Rotational correlation times of peptides determined by perturbed angular correlations of γ-rays

The technique of Perturbed Angular Correlations of -rays has been used to study the rotational correlation times in aqueous solution of the peptides: oxytocin, glycyltryptophan, cholecystokinin and the glycopeptide ristocetin. These peptides were labelled with excited ^111mCd through the covalent coupling of the metal chelator diethylenetriaminepentaacetic acid (DTPA) to the primary amines-of the peptides. The experimental correlation times are in good accordance with calculations based on the molecular weight. This indicates that the ^111mCd-DTPA is rigidly bound to the molecules. In the case of ristocetin, the correlation time was measured at 2°C, 25°C and 38°C. These experiments show the expected linear dependence on the viscosity divided by temperature. The feasibility of determining rotational correlation times for peptides without lysines and with correlation times in the ns region is thus demonstrated. Also, the correlation time of ^111mCd-DTPA coupled to the lysines of bovine serum albumin was determined. The measured correlation time is about 5 times less than the calculated correlation time. This effect is assigned to local motion. In spite of this, experiments show that ^111mCd-DTPA-bovine-serum-albumin is significantly immobilised by aggregation with immunoglobulins. The nuclear quadrupole interactions, necessary for determining the correlations times, were determined for ^111mCd-DTPA-ristocetin and ^111mCd-DTPA-bovine-serum-albumin by adding sucrose to a concentration of 63% and cooling to 2°C. This showed a small but significant difference between the two molecules. We interpret this as due to different conformations, possibly different coordination numbers. Offprint requests to: E. Danielsen     

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.     

Protein and other compositional differences of the extracellular material from slimy and non-slimy colonies of non-mucoid Pseudomonas aeruginosa

The composition of extracellular material produced by non-mucoid strains of Pseudomonas aeruginosa differed when grown on surfaces that either did or did not induce the formation of slime under conditions where the medium was identical. The nature of the changes in protein composition indicated that protein expression differed in the course of growth on the two surfaces, and hence that there were physiological consequences associated with growth under conditions which do or do not lead to slime formation. The compositional differences also included elevated levels in extracellular material from the slimy colonies of two virulence factors, protease and rhamnolipids.     

Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection

Cysteamine is currently used to treat children with the inherited disorder nephropathic cystinosis. A method for the quantitative determination of this aminothiol in human plasma is presented. Whole plasma was reduced with sodium borohydride to convert disulfides to thiols. Cysteamine was then separated by high-performance liquid chromatography and detected electrochemically. The recovery of standard cysteamine added to plasma was 96.6 +/- 1.9%. In a patient with cystinosis, an oral dose of cysteamine was absorbed rapidly, with plasma cysteamine reaching a maximum of 56 microM 1 h after the dose. By 1.8 h the plasma cysteamine concentration had decreased to one-half the maximum value.     

Participation of mitochondrial proliferation in morphological differentiation of murine mastocytoma cells

Proliferation of a cold-sensitive cell-cycle mutant isolated from an undifferentiated murine mastocytoma line is reversibly arrested at the nonpermissive temperature of 33 degrees C, and the arrested cells undergo morphological differentiation as expressed by the formation of metachromatic granules. Following transfer of these mutant cells from the permissive temperature of 39.5 to 33 degrees C, a transient increase in both cytochrome c oxidase and DNA polymerase gamma was observed, the ratio of total mitochondrial volume to cell volume nearly doubled within 6 days, and numbers of mitochondrial cross-sections per cellular cross-section as determined in electron micrographs underwent a threefold increase. Addition of chloramphenicol (100 micrograms/ml) to the mutant cell cultures 6 days prior to transfer from 39.5 to 33 degrees C prevented the increase in the ratio of total mitochondrial to cell volume. Furthermore, chloramphenicol markedly inhibited the increase in granule number per cell that normally is observed after transfer of cultures to 33 degrees C or during treatment with 1 mM butyrate, suggesting that mitochondrial proliferation may be an obligatory step in the process of morphological differentiation of these mastocytoma cells.     

Transfer and Expression of Lithoautotrophy and Denitrification in a Host Lacking These Metabolic Activities

The conjugative 450-kilobase-pair megaplasmid pHG1 from Alcaligenes eutrophus H16 was transferred to the herbicide-degrading soil bacterium A. eutrophus JMP134. This transfer was achieved by means of RP4 mobilization and a Tn5-Mob insertion provided in trans on the megaplasmid replicon. Although kanamycin-resistant transconjugants also occurred with other gram-negative species such as Rhizobium, Agrobacterium, and thiobacteria, A. eutrophus JMP134 was the only recipient which stably maintained the megaplasmid. pHG1-containing transconjugants derived from JMP134 expressed all metabolic functions associated with the plasmid: the ability to oxidize hydrogen through catalysis of two hydrogenases, to assimilate carbon dioxide via the Calvin cycle pathway, and to grow with nitrate anaerobically. All of these metabolic activities were absent in the original strain JMP134.     

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

beta-Hydroxyaspartic acid or beta-hydroxyasparagine in bovine low density lipoprotein receptor and in bovine thrombomodulin

All of the vitamin K-dependent plasma proteins with domains that are homologous to the epidermal growth factor (EGF) precursor have 1 hydroxylated aspartic acid residue in the NH2-terminal EGF-homology region. In addition, protein S has 1 hydroxylated asparagine residue in each of the three COOH-terminal EGF-homology regions. All of these proteins have been found to have the amino acid sequence, CX(D or N)XXXX(F or Y)XCXC (corresponding to residues 20 to 33 in EGF), where the Asp or Asn residue is hydroxylated. This sequence also appears in two of the three EGF-homology regions of the human low density lipoprotein receptor and in two of the six EGF-homology regions of bovine thrombomodulin so far identified, suggesting that they may have the modified amino acid. We have now identified beta-hydroxyaspartic acid in acid hydrolysates of both these proteins.