Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.     

Selective systems for obtaining recessive ribosomal suppressors in saccharomycete yeasts

Recessive mutations only occurring in two genes (ribosomal suppressors sup1 and sup2) can be obtained using special selective system. We demonstrate that the absolute selectivity of the system is based on selection for simultaneous reversions to prototrophy in mutants requiring adenine and histidine in haploids marked by two different nonsense mutations--his7-1 (UAA) and ade1-14 (UGA, this being identified in the present study). In support to this conclusion, we developed an analogous system utilising his7-1 (UAA) and lys2-87 (UGA). The selectivity of the system is shown to be influenced both by the choice of nonsense alleles and by genotypic background.     

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.     

Side populations of glioblastoma cells are less sensitive to HSV-TK/GCV suicide gene therapy system than the non-side population

Side populations of glioblastoma cells are resistant to chemotherapy basically due to ABCG2-mediated efflux of small-molecule drugs. The herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy system is one of the best-characterized strategies for malignant tumors including glioblastoma. Since this system involves a small-molecule drug ganciclovir, we wonder if glioblastoma side population cells are able to “pump out” ganciclovir and thus resistant to this suicide gene therapy. By 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, we found that side populations are more resistant to this system than non-side populations. By flow cytometry and competition assay, we found that ganciclovir is a substrate for ABCG2.     

血管紧张素-(1-7)的舒血管效应

实验采用兔外周动脉离体标本,在预收缩血管后,用血管紧张素[angiotensin-(1-7),Ang-(1-7)]舒张血管,比较Ang-(1-7)对外周各血管床的舒张效应并分析其产生机制。结果显示：(1)Ang-(1-7)可剂量依赖性舒张血管,但舒张作用有所不同;(2)Ang-(1-7)的舒张作用在很大程度上依赖于内皮的NO系统;(3)Ang-(1-7)的舒张血管效应不通过AT1和AT2受体。上述结果提示：Ang-(1-7)可能作用于内皮上的非AT1和AT2受体,通过调节NO释放而起舒血管作用。     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis

Up-regulation of the high-affinity transport system (HATS) for NO(3)(-) and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO(3)(-) HATS by nitrogen starvation is suppressed in the atnrt2.1-1 mutant of Arabidopsis (Arabidopsis thaliana), deleted for both NRT2.1 and NRT2.2 nitrate transporter genes. We then used this mutant to determine whether lack of HATS stimulation affected the response of the root system architecture (RSA) to low NO(3)(-) availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO(3)(-) medium resulted in contrasting responses of RSA, depending on the level of nitrogen limitation. Moderate nitrogen limitation (transfer from 10 mm to 1 or 0.5 mm NO(3)(-)) mostly led to an increase in the number of visible laterals, while severe nitrogen stress (transfer from 10 mm to 0.1 or 0.05 mm NO(3)(-)) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO(3)(-) was markedly different. After transfer from 10 to 0.5 mm NO(3)(-), the stimulated appearance of LRs was abolished in atnrt2.1-1 plants, whereas the increase in mean LR length was much more pronounced than in Ws. These modifications of RSA mimicked those of Ws plants subjected to severe nitrogen stress and could be fully explained by the lowered NO(3)(-) uptake measured in the mutant. This suggests that the uptake rate of NO(3)(-), rather than its external concentration, is the key factor triggering the observed changes in RSA. However, the mutation of NRT2.1 was also found to inhibit initiation of LR primordia in plants subjected to nitrogen limitation independently of the rate of NO(3)(-) uptake by the whole root system and even of the presence of added NO(3)(-) in the external medium. This indicates a direct stimulatory role for NRT2.1 in this particular step of LR development. Thus, it is concluded that NRT2.1 has a key dual function in coordinating root development with external NO(3)(-) availability, both indirectly through its role as a major NO(3)(-) uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.     

Nonopioid placebo analgesia is mediated by CB1 cannabinoid receptors

Placebo analgesia is mediated by both opioid and nonopioid mechanisms, but so far nothing is known about the nonopioid component. Here we show that the specific CB1 cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (rimonabant or SR141716) blocks nonopioid placebo analgesic responses but has no effect on opioid placebo responses. These findings suggest that the endocannabinoid system has a pivotal role in placebo analgesia in some circumstances when the opioid system is not involved.     

