Ion channel regulation by calmodulin binding

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca²⁺-calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca²⁺-release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N-lobe of calmodulin specifically interacts with one class of ion-channel proteins and the C-lobe with another.     

L-Alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na⁺-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na⁺-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 10.62 ±0.25 (xxxsd) (short-circuit current) or 10.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, -aminoisobutyric acid, and -alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.This study was supported by the Wellcome Trust (to D.T.T. and N.L.S.) and the LINK Programme in Selective Drug Delivery and Targeting (funded by the SERC/MRC/DTI and Industry). Charlotte Ward gave excellent technical assistance.     

Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.     

Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

[3H] CGP 39653 Binding to the Agonist Site of the N-Methyl- D-Aspartate Receptor Is Modulated by Mg2+ and Polyamines Independently of the Arcaine-Sensitive Polyamine Site

Abstract: This study investigated the binding of [³H] CGP 39653, a novel high-affinity antagonist of the N-methyl-D- aspartate (NMDA) recognition site of the NMDA receptor complex. [³H] CGP 39653 bound to the NMDA receptor in well washed rat brain membranes with an affinity of about 15 nM. Other NMDA site drugs inhibited [³H] CGP 39653 binding with the following order of potency: DL-(tetrazol-5- yl)glycine > glutamate > CGS 19755 > DL-2-amino-5- phosphonovalerate (DL-AP5) > NMDA. Glycine and 5, 7- dichlorokynurenate partially inhibited binding. The poly-amines spermine and spermidine increased [³H] CGP 39653 binding (EC₅₀ values of 10 and 22 μM, respectively). This effect was mimicked by arcaine, 1, 5-diethylaminopiperidine, diaminodecane, diethylenetriamine, and Mg²⁺. The increase in [3H] CGP 39653 was a result of an increased affinity of the binding site for the ligand with very little effect on binding site density. Spermine and Mg²⁺also increased the affinity of the antagonists DL-AP5 and CGS 19755, but had only minor effects on the affinity of glutamate and NMDA. Arcaine did not reverse the enhancement of [3H] CGP 39653 binding by spermine, spermidine, or Mg²⁺. Channel-blocking dissociative anesthetics, including dizocilpine and ketamine, did not alter basal or Mg²⁺-stimulated [3H] CGP 39653 binding. Spermine did not alter either the enhancement of [³H]- dizocilpine by glutamate or the inhibition of [³H]dizocilpine by DL-AP5 or CGS 19755. These studies show that poly-amines and divalent cations selectively enhance the affinity of antagonists for the agonist binding site on the NMDA receptor complex. However, this effect is mediated by a site independent of the primary polyamine site defined using [³H] dizocilpine binding.     

ISL2, a new mobile genetic element in Lactobacillus helveticus

Spontaneous, phenotypically stable mutations at the -galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 by long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1 % identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4–21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.     

Physiological effects of five months exposure to low concentrations of O3 and NH3 on Douglas fir (Pseudotsuga menziesii)

Three years old seedlings of Douglas fir (Pseudotsuga menziesii) were exposed lo filtered air, O₃ (day and night concentrations of 78 and 30 μgm^?3: respectively). NH₃ (54 μg m^?3) and to a mixture of NH₃+O₃ (day and night concentrations of 49 + 83 and 49 + 44 μg m^?3 respectively), for 5 months in fumigation chambers. Both gas exchange and chlorophyll fluorescence were measured on shoots which had sprouted at the beginning of the exposure period. After 4. 8, 10 and 20 weeks of exposure, light response curves of electron transport rate (J) were determined, in which J was deduced from chlorophyll fluorescence. Net CO₂ assimiialion was measured at maximum light intensity of 560) μmol m^?2 S^?1 (P_n.560). After 8 and 10 weeks of exposure also light response curves of CO₂ assimilation were assessed. Shoots exposed to O₃ showed a reduction in net CO₂ assimilation as compared to the control shoots during the entire exposure period. The reduction was related lo a lower chlorophyll content and a lower electron transport rate, whereas no effect on quantum yield efficiency (qy) was observed. In contrast, shoots exposed to NH₃ showed a positive effect on photosynthesis. Shoots exposed to NH₃. + O₃ showed a rapid increase in P_n.560, in the period between 4 and 8 weeks to a level equal of that of the NH₃-treatment. After this period a decline in P_n.560 was observed. After 10 weeks of exposure shoots exposed to O₃ showed an increased transpiration rate in the dark as compared to the control shoots. In addition, water use efficiency (WUE) declined as a result of an increase in leaf conductance. Both observations indicate that the stomatal apparatus was affected by O₃. A high transpiration rate in the dark was also found for shoots esposed to NHX. However, shoots exposed to NH₃+ O₃ showed neither an effect on WUE, nor an effect on transpiration rate in the dark. The possibility that NH₃ delayed the O₃ induced effects on photosynthesis and stomatal conductance is discussed.     

Relationship between soluble tumor necrosis factor (TNF) receptors and TNFα during immunotherapy with interleukin-2 and/or interferon α

Eleven metastatic cancer patients were studied during three different regimens of immunotherapy with interleukin-2 (IL-2) and/or interferon (IFN): group A received 4 days of IL-2 i.a. infusion (n=3), group B IFN s.c. during 5 days (n=4), followed on day 3 by 5 days of a continuous IL-2 i.v. infusion, and group C had 4 days of IL-2 i.v. infusion together with s.c. IFN on days 1 and 4 (n=4). Soluble tumor necrosis factor receptors (sTNFR) p55 and p75 and TNF concentrations in serum were analyzed before therapy and daily during 8 days of the first therapy cycle. sTNFR was measured by radioimmunoassay. sTNFR p55 increased in all patient groups from a baseline value of 5.2±0.9 ng/ml to a maximum of 13.6±1.2 ng/ml by days 3–4 (P=0.003). sTNFR p75 increased from 7.6±1.1 ng/ml to peak values of 30.1±2.6 ng/ml in groups A and B (P=0.02). In group C the sTNFR p75 response was weak (NS). In group B, the increase of both p55 and p75 occurred only after addition of IL-2 to IFN. TNF increased weakly during treatment with IFN alone (group B); it rose strongly during IL-2 and the combined treatment (groups A-C) from 8±2 pg/ml to 115±13 pg/ml (P=0.003). In group B, it reached the maximum 24 h after addition of IL-2 to IFN and decreased thereafter. there was a significant relationship between TNF and sTNFR p55 or sTNFR p75 in groups A and C, (P=0.001), but not in group B. Group C was also investigated during the third therapy cycle. The increase of sTNFR p75 was stronger (P=0.01) and that of TNF weaker than in the first cycle; the sTNFR p55 response was similar in both cycles. In conclusion sTNFR p55 and p75 are rapidly induced during IL-2 and IL-2+IFN treatment, the increase of sTNF receptors parallels or exceeds that of TNF and may influence the immunomodulatory effects of TNF during cytokine therapy.