Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

Human epithelial teratocarcinoma cells (P3): radiobiological characterization, DNA damage, and comparison with other rodent and human cell lines

Survival parameters and immediate DNA damage induced by 60Co gamma rays, 50-kVp X rays, and Janus fission-spectrum neutrons in human epithelial P3 cells (derived from an embryonic teratocarcinoma) are compared with those for Chinese hamster lung V79 cells. DNA damage caused by X and gamma irradiation, measured by alkaline elution methods, is the same in both cell types, whereas the P3 cells are about two times more sensitive (as measured by Do ratios of the final survival curve slope) to the lethal effects of these radiations than are the V79 cells. Human P3 cells are also more sensitive to the lethal effects of fission-spectrum neutrons than V79 cells. Survival experiments with split radiation doses and hypertonic salt treatment indicate that both P3 cells and V79 cells can recover from radiation-induced damage efficiently.     

Measurement of double-strand breaks in Chinese hamster cell DNA by neutral filter elution: calibration by 125I decay

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.     

Short-term entrainment of ventilation to the walking cycle in humans

We describe a breath-by-breath method to test for entrainment of breathing and walking cycles. Thirty-eight normal subjects walked comfortably on a treadmill while breathing through a pneumotachograph. We analyzed the time intervals between heel strikes and the onset of inspiration (or expiration) for evidence of phase locking between steps and breaths, using Monte Carlo simulation to model the probability that n consecutive inspirations (or expirations) would begin at a constant time interval +/- 0.10 s from heel strikes by chance. We developed empirical criteria for rhythm synchronization during series of four or more breaths, while maintaining an estimated specificity of 95%. The majority of subjects showed some evidence of entrainment (29 +/- 23% of breaths on average), which occurred intermittently, usually lasting less than 10 breaths at a time. The precision of phase locking during spontaneous entrainment was similar to that in 10 subjects who attempted to maintain deliberate entrainment. The results suggest that the walking cadence provides a persuasive, but not dominant, input to the central breathing pattern generator. The present method can detect entrainment even when it occurs sporadically or with varying coupling pattern.     

Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) has been used to detect Listeria monocytogenes in whole milk at a level of 0.1 cfu per 30 ml. This high degree of sensitivity has been achieved following enzymatic digestion, polysulphonone membrane filtration and amplification of a nucleotide sequence within the promoter region of hlyA. Key elements of the procedure are the absence of enrichment culture and a complete solubilization of the membrane filter, ensuring total nucleic acid recovery. The simplicity of the protocol coupled with high sample volumes and exquisite sensitivity extends the relevance of PCR within food and environmental microbiology.     

Genetic,biochemical and immunological evidence for the involvement of two alcohol dehydrogenases in the metabolism of propane by Rhodococcus rhodochrous PNKb1

NAD⁺-dependent propan-1-ol and propan-2-ol dehydrogenase activities were detected in cell-free extracts of Rhodococcus rhodochrous PNKb1 grown on propane and potential intermediates of propane oxidation. However, it was unclear whether this activity was mediated by one or more enzymes. The isolation of mutants unable to utilize propan-1-ol (alcA^-) or propan-2-ol (alcB^-) as sole carbon and energy sources demonstrated that these substrates are metabolized by different alcohol dehydrogenases. These mutants were also unable to utilize propane as a growth substrate indicating that both alcohols are intermediates of propane metabolism. Therefore, propane is metabolized by terminal and sub-terminal oxidation pathways. Westernblot analysis demonstrated that a previously purified NAD⁺-dependent propan-2-ol dehydrogenase (Ashraf and Murrell 1990) was only synthesized after growth on propane and sub-terminal oxidation intermediates (but not acetone), and not propan-1-ol or terminal oxidation intermediates. Therefore, our evidence suggest that another dehydrogenase is involved in the metabolism of propan-1-ol and this agrees with the isolation of the alcA^- and alcB^- phenotypes. The previously characterized NAD⁺-dependent propan-2-ol dehydrogenase from R. rhodochrous PNKb1 is highly conserved amongst members of the propane-utilizing Rhodococcus-Nocardia complex.     

Cyclic behavior of plum curculio,Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), within caged dwarf apple trees in spring

From 12 to 19 May 1987, during Morspur apple bloom, 21 radioactively labeled (⁶⁵ Zn) adult plum curculios, Conotrachelus nenuphar (Herbst), were released within a field cage containing four dwarf apple trees and located three times a day. A technique was developed for quickly obtaining (x, y, z)coordinates of location for adults foraging within apple trees. Cyclic patterns of behavior were detected using spectral analysis procedures. Over 70% of plum curculios exhibited diel periodicity with respect to activity and rate of movement, 36% exhibited such periodicity with respect to presence in the trees, and 27% with respect to movements from the center to the periphery of the canopy. Presence in fruit clusters, height in the trees, and movements along east-west and north-south axes showed little or no periodicity. Factors triggering cyclic behavior and practical implications of the results are discussed.