Properties of aminoglycoside-3'-phosphotransferases determined by kanamycin transposones Tn 601 and Tn 5

Aminoglycoside-3'-phosphotransferase I and II (APT-3'-I and APT-3'-II) has been purified to homogenity from the cells of E. coli containing the plasmids R6 and JR67, respectively. The purification procedure involved competitive affinity chromatography on neomycin-sepharose and gel-filtration on Sephadex G-100. The specific activity of APT-3'-I with the substrates--lividomycin A, neomycin B, paromycin, ribostamycin, kanamycins A and B--are 4.3, 2.8, 2.1, 1.6, 0.9 and 0.8 mol/min. mg protein, respectively. The specific activity of APT-3'-II with the substrates--ribostamycin, paromycin, kanamycins A and B, neomycin B--are 8.0, 7.2, 4.0, 4.5 and 3.6, respectively. Mg2+ is required for the activity of both enzymes. Co2+, Zn2+ and Mn2+ are active in case of APT-3'-I; however, these cations are less active than Mg2+. The pH-optimum of APT-3'-I and APT-3'-II is 7.0--7.5. High ionic strength is required for the activity of both enzymes. The molecular weights of APT-3'-I and APT-3'-II are about 36 000 and 26 000, respectively. The amino acid composition of APT-3'-I and APT-3'-II was determined. Both enzymes contain tryptophane residues whose fluorescence intensity decreased when ATP, but not amino-glycoside antibiotics, is added. The interrelationship between the molecular weights of these enzymes and the sizes of the loops of transposones Tn 601 and Tn 5, encoding APT-3'-I and APT-3'-II, is discussed.     

Ultracentrifugal analysis of the junction complexes of the red cell membrane cytoskeletal network: application to hereditary spherocytosis and metabolically depleted cells

A method has been developed for the assessment of the number of spectrin dimer units associated with each actin protofilament junction, in the membrane cytoskeletal network (i.e. the degree of branching) of the red cell. Ghosts are first exposed to elevated temperature at low ionic strength to dissociate some 65% of the spectrin tetramers (that link the network junctions) into dimers, without causing their release from the actin filaments. Non-ionic detergent is then added to solubilize the membrane itself with its intrinsic proteins, so as to liberate the cytoskeletal material, and the mixture is immediately examined in the analytical ultracentrifuge. The predominant components observed are isolated junctions (20 S), free spectrin dimers and the residual undissociated cytoskeletal material, with very minor components, probably corresponding to multiple junctions, linked by spectrin tetramers. The junction boundary is homogeneous within the accuracy of measurement and is taken to correspond to a complex containing six spectrin dimers, known to predominate in situ. About 17% of the total network is liberated in this form and 12% as free spectrin dimers. In hereditary spherocytosis both the size of the junction complex (as reflected by its sedimentation coefficient) and the proportion of the complex and of free spectrin liberated are indistinguishable from normal values. We conclude that the reported deficit of spectrin in hereditary spherocytosis is not reflected by a lower degree of branching of the network, and, if the membrane area is not correspondingly reduced, this must mean that the junctions are more widely spaced and the spectrin tetramers therefore more extended. In metabolically depleted cells, in which the cytoskeletal proteins are known to be extensively dephosphorylated, there is no change in the sedimentation pattern and thus no detectable loss of spectrin from the junctions or weakening in the cohesion of the cytoskeletal network.     

Lipid composition of spinal bone tissue in rabbits of different ages

Studies have been made on the lipid composition of total lipids, triglycerides and their fatty acids, cholesterol and phospholipids in the vertebral column of young and adult rabbits. It was shown that the content of total lipids and triglycerides increases, whereas that of cholesterol and phospholipids decreases with age. The content of total lipids in the vertebral column is 10 times higher as compared to that in the bones of the extremities. Mid-thoracic part of the vertebral column exhibits higher lipid content than other thoracic parts of the column. Lipid content of the vertebral processes is lower than that of the vertebral bodies. These data indicate lipid specificity and heterogeneity of bone tissue of the vertebral column. The main fatty acids of vertebral triglycerides are presented by those with 14-18 carbon atoms (90%), no acids with 22 atoms were found. Higher content of the linoleic acid (19%) and higher total unsaturation of triglycerides were found in the bone tissue of rabbits in comparison with those of man.     

