Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.     

Behavior of nursery/peer-reared and mother-reared rhesus monkeys from birth through 2 years of age

The behavior of 8 nursery/peer-reared and 16 mother-only reared rhesus macaques was observed between birth and 5 months of age, with follow-up studies conducted when the animals were 10–21 months old and living in large social groups. Nursery-reared neonates were more awake, active, and irritable than mother-only reared monkeys. From 1 to 5 months of age the nursery/peer-reared animals exhibited a greater variety of behaviors than the mother-only reared infants, which spent the majority of the time in ventral contact with mothers. As juveniles the groups were indistinguishable with the exception of more self-directed behaviors observed in the nursery/peer-reared monkeys. Both rearing conditions, by virtue of their atypicality, imposed restrictions on social development. The behavioral similarity of the juveniles while in the large social group may be a function of maturation or due to the rehabilitative effect of the large social group.     

Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

Functional torque-velocity and power-velocity characteristics of elite athletes

Technical limitations of some isokinetic dynamometers have called into question the validity of some data on human muscle mechanics. The Biodex dynamometer has been shown to minimize the impact artefact while permitting automatic gravity correction. This dynamometer was used to study quadriceps muscle torque and power generation in elite power (n = 6) and elite endurance (n = 7) athletes over 12 randomly assigned isokinetic velocities from 30 degrees.s-1 to 300 degrees.s-1. The angle at peak torque varied as a negative, linear function of angular velocity, with the average angle across test velocities being 59.5 degrees (SD 10.2 degrees). Power athletes developed greater peak torque at each angular velocity (P less than 0.05) and experienced a 39.7% decrement in torque over the velocity range tested. Endurance athletes encountered a 38.8% decline in peak torque. Torques measured at 60 degrees of knee flexion followed a similar trend in both groups; however the greatest torques were recorded at 60 degrees.s-1 rather than at 30 degrees.s-1. Leg extensor muscle power increased monotonically with angular velocity in both power (r2 = 0.728) and endurance athletes (r2 = 0.839); however these curves diverged significantly so that the power athletes produced progressively more power with each velocity increment. These inter group differences probably reflected a combination of natural selection and training adaptation.     

A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland

A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.     

Nucleotide sequence analysis of tetracycline resistance gene tetO from Streptococcus mutans DL5.

Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.