of human chromosome 8.

The host range of retroviruses is determined primarily by the presence of specific receptors on target cells which are recognized by the retroviral envelope glycoprotein. Somatic cell hybrids have been used to determine the chromosomal locations of several retroviral receptors in mice prior to their molecular cloning. Here we report that by using human-Chinese hamster somatic cell hybrids and a retroviral vector, we have mapped the receptor for the amphotropic murine leukemia virus to the pericentromeric region of human chromosome 8.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.