Activation of NADPH-dependent superoxide production in a cell-free system by sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) elicits the production of superoxide (O2-) by a cell-free system represented by sonically disrupted guinea pig peritoneal macrophages. O2- generation requires NADPH and a heat-sensitive cellular component, is proportional to the amount of macrophage protein, and exhibits a pH optimum of 6.5-7. The kinetic parameters of the SDS-stimulated enzyme are: Km (+/- S.E.) = 0.0367 +/- 0.003 mM NADPH and Vmax (+/- S.E.) = 73.46 +/- 9.09 nmol O2-/mg of protein/min. O2- production is dependent on the cooperation between a particulate subcellular component sedimentable at 48,000 X g and a cytosolic factor present in the 48,000 X g supernatant. The activity of both components is destroyed by heating at 80 degrees C. Pretreatment of intact macrophages with phorbol myristate acetate results in the partial removal of the requirement for cytosolic factor; SDS is now capable of activating the isolated 48,000 X g pellet. Among a large number of anionic, cationic, and nonionic detergents tested, only the anionic detergents SDS and sodium dodecyl sulfonate are capable of eliciting O2- production in the cell-free system, SDS being the more potent stimulant. It is proposed that the structural requirements that make these compounds capable of activating the O2- forming NADPH oxidase in a cell-free system are the presence of an anionic polar head and a long hydrophobic alkyl tail. We suggest that sodium salts of long chain unsaturated fatty acids that were found by us to be capable of stimulating O2- production in a cell-free system (Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221) owe their activity to the fact that they function as anionic detergents.     

Interaction of diiodo-L-tyrosine and triiodophenol with bovine serum albumin. Circular dichroism and fluorescence studies.

As a model study to investigate the binding mechanism between thyroid hormones and carrier protein, the interaction of diiodo-L-tyrosine (DIT) and triiodophenol (I3phi) with bovine serum albumin (BSA) was investigated by circular dichroism (CD) and fluorescence methods. In both the DIT-BSA system and the I3phi-BSA system, induced Cotton effect was observed in the wavelength region near 320 nm. This induced Cotton effect was measured at various molar ratios of ligands to BSA (L/P). The value of the ellipticity at 319 nm, [theta]319, in the I3phi-BSA system was remarkably large compared with that of the DIT-BSA system, and [theta]319 at an L/P ratio of one was -1.96 X 10(4) (degree cm2 decimole-1) for the I3phi-BSA system and -0.1 X 10(4) for the DIT-BSA system. The binding constants for the combination of BSA with a single molecule of ligand, calculated by measuring the quenching of the fluorescence of the protein, were 1.33 X 10(5) M(-1) at 15 degrees for the DIT-BSA system and 1.6 X 10(9) M(-1) at 28 degrees for the I3theta-BSA system. These results suggest that the binding of I3theta to BSA is stronger than that of DIT and a cleft may exist more congruent with the molecular dimensions of I3theta than with those of DIT.     

Myo-Inositol transport in Klebsiella aerogenes. scyllo-Inositol, a non-metabolizable substrate for the study of the myo-inositol transport system.

1. Scyllo-Inositol is transported against a concentration gradient by Klebsiella aerogenes, but is not metabolized. 2. The apparent KT of scyllo-inositol is 0.05 X 10(-3) M while that of myo-inositol is 0.23 X 10(-3) M. The V values are respectively 32 and 12 nmol min(-1) mg cells(-1) (dry weight). 3. Both cyclitols bind to the same carrier, since typical competitive inhibition kinetics are observed. 4. Neither phosphoenolpyruvate phosphotransferase nor a periplasmic binding protein seems to be involved in the scyllo-inositol transport system. 5. The induction of the transport system is not repressed by malate, furmarate, citrate, succinate or pyruvate; however, acetate represses the induction. The activity of the transport system is increased when the growth medium is supplemented with adenosine 3':5'-monophosphate.     

Eosinophil peroxidase oxidation of thiocyanate. Characterization of major reaction products and a potential sulfhydryl-targeted cytotoxicity system

Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.     

