What determines the substrate specificity of the multi-drug-resistance pump?

Multi-drug-resistance protein (P-glycoprotein) turns out to be an ATP-hydrolysing transmembrane pump that increases the resistance of cells in which it is expressed by actively extruding toxic chemicals. The baffling question is how does the pump know which chemicals to extrude? Common features among its substrates are still elusive. The question raised here concerns the relationship between this pump and that known for many years as capable of extruding glutathionyl and cysteinyl S-conjugates of xenobiotics. Are excreted drugs conjugated before excretion? Does the multi-drug-resistance pump recognize a simple chemical tag put on xenobiotics by a family of transferase enzymes?     

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Are olfactory cues involved in nest recognition in two social species of estrildid finches?

Reliably recognizing their own nest provides parents with a necessary skill to invest time and resources efficiently in raising their offspring and thereby maximising their own reproductive success. Studies investigating nest recognition in adult birds have focused mainly on visual cues of the nest or the nest site and acoustic cues of the nestlings. To determine whether adult songbirds also use olfaction for nest recognition, we investigated the use of olfactory nest cues for two estrildid finch species, zebra finches (Taeniopygia guttata) and Bengalese finches (Lonchura striata var. domestica) during the nestling and fledgling phase of their offspring. We found similar behavioural responses to nest odours in both songbird species. Females preferred the odour of their own nest over a control and avoided the foreign conspecific nest scent over a control during the nestling phase of their offspring, but when given the own odour and the foreign conspecific odour simultaneously we did not find a preference for the own nest odour. Males of both species did not show any preferences at all. The behavioural reaction to any nest odour decreased after fledging of the offspring. Our results show that only females show a behavioural response to olfactory nest cues, indicating that the use of olfactory cues for nest recognition seems to be sex-specific and dependent on the developmental stage of the offspring. Although estrildid finches are known to use visual and acoustic cues for nest recognition, the similar behavioural pattern of both species indicates that at least females gain additional information by olfactory nest cues during the nestling phase of their offspring. Thus olfactory cues might be important in general, even in situations in which visual and acoustic cues are known to be sufficient.     

Are rates of species diversification correlated with rates of morphological evolution?

Some major evolutionary theories predict a relationship between rates of proliferation of new species (species diversification) and rates of morphological divergence between them. However, this relationship has not been rigorously tested using phylogeny-based approaches. Here, we test this relationship with morphological and phylogenetic data from 190 species of plethodontid salamanders. Surprisingly, we find that rates of species diversification and morphological evolution are not significantly correlated, such that rapid diversification can occur with little morphological change, and vice versa. We also find that most clades have undergone remarkably similar patterns of morphological evolution (despite extensive sympatry) and that those relatively novel phenotypes are not associated with rapid diversification. Finally, we find a strong relationship between rates of size and shape evolution, which has not been previously tested.     

Are polyamines involved in the induction and regulation of the Crassulacean acid metabolism?

Leaves of plants with Crassulacean acid metabolism (CAM) were analyzed for variation in the content of polyamines in connection with the metabolism of malic acid in the dark and in the light, and with the induction of full-CAM activity. Under conditions (long days) resulting in extremely low CAM activity, young leaves of K. blossfeldiana have very low content in the polyamine-precursor arginine and in putrescine. The content in these two substances was increased dramatically by full-CAM induction with short days. During the course of the night/day cycle two peaks of putrescine content were observed in leaves of Kalanchoe blossfeldiana Poelln. Tom Thumb performing full-CAM operation: a large increase occurs toward the end of the day and the first half of the night, and its kinetics corresponds to the increase in the rate of malic acid synthesis; another peak, very sharp, appears during the first hours of the day, concomitant with the time of release of malic acid from the vacuole into the cytoplasm. In the case of Bryophyllum daigremontianum Berger similar variations were observed for the content in spermidine. These results support the hypothesis that polyamines could be involved in countering the tendency toward acidification of the cytoplasm at those moments of CAM operation at which the local concentration of malic acid is increased (i.e., during active synthesis in the dark and during the efflux from the vacuole in the light).Abbreviation CAM Crassulacean acid metabolism     

Are accessory nuclei involved in the establishment of developmental gradients in hymenopteran oocytes?