Proliferation of AXC/SSh rat prostate cancer cells in vitro is androgen modulated

We used heterogeneous parental cultures of AXC/SSh rat prostate cancer cells to isolate clonally derived prostate cancer cell lines. Light and electron microscopic analyses established that parental and clonally isolated cells possess features characteristic of secretory epithelium. Biochemical analyses showed that these cells contained androgen receptors and acid phosphatase and 5 alpha-reductase activity; phenotypic markers characteristic of differentiated prostate epithelium. Content of these prostate epithelial cell markers was variable and cell line specific. We used selected cell lines to examine androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro. We found that proliferation of C-family or D-family cells, those respectively maintained on medium without additions or medium containing 10(-7) M 5 alpha-dihydrotestosterone, was not affected by changes in medium testosterone concentration through the range 10(-6)-10(-9) M. In contrast, testosterone modified proliferation of T-family cells, those maintained on medium containing 10(-7) M testosterone, and effects were antagonized by the anti-androgen RU 23908. Preliminary studies established that AXC/SSh rat prostate cancer cells elaborate polypeptide components which stimulate in vitro cell proliferation. Both the ability to elaborate these components and their effects on in vitro cell proliferation appeared to be cell line specific.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

Genetic variation maintained in multilocus models of additive quantitative traits under stabilizing selection.

Stabilizing selection for an intermediate optimum is generally considered to deplete genetic variation in quantitative traits. However, conflicting results from various types of models have been obtained. While classical analyses assuming a large number of independent additive loci with individually small effects indicated that no genetic variation is preserved under stabilizing selection, several analyses of two-locus models showed the contrary. We perform a complete analysis of a generalization of Wright's two-locus quadratic-optimum model and investigate numerically the ability of quadratic stabilizing selection to maintain genetic variation in additive quantitative traits controlled by up to five loci. A statistical approach is employed by choosing randomly 4000 parameter sets (allelic effects, recombination rates, and strength of selection) for a given number of loci. For each parameter set we iterate the recursion equations that describe the dynamics of gamete frequencies starting from 20 randomly chosen initial conditions until an equilibrium is reached, record the quantities of interest, and calculate their corresponding mean values. As the number of loci increases from two to five, the fraction of the genome expected to be polymorphic declines surprisingly rapidly, and the loci that are polymorphic increasingly are those with small effects on the trait. As a result, the genetic variance expected to be maintained under stabilizing selection decreases very rapidly with increased number of loci. The equilibrium structure expected under stabilizing selection on an additive trait differs markedly from that expected under selection with no constraints on genotypic fitness values. The expected genetic variance, the expected polymorphic fraction of the genome, as well as other quantities of interest, are only weakly dependent on the selection intensity and the level of recombination.     

N-Methyl-D-Aspartate receptors in acquisition of the short-term memory in the honeybeeApis mellifera

Role of NMDA receptors in the process of associative learning has been studied in the honeybeeApis mellifera L. in behavior experiments, using method of conditional reflexes. To determine pharmacological profile of NMDA receptors, effects of Mg²⁺ ions, NMDA, glycine (Gly), antagonist of the glycine site 5,7-dichlorokynurenic acid (DCK), competitive antagonists of NMDA receptors: D,L-2-aminophosphovalerate (APV), L-2-aminophosphobutyrate (APB), and D-glutamyl-aminomethylphosphonic acid (GAMP) as well of antagonists of NMDA receptor ion channels MK-801 and ketamine (Ket) were studied on acquisition of alimentary conditional reflex and its retention in memory. NMDA increased capacity for learning by stimulating shortterm memory. The NMDA receptor co-agonist Gly activated this NMDA effect. DCK eliminated the NMDA and Gly stimulatoty effects. All tested antagonists at millimolar concentrations inhibited associative function. The data obtained confirm our hypothesis about participation of NMDA receptors in processes of formation of short-term memory in the honeybeeApis mellifera and suggest that functional characteristics of the NMDA receptors involved in the process of associative learning in the honeybee resemble those in mammals.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).     

Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin.

The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy