Prerequisite for His4 in deltorphin A for highδ opioid receptor selectivity

Summary Analysis of deltorphin A position 4 analogues included: backbone constrained N MeHis, spinacine (Spi), N MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His⁴ enantiomers. High selectivity was lost with the following replacements: N MeHis⁴, N MePhe⁴ and Phg⁴ reduced binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi⁴ or Tic⁴ increasedµ affinity. Imidazole methylation of His⁴ marginally affected opioid binding and doubled selectivity; alkylatedd-His⁴-derivatives generally maintained selectivity in spite of decreased affinities. Thus, His⁴ imidazole preserves selectivity by facilitating high binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His⁴ imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr¹ suggestive that this conformation might contribute to high affinity and selectivity.     

Some biochemical properties of an acyclic oligonucleotide analogue. A plausible ancestor of the DNA?

As acyclic oligonucleotides have been suggested as a primitive model of DNA or RNA in prebiotic times, we compared some biochemical properties of these analogues to that of natural ones. Firstly, an acyclic analogue of deoxyribonucleoside triphosphates was tested as a potential substrate of enzymes intervening in nucleic acids synthesis. GlyTTP, a dTTP analogue with a missing 2-methylene group is notaccepted as a substrate by either DNA polymerase or deoxynucleotidyl terminal transferase (TdT). Secondly, themodified dodecathymidylate (GlyT)₁₂, the racemic acyclic sugar analogue of (dT)₁₂, proved to be anefficient primer for DNA polymerase and TdT, though the associative properties of (GlyT)₁₂ are very weak as shown by UV spectroscopy in phosphate buffer without magnesium chloride. But (GlyT)₁₂ has the advantage to be 500-times more stable against hydrolysis by snake venom phosphodiesterase than the corresponding oligothymidylate.     

Propionyl-CoA elimination may be a rate-determining step of selective cleavage of sterol side chain

Summary The effect of propionate on the growth and 4-androstene-3, 17-dion (AD) yield was investigated in cultures of Mycobacterium sp. NRRL B-3805 growing in minimal medium containing -sitosterol as substrate for selective side chain cleavage. Although the addition of propionate (PA) resulted in a concentration-dependent inhibition of growth at the beginning of fermentation, cultures started to grow in the presence of 0.1% of propionic acid reached an AD concentration 38% higher than the cultures growing in the absence of propionate during two day cultivation. After three days of incubation, the AD yields in cultures containing 0, 0.1 and 0.2% propionate at the inoculation were 68, 79 and 73%, while the protein levels were 2.01, 2.11 and 2.60 mg/ml, respectively. Our data showed that the positive effect of PA on the AD production from sterols by Mycobacterium sp. NRRL B-3805 could be explained by the induction of the enzymes of the methylmalonate pathway. The activity of propionyl-CoA carboxylase was about 30% higher in the crude extracts from the induced cultures growing in minimal medium, after 20 hours of growth, than in those from the controls (18.2 and 14.1 mU/mg, respectively, using propionyl-CoA as substrate). The distribution of the acid-stable ¹⁴C-radioactivity which built into methylmalonate, succinate and fumarate indicated that methylmalonyl-CoA mutase was also induced. Our data demonstrated that elimination of the toxic propionyl-CoA released from the side chain of the sterol is likely the rate-determining step of the AD production, at least at the beginning of the process.     

Repetitive DNA variation and pivotal-differential evolution of wild wheats.

Several polyploid species in the genus Triticum contain a U genome derived from the diploid T. umbellulatum. In these species, the U genome is considered to be unmodified from the diploid based on chromosome pairing analysis, and it is referred to as pivotal. The additional genome(s) are considered to be modified, and they are thus referred to as differential genomes. The M genome derived from the diploid T. comosum is found in many U genome polyploids. In this study, we cloned three repetitive DNA sequences found primarily in the U genome and two repetitive DNA sequences found primarily in the M genome. We used these to monitor variation for these sequences in a large set of species containing U and M genomes. Investigation of sympatric and allopatric accessions of polyploid species did not show repetitive DNA similarities among sympatric species. This result does not support the idea that the polyploid species are continually exchanging genetic information through introgression. However, it is also possible that repetitive DNA is not a suitable means of addressing the question of introgression. The U genomes of both diploid and polyploid U genome species were similar regarding hybridization patterns observed with U genome probes. Much more variation was found both among diploid T. comosum accessions and polyploids containing M genomes. The observed variation supports the cytogenetic evidence that the M genome is more variable than the U genome. It also raises the possibility that the differential nature of the M genome may be due to variation within the diploid T. comosum, as well as among polyploid M genome species and accessions.     

Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10⁻⁴ or 10⁻⁵ M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with ³H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine (nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.     

Comparison of the effect of calcium(II) and manganese(II) ions on trypsin autolysis

The effect of Mn²⁺ and Ca²⁺ ions on the rate of trypsin autolysis was studied at pH 7.0 and at 34.4-60.2°C. For comparison, the kinetic constants of esterolytic activity of trypsin in the presence of the metal ion were determined at pH 7.4 and at 36° and 40°C. There was no significant difference in the rate of autolysis between Mn²⁺ and Ca²⁺ in the temperature range 34-47°C, but at 56.8° and 60.2° autolysis was slightly more rapid in the presence of Mn²⁺. The Mn²⁺ or Ca²⁺ ion bound to trypsin is supposed to control the conformation and thereby the stability and the activity of the enzyme. This indirect effect of Mn²⁺ and Ca²⁺ is discussed on a structural basis of the enzyme molecule.     

The nature of absorbance changes following the addition of ADP to mitochondria: ATP synthesis from intramitochondrial inorganic phosphate

The transient absorbance increase induced by ADP in phosphate-loaded respiring mitochondria becomes stable and greatly amplified by inhibitors of phosphate transport. The absorbance changes are sensitive to oligomycin and to aurovertin and their extent is proportional to the amount of ADP added. Simultaneously with the ADP-dependent increase in absorbance the inorganic phosphate and K⁺-ion content of the matrix decreases. It is concluded that the optical change reflects contraction of the matrix compartment secondary to intramitochondrial solute changes.     

Monthly incidence of Theileria cervi and seroconversion to Babesia odocoilei in white-tailed deer (Odocoileus virginianus) in Texas.

Monthly monitoring of fawns collected from an area in Texas endemic for Theileria cervi and Babesia odocoilei showed that transmission of T. cervi occurred during July and August, a time period consistent with the occurrence of Amblyomma americanum. Seroconversion to B. odocoilei occurred during October to December and possibly continued through January and February. The time of seroconversion was more suggestive of transmission of B. odocoilei by Ixodes scapularis than by Amblyomma americanum.