Summary In the oocytes ofTenthredo olivacea, accessory nuclei (AN) are formed by budding from the nuclear envelope of the oocyte nucleus. Newly formed AN contain electron-dense material of nuclear origin and are surrounded by a double envelope devoid of pores. Such structures are subsequently transported to the peripheral ooplasm (periplasm), where they grow to reach a final diameter of 5 µm. In the envelopes of advanced AN nuclear pores arise. Through these pores nuage material is extruded into the surrounding periplasm. These findings are discussed with respect to a possible involvement of AN in the establishment of developmental gradients in hymenopteran oocytes.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.     

Are prostaglandins involved in the regulation of coronary blood flow? A review of the evidence

Prostaglandins (PG's) fulfil most of the criteria required for the metabolic "coupler" linking increases in myocardial activity with increases in coronary blood flow. They are synthesized by the heart and coronary vessels, profoundly modify coronary blood flow in low concentrations and are released under conditions of hypoxia and myocardial ischaemia. Studies with inhibitors of PG-synthetase however provide no firm evidence that PG's are involved in the physiological regulation of myocardial blood flow. These studies are reviewed.     

Are separable aromatase systems involved in hormonal regulation of the male brain?

In vitro study of testosterone (T) metabolism shows that formation of estradiol-17β (E₂) is regionally specific within the preoptic area (POA) of the male ring dove. The POA is known to be involved in the formation of E₂ required for specific components of male sexual behavior. Two sub-areas of high aromatase activity, anterior (aPOA) and posterior preoptic (pPOA) areas, have been identified. Aromatase activity is higher in aPOA than in pPOA. The aromatase activity within the aPOA is also more sensitive to the inductive effects of low circulating T, derived from subcutaneous silastic implants, than the enzyme activity in pPOA. Kinetic analysis of preoptic fractions indicates that a similar high-affinity enzyme occurs in both areas (apparent K_m < 14nM), but the V_max of aPOA enzyme activity is higher than pPOA. Cells containing estrogen receptors (ER) are localized in areas of high aromatase activity. There is overlap between immunostained cells in the aPOA and in samples containing inducible aromatase activity measured in vitro. Within the aPOA there is a higher density of ER cells in the nucleus preopticus medialis. The pPOA area also contains ER, notably in the nucleus interstitialis, but at a lower density. We conclude that the hormonal regulation of the male preoptic-anterior hypothalamic region, which is a target for the behavioral action of T, involves at least two inducible aromatase systems with associated estrogen receptor cells.     

Are separable aromatase systems involved in hormonal regulation of the male brain?

In vitro study of testosterone (T) metabolism shows that formation of estradiol-17 beta (E2) is regionally specific within the preoptic area (POA) of the male ring dove. The POA is known to be involved in the formation of E2 required for specific components of male sexual behavior. Two sub-areas of high aromatase activity, anterior (aPOA) and posterior preoptic (pPOA) areas, have been identified. Aromatase activity is higher in aPOA than in pPOA. The aromatase activity within the aPOA is also more sensitive to the inductive effects of low circulating T, derived from subcutaneous silastic implants, than the enzyme activity in pPOA. Kinetic analysis of preoptic fractions indicates that a similar high-affinity enzyme occurs in both areas (apparent Km less than 14 nM), but the Vmax of aPOA enzyme activity is higher than pPOA. Cells containing estrogen receptors (ER) are localized in areas of high aromatase activity. There is overlap between immunostained cells in the aPOA and in samples containing inducible aromatase activity measured in vitro. Within the aPOA there is a higher density of ER cells in the nucleus preopticus medialis. The pPOA area also contains ER, notably in the nucleus interstitialis, but at a lower density. We conclude that the hormonal regulation of the male preoptic-anterior hypothalamic region, which is a target for the behavioral action of T, involves at least two inducible aromatase systems with associated estrogen receptor cells.     

Are RB proteins a potential substrate of Pin1 in the regulation of the cell cycle?