Major alterations of the regulation of root NO(3)(-) uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis

The role of AtNrt2.1 and AtNrt2.2 genes, encoding putative NO(3)(-) transporters in Arabidopsis, in the regulation of high-affinity NO(3)(-) uptake has been investigated in the atnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M.F. Dorbe, M. Cerezo, M. Orsel, F. Granier, A. Gojon, F. Daniel-Vedele [2001] FEBS Lett 489: 220-224) demonstrated that root NO(3)(-) uptake is affected in this mutant due to the alteration of the high-affinity transport system (HATS), but not of the low-affinity transport system. In the present work, we show that the residual HATS activity in atnrt2 plants is not inducible by NO(3)(-), indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO(3)(-) uptake in this genotype is likely to be due to the constitutive HATS. Root (15)NO(3)(-) influx in the atnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO(3)(-) availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO(3)(-) uptake in NO(3)(-)-fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH(4)(+) in the nutrient solution fails to inhibit (15)NO(3)(-) influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH(4)(+) uptake systems in the mutant. These results collectively indicate that AtNrt2.1 and/or AtNrt2.2 genes play a key role in the regulation of the high-affinity NO(3)(-) uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment.     

Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli.

Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).     

A perfluorochemical loss/restoration (L/R) system for tidal liquid ventilation

Tidal liquid ventilation is the transport of dissolved respiratory gases via volume exchange of perfluorochemical (PFC) liquid to and from the PFC-filled lung. All gas-liquid surface tension is eliminated, increasing compliance and providing lung protection due to lower inflation pressures. Tidal liquid ventilation is achieved by cycling fluid from a reservoir to and from the lung by a ventilator. Current approaches are microprocessor-based with feedback control. During inspiration, warmed oxygenated PFC liquid is pumped from a fluid reservoir/gas exchanger into the lung. PFC fluid is conserved by condensing (60-80% efficiency) vapor in the expired gas. A feedback-control system was developed to automatically replace PFC lost due to condenser inefficiency. This loss/restoration (L/R) system consists of a PFC-vapor thermal detector (+/- 2.5%), pneumatics, amplifiers, a gas flow detector (+/- 1%), a PFC pump (+/- 5%), and a controller. Gravimetric studies of perflubron loss from a flask due to evaporation were compared with experimental L/R results and found to be within +/- 1.4%. In addition, when L/R studies were conducted with a previously reported liquid ventilation system over a four-hour period, the L/R system maintained system perflubron volume to within +/- 1% of prime volume and 11.5% of replacement volume, and the difference between experimental PFC loss and that of the L/R system was 1.8 mL/hr. These studies suggest that the PFC L/R system may have significant economic (appropriate dosing for PFC loss) as well as physiologic (maintenance of PFC inventory in the lungs and liquid ventilator) impact on liquid ventilation procedures.     

Relationship of bacterioplankton production with primary production and respiration in a shallow estuarine system (Ria de Aveiro, NW Portugal)

The response of bacterial growth to phytoplankton production and planktonic respiration (RESP) variation was examined over different stations and dates in the shallow estuarine system Ria de Aveiro. The temporal and spatial profiles of bacterial productivity (2.7-744.2mg Cm(-3)d(-1)) did not coincide with those of primary production (PP) (0.2-19.1 g Cm(-3)d(-1)) and RESP (0.1-8.2 g Cm(-3) d(-1)). The bacterioplankton production/PP ratio varied differently, depending on the season and location. The heterotrophic zones, with the lowest values of PP, exhibited the most intense bacterial secondary production. Moreover, the variation of PP in the system was rather small when compared with that of bacterial secondary production. These suggest that, in a large extension of the lagoon and throughout the year, bacterioplankton growth is largely dependent on non-phytoplanktonic carbon sources. Benthic PP and/or allochtonous organic matter from land have a fundamental role in the dynamics of the planktonic compartment of the estuarine system.     

The Y1 receptor for NPY: a key modulator of the adaptive immune system

Growing evidence suggests that the neuropeptide Y (NPY) system plays an important role in the immune system. Yet, little is known about the expression of NPY and receptors in the immune system. Moreover, original contradicting results have confused the picture and hampered a clear understanding of its role in the immune system. The use of Y(1) receptor-deficient mice, combined with advanced methods to investigate immune functions, have provided the solution to the problem raised by previous disparities. From results obtained using Y(1)-deficient mice (Y(1)(-/-)), we uncovered a bimodal role for Y(1) on immune cells. Y(1) expression on antigen-presenting cells (APC) is essential for their function as T cell priming elements. Conversely, Y(1) signaling in T cells plays a regulatory role without which T cells are hyper-responsive. The opposite role of Y(1) on APC and T cells has reconciled previous disparities by showing that signaling via Y(1) protects against inflammation by inhibiting T cell responses, whereas Y(1)(-/-) mice are protected in the same inflammatory models due to defective APCs.