Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

How has extracorporeal shock-wave lithotripsy changed the treatment of urinary stones in Quebec?

OBJECTIVES: To determine the number of people who underwent treatment of urinary stones in Quebec before and after the introduction of extracorporeal shock-wave lithotripsy (ESWL) and to determine how the introduction of ESWL influenced resource utilization. DESIGN: Before-after study; data were obtained from administrative databases and hospital-based cost estimates. SETTING: The 68 acute care hospitals in Quebec in which treatment of urinary stones is undertaken. PATIENTS: Quebec residents admitted to hospital for treatment of urinary stones between the fiscal years 1984 and 1992. OUTCOME MEASURES: Number of people treated for urinary stones per year, total number of procedures per year (including open surgery, percutaneous procedures, retrograde procedures and ESWL), and annual resources (including number of hospital bed-days and direct costs) for treatment of urinary stones used overall and in hospitals with and without ESWL services. RESULTS: Over the study period the number of people treated for urinary stones increased by 59%. As well, the combined frequency of ESWL and surgery (the two main treatment methods) increased by 107%. These increases were largely due to rates of treatment that grew by 52% among women and by 34% among men. The total number of hospital bed-days decreased by 28%, which reflected shorter hospital stays for ESWL. However, despite this decrease, the total direct annual costs were 7% higher in 1992 than in 1984 because of the increased numbers of people treated and procedures performed. In the three hospitals that offered ESWL the number of hospital bed-days and the direct costs of treating urinary stones increased by 49% and $2.5 million respectively. In the 65 other hospitals these figures decreased by 41% and about $2.9 million respectively. CONCLUSIONS: Because of increased intervention rates the total cost of treating urinary stones has risen since the introduction of ESWL. The introduction of ESWL has also been associated with a shift in the use of resources for treating urinary stones to hospitals with a lithotriptor. The reasons for the increased intervention rates are unknown. However, given the possibility of negative health effects and the increased costs, studies to determine whether the increased rates improve health outcomes are warranted.     

Transferable clastogenic activity in plasma from patients with Fanconi anemia

The present study was conducted on 13 patients with Fanconi anemia, 25 parents and 12 siblings. The chromosomal instability characteristic of this congenital breakage syndrome was associated with the presence of transferable clastogenic material in the plasma, as also reported previously for ataxia telangiectasia and Bloom's syndrome. While all plasma ultrafiltrates from homozygotes had chromosome damaging properties, the clastogenic material had to be concentrated in most heterozygotes to reach detectable levels. The clastogenic effect was exerted via the intermediacy of superoxide radicals, since it was regularly inhibited by superoxide dismutase (SOD). This adds further evidence for a prooxidant state in this hereditary disease. The autosustained clastogenic activity possibly plays a role in the progressive impairment of blood cell-producing bone marrow and may predispose patients to develop cancer and leukemia. Prophylactic use of antioxidants may be recommended, using clastogenic plasma activity as a guide.     

Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Reduced maximum capacity of glycolysis in brown adipose tissue of genetically obese, diabetic (db/db) mice and its restoration following treatment with a thermogenic beta-adrenoceptor agonist

The maximal activities of the key glycolytic enzymes hexokinase and 6-phosphofructokinase, were reduced in brown adipose tissue in db/db mice compared to their lean littermates. Treatment of db/db mice with the thermogenic beta-adrenoceptor agonist, BRL 26830, restored normoglycaemia. The only significant increase in activity of hexokinase and 6-phosphofructokinase in the BRL 26830-treated db/db mice occurred in brown adipose tissue where the total tissue activity increased 10- and 11-fold respectively. These changes together with increased 2-deoxyglucose uptake in vivo suggest that brown adipose tissue can play a quantitatively important role in the removal of glucose from the blood.     

The influence of glycyl residues on the flexibility of peptide hormones in solution. A 13C-nuclear-magnetic-resonance study of luteinizing hormone-releasing hormone (luliberin) and its des-glycinamide10 N-ethylamide analog.

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.     

Labeling of glucagon binding components in hepatocyte plasma membranes.

A photosensitive derivative of glucagon, ¹²⁵I-N^?-4-azido-2-nitrophenyl-glucagon, has been synthesized and used to specifically label glucagon binding proteins in hepatocyte plasma membranes. Photolysis of the derivative in the presence of a membrane suspension results in the incorporation of radioactivity primarily into membrane components with a molecular weight range of 23,000–25,000. The binding properties of the derivative are essentially identical to that observed for glucagon. The binding of ¹²⁵I-NAP-glucagon was completely inhibited in the presence of glucagon (3 μM) while greater than 90% of the covalent labeling was also inhibited in the presence of glucagon. These studies suggest that the labeled membrane protein may be a component of the glucagon receptor.