RB family members are post-transductionally regulated proteins and phosphorylation at Ser/Thr residues leads to their gradual inactivation. Cyclin/cdk complexes are mainly responsible for the regulation of these pocket proteins, which is crucial for release of E2F factor. Despite the fact that E2F release is a phosphorylation-dependent process, it is still not evident how phosphorylation physically determines the shift from the active to the inactive feature of RB molecules. We would like to put forward the hypothesis that Pin1 is involved in RB proteins phosphorylation and E2F release, suggesting an additional post-translational level of control on this family of molecules.     

Are invasive species the drivers of ecological change?

Invasive species are widely accepted as one of the leading direct causes of biodiversity loss. However, much of the evidence for this contention is based on simple correlations between exotic dominance and native species decline in degraded systems. Although appealing, direct causality is not the only possible interpretation. A plausible alternative hypothesis is that exotic dominance could be the indirect consequence of habitat modification driving native species loss. In a new paper, MacDougall and Turkington now provide the first direct test of whether invasive species are the drivers of community change, or merely 'passengers' along for the environmental ride.     

Are caveolae involved in clathrin-independent endocytosis?

In addition to endocytosing molecules via clathrin-coated pits, cells also internalize membrane and fluid by a clathrin-independent endocytic mechanism. In this article we search for the equivalent of clathrin-coated pits in clathrin-independent endocytosis, and discuss some pitfalls in the interpretation of electron micrographs. We also discuss how the early steps in clathrin-independent endocytosis might be analysed morphologically, and we argue that caveolae are not involved in clathrin-independent endocytosis.     

Are polyphosphoinositides involved in platelet activation?

In human platelets, the amounts of triphosphoinositides (TPI) and diphosphoinositides (DPI) increase after 30 sec and level off after 120 sec of thrombin stimulation. After 180 sec of thrombin challenge, TPI and DPI increase accounts for 66 and 80%, respectively. Polyphosphoinositide changes roughly parallel the release of N-acetyl-beta-D-glucosaminidase and appear as a later event compared to aggregation and serotonin secretion. It is concluded that an increased phosphorylation of polyphosphoinositides might participate in platelets to the process of stimulus-activation coupling and might be linked to thrombin receptor occupancy. A role of DPI in platelet activation is suggested by the observation that DPI promote platelet aggregation, the mechanism of which is discussed.     

Are isocitrate dehydrogenases and 2-oxoglutarate involved in the regulation of glutamate synthesis?

In plants, nitrogen assimilation into amino acids relies on the availability of the reduced form of nitrogen, ammonium. The glutamine synthetase–glutamate synthase pathway, which requires carbon skeletons in the form of 2-oxoglutarate, achieves this. To date, the exact enzymatic origin of 2-oxoglutarate for plant ammonium assimilation is unknown. Isocitrate dehydrogenases synthesize 2-oxoglutarate. Recent efforts have concentrated on evaluating the involvement of different isocitrate dehydrogenases, distinguished by co-factor specificity and sub-cellular localization. Furthermore, several observations indicate that 2-oxoglutarate is likely to be a metabolic signal that regulates the coordination of carbon:nitrogen metabolism. This is discussed in the context of recent advances in bacterial signalling processes.     

Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.     

Probing the donor and acceptor substrate specificity of the γ-glutamyl transpeptidase

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.     

Are species of bacteria unclassifiable?

Summary Every one of eleven different strains randomly selected from 10 different randomly selected genera have shown the same high frequency of occurrence of colony mutants as did almost all strains ofAcetobacter (previously considered outstanding in this respect). Correlation of other properties with such mutant colony forms was not specifically studied, but in 4 strains correlation was noticed, suggesting its presence in the others, as was so often found inAcetobacter. It is suggested from this, that a similar study of strains of other genera might reveal a similarly high frequency of occurrence of mutants, most so-called pure cultures being thus probably mixtures of different cells with different properties. Also the proportion of each cell-type in the culture may vary from predominance to extinction according to the biochemical and other tests applied for the purpose of the ‘characterization’ of the species for taxonomic purposes. If the classification of such varying mixtures is considered of doubtful use, then it seems to follow that ‘species’ of bacteria are virtually unclassifiable, and that even the conception of a genus should be on a broader basis than is often the case at present.