Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.     

6-Aminohexanoic acid-plasminogen interactions studied by fluorescence

Circular polarization of luminescence spectra of human plasminogen and of its derivatives were measured in solutions of ligand-free proteins and with saturating amounts of 6-aminohexanoic acid. Spectroscopic changes induced by the ligand reveal similar perturbations of the binding sites in all the protein derivatives. It is concluded that the gross conformational change induced by 6-aminohexanoic acid binding to the native plasminogen involves changes of sterical relations of entire protein domains.     

Accessibility of adenine binding sites in dehydrogenases to small molecules studied by fluorescence quenching.

Quenching of the fluorescence of ethenoadenine derivatives by iodide ions and by methionine was studied in solution and when the nucleotides were bound to several dehydrogenases. The fluorescence of epsilonADPR in neutral aqueous solution is dynamically quenched by both quenching agents. The quenching of free epsilonNAD+ by methionine was found to be predominantly static and was satisfactorily described to result from complex formation between quencher and dinucleotide. The rat constant for quenching by iodide of epsilonNAD+ in the ternary complex with LADH and pyrazole is comparable to that of free epsilonADPR or epsilonADP. it is concluded that the bound epsilon-adenine ring is partially exposed to the solvent. The opening, to the solvent, of the adenine binding site is not large enough to allow free methionine diffusion since the rate constant for quenching of bound coenzyme by this quenching agent is relatively small. The difference between the rate constants for quenching of free and enzyme bound nucleotide was used to evaluate the binding constants of epsilonADPR to GPDH, epsilonNAD+ to LDH, and oxalate to the LDH:epsilonNAD+ complex. This technique may prove to be particularly useful when the binding of a fluorescent ligand to a protein is not accompanied by significant changes in its fluorescence.     

A tale of three acaropathogenic fungi in Israel: Hirsutella, Meira and Acaromyces

We review published and unpublished studies conducted in Israel with six acaropathogenic fungi, assayed in order to control the citrus rust mite, Phyllocoptruta oleivora (Ashmead) (CRM). Hirsutella thompsonii Fisher was introduced twice, killed 80-90% of the exposed mites, but due to its requirements for near-saturation humidities was deemed unsuitable for local outdoors conditions. Hirsutella kirchneri (Rostrup) Minter et al. and Hirsutella necatrix Minter et al. were also introduced and assayed against CRM and spider mites, but their efficacy was unsatisfactory. Three indigenous fungi found to be associated with mites, Meira geulakonigii, Meira argovae and Acaromyces ingoldii--all three recently described by Boekhout, Gerson, Scorzetti & Sztejnberg--were assayed against several mites. Meira geulakonigii killed 80-90% of several spider mites and of the CRM, and caused some mortality of Iphiseius degenerans (Berlese), one out of three phytoseiid predators assayed. Mortality was not due to parasitization; extracts from the media in which the fungi had developed caused considerable mite death, suggesting that it was a result of fungal toxins. Data from a field study indicated that spraying blastoconidia of M. geulakonigii on grapefruits infested by CRM significantly reduced pest-incurred damage from 23 to 13%. Applying qRT-PCR methodology indicated that M. geulakonigii was endophytic within sealed grapefruit flowers and in the flavedo of the fruits' peel. Neither in the laboratory nor in the field was any evidence ever obtained that this fungus damaged the plants, leading us to hypothesize that M. geulakonigii serves as a "body guard" of grapefruits (and perhaps other plants as well). All three fungi suffered very little mortality after being exposed to various insecticides and acaricides that are in current local use (with the exception of sulfur). The ability of M. geulakonigii to reduce mite numbers without affecting the host plant, the minimal fungal effect on some predatory mites, its endophytic nature along with the apparent tolerance of M. geulakonigii to many insecticides and acaricides, suggest that this fungus could be suitable for integrated pest management (IPM) program.     

Localizing protein-protein interactions by bimolecular fluorescence complementation in planta

The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being developed. BiFC is a powerful tool which enables not only detection, but also visualization and subcellular localization of protein–protein interactions in living cells. Here we describe the application of BiFC in plant cells while focusing on the use of our versatile set of vectors which were specifically designed to facilitate the transformation, expression and imaging of protein–protein interactions in various plant species. We discuss the considerations of using our system in various plant model systems, the use of single versus multiple expression cassettes, the application of our vectors using various transformation methods and the use of internal fluorescent markers which can assist in signal localization and easy data acquisition in living cells.     

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model

Introduction Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. Material and methods Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFκB (IκBα) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. Results Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFκB (IκBα) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. Conclusion Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFκB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation. Edith Hochhauser and Orit Pappo are first two coauthors.     

Inhibition of vesicular uptake of monoamines by hyperforin

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.     

The stimulatory effect of cannabinoids on calcium uptake is mediated by Gs GTP-binding proteins and cAMP formation

Cannabinoids are neurodepressive drugs that convey their cellular action through G(i/o) GTP-binding proteins which reduce cAMP formation and Ca(2+) influx. However, a growing body of evidence indicates that the stimulatory effects of cannabinoids include the elevation in cAMP and cytosolic Ca(2+) concentration. The present study expands our previous findings and demonstrates that, in N18TG2 neuroblastoma cells, the cannabinoid agonist desacetyllevonantradol (DALN) stimulates both cAMP formation and Ca(2+) uptake. The stimulatory effect of DALN on cAMP formation was not eliminated by blocking Ca(2+) entry to the cells, while its stimulatory effect on Ca(2+) uptake was abolished by blocking cAMP-dependent protein kinase. Furthermore, elevating cAMP by forskolin stimulated calcium uptake, while elevating the intracellular Ca(2+) concentration by ionomycin or KCl failed to stimulate cAMP formation. These findings suggest that cAMP production precedes the influx of Ca(2+) in the cannabinoid stimulatory cascade. The stimulatory effect of DALN on calcium uptake resisted pertussis toxin treatment, and was completely blocked by introducing anti-G(s) antibodies into the cells, indicating that the stimulatory activity of cannabinoids is mediated by G(s) GTP-binding proteins. The relevance of the cellular stimulatory activity of DALN to the pharmacological profile of cannabinoid drugs is discussed.