The biosynthetic pathway of new polyamines in Caldariella acidophila

A spontaneous mutant of Escherichia coli (strain AB2847), selected for resistance to the aminoglycoside antibiotic neamine, shows severe restriction of amber suppressors in vivo. Ribosomes isolated from the mutant exhibit only low misreading in vitro in the presence of the antibiotic. Genetic and biochemical analyses indicate that the neamine-resistant phenotype is the result of two distinct mutations. The first, res3128, appears to affect the gene (strA) coding for the ribosomal protein S12. Although it leads to a restrictive phenotype it does not, however, confer resistance to streptomycin. The second mutation, X3128, is located between the sirA and AROB loci and is lethal when segregated from the res3128 mutation. It may affect the ribosome at the level of a post-translational modification.     

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

Structure of F1-ATPases

F₁-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F₁-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type ₃₃ and ₂₂₂₂₂ were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F₁-ATPases.     

Analysis of prednisone,prednisolone and their 20β-hydroxylated metabolites by high-performance liquid chromatography

A high-performance liquid chromatographic technique primarily developed for use on samples from kidney perfusion studies is presented for simultaneous determination of prednisone, prednisolone and their 20β-hydroxylated metabolites. The technique employs 6β-hydroxycortisol as the internal standard. Samples are extracted with ethyl acetate, washed with sodium hydroxide and water and injected onto a silica gel column with UV detection at 254 nm. Inter- and intraday variability of the assay was determined at two concentrations of each steroid and was less than 10%. Assay steroid recovery ranged from 54.1% for prednisone to 63.2% for 20β-hydroxyprednisone. Sensitivity is 4–10 ng/ml for the steroids measured. The chromatographic conditions may be modified to permit quantitation of these steroids from plasma samples. This method may alternatively be used for quantitation of 6β-hydroxycortisol, an endogenous indicator of enzyme induction. A perfusate concentration-time profile is presented from a kidney perfusion study using prednisolone.     

A METHOD OF CORONARY RETROPERFUSION FOR THE TREATMENT OF ACUTE MYOCARDIAL ISCHEMIA

A method of retrograde perfusion of the myocardium has been developed in dogs. It consists of a double lumen balloon-tipped catheter inserted transvenously into the coronary sinus, with one lumen connected to a roller pump, the other to a helium counterpulsing pump. Oxygenated heparinized blood is obtained from the femoral artery and pumped continuously into the coronary sinus at a pressure of 50-75 mm Hg. The balloon is inflated during diastole, sealing the coronary sinus and promoting retrograde flow, and is deflated during systole, allowing blood drainage into the right atrium and preventing venous congestion. Thirteen anesthetized open-chest dogs were subjected to 15 minutes of proximal LAD artery occlusion and 30 minutes of diastolic coronary sinus perfusion (DCSP). The area of ischemia was mapped by means of platinum electrodes capable of simultaneously measuring myocardial tissue oxygen tension M(p)O(2)) and electrograms. Reduction of M(p)O(2) with simultaneous elevation of the ST segment on the corresponding electrogram was considered an indication of ischemia. Diastolic coronary sinus perfusion improved myocardial oxygen tension in the ischemic myocardium, reduced ST segment elevation, and tended to restore arterial blood pressure. Histologically, there was no intramyocardial hemorrhage.     

In vitro flowering in cassava (Manihot esculenta Crantz)

Meristem-derived plantlets of cassava (Manihot esculenta Crantz) were induced to flower in vitro. Five genotypes out of 13 consistently responded to our culture conditions giving rise to male or female flowers. Male flowers contained anthers in which meiosis occurred and apparently normal pollen grains were formed.     

Effects of Intranigral Substance P and Neurokinin A Injections on Extracellular Dopamine Levels Measured with Microdialysis in the Striatum and Frontoparietal Cortex of Rats

Extracellular levels of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the striatum and frontoparietal (sensorimotor) cortex in halothane-anesthetized rats were analyzed simultaneously using in vivo microdialysis. Basal DA levels, measured from the microdialysis perfusate, were 6.4 +/- 0.8 nM (n = 15) in the striatum and 0.9 +/- 0.1 nM (n = 15) in the frontoparietal cortex. Subcutaneous injections of d-amphetamine (2 mg/kg) increased DA levels 10-fold in the striatum and fivefold in the cortex. Injections of substance P (0.07 nmol/0.2 microliters) into the substantia nigra pars reticulata (SNR) increased DA and DOPAC levels approximately 30% in the ipsilateral striatum and approximately 50% in the ipsilateral frontoparietal cortex. Injections of neurokinin A (0.09 nmol/0.2 microliter) into the SNR increased DA and DOPAC levels approximately 30% in the ipsilateral striatum but did not significantly affect DA levels in the ipsilateral frontoparietal cortex, although DOPAC levels were increased by approximately 50%. It is suggested that striatal and cortical DA release is regulated differently by nigral substance P and neurokinin A terminals.     

Steroid modulation of oxytocin/vasopressin receptors in the uterus

Oxytocin (OT) and V1 vasopressin (VP) receptors are present simultaneously in several tissues, including the uterus. In myometrium these receptors mediate contractility, while in endometrium they mediate the release of other uterotonic substances as endothelin (ET). In rabbit myometrium, estrogens increase, while progesterone blunts neurohypophysial hormone receptors. However, the action of sex steroids on OT and V1 VP receptors differs in terms of the ED₅₀ and maximal effect. Therefore, at parturition, only OT receptors show a dramatic rise, while V1 VP receptors do not change, suggesting a major role for OT in labor. ET is a potent stimulator of uterine activity acting through specific receptors present on myometrial cells. These receptors as well as the endometrial localization of ET are modulated by sex steroids, indicating that ET might represent a paracrine regulator of uterine activity. In humans, OT but not V1 VP receptors increase as pregnancy progresses, confirming the primary relevance of OT in timing delivery.     

Quaternary structure of ATP synthases: Symmetry and asymmetry in the F1 moiety

It has been proposed that during ATP synthesis/hydrolysis F₁ ATPases experience a complex pattern of nucleotide binding and release during the catalytic cycle (binding change mechanism). This type of mechanism has implications that can be correlated with the structure of the enzyme. F₁-ATPases (stoichiometry ₃₃) are essentially a symmetrical trimer of pairs of the major subunits ( and ); the minor subunits (, and ) are in single copies and interact with the trimer in an asymmetrical fashion. The asymmetry introduced by the minor subunits has important structural and functional consequences: (1) it introduces differences between the potentially equivalent binding and catalytic sites in the major subunits, (2) it restricts the ways in which a binding change mechanism can occur, and (3) it governs the way in which the F₁ interacts with the (asymmetrical) F₀ sector.