Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria.

Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Oligomerization of the heptahelical G protein coupling receptors: a case for association using transmembrane helices.

The heptahelical G protein coupling receptors oligomerize extensively via transmembrane domains, in association with heterotrimeric G proteins. This provides higher affinity for agonists, conformational stability necessary for signal transduction, and protection from intracellular proteinases. The oligomerization is relevant to organismic pathophysiology and could be targeted by natural or modified agonists.

Dimers of the neuropeptide Y (NPY) Y2 receptor show asymmetry in agonist affinity and association with G proteins.

In conditions precluding activation of G proteins, the binding of agonists to dimers of the neuropeptide Y (NPY) Y2 receptor shows two components of similar size, but differing in affinity. The dimers of all NPY receptors are solubilized as approximately 180-kDa complexes containing one G protein alpha beta gamma trimer. These heteropentamers are stable to excess agonists, chelators, and alkylators. However, dispersion in the weak surfactant cholate releases approximately 300-kDa complexes. These findings indicate that both protomers in the Y2 dimer are associated with G protein heterotrimers, but the extent of interaction depends on affinity for the agonist peptide. The G protein in contact with the first-liganded, higher-affinity protomer should have a stronger interaction with the receptor and a larger probability of activation.

An ion-responsive motif in the second transmembrane segment of rhodopsin-like receptors.

A L(M)xxxD(N, E) motif (x=a non-ionic amino acid residue, most frequently A, S, L or F; small capitals indicating a minor representation) is found in the second transmembrane (tm2) segment of most G-protein coupling metazoan receptors of the rhodopsin family (Rh-GPCRs). Changes in signal transduction, agonist binding and receptor cycling are known for numerous receptors bearing evolved or experimentally introduced mutations in this tm2 motif, especially of its aspartate residue. The [Na(+)] sensitivity of the receptor-agonist interaction relates to this aspartate in a number of Rh-GPCRs. Native non-conservative mutations in the tm2 motif only rarely coincide with significant changes in two other ubiquitous features of the rhodopsin family, the seventh transmembrane N(D)PxxY(F) motif and the D(E)RY(W,F) or analogous sequence at the border of the third transmembrane helix and the second intracellular loop. Native tm2 mutations with Rh-GPCRs frequently result in constitutive signaling, and with visual opsins also in shifts to short-wavelength sensitivity. Substitution of a strongly basic residue for the tm2 aspartate in Taste-2 receptors could be connected to a lack of sodium sensing by these receptors. These properties could be consistent with ionic interactions, and even of ion transfer, that involve the tm2 motif. A decrease in cation sensing by this motif is usually connected to an enhanced constitutive interaction of the mutated receptors with cognate G- proteins, and also relates to both the constitutive and the overall activity of the short-wavelength opsins.

Neuropeptide Y (NPY) Y2 receptors of rabbit kidney cortex are largely dimeric.

The neuropeptide Y (NPY) Y2 receptors and the pancreatic polypeptide Y4 receptors from rabbit kidney cortex are isolated largely as approximately 180 kDa complexes constituted of one receptor dimer and one G-protein heterotrimer, similar to NPY receptors expressed in the Chinese hamster ovary (CHO) cells. As expected, kidney and CHO cell Y2 dimers are converted into monomers by increasing concentrations of a selective agonist. Prevalence of dimeric Y2 receptors in the kidney could be related to low plasma levels of Y2 agonists, and possibly also to a relatively low concentration of Gi alpha subunits.

Self-regulation of agonist activity at the Y receptors.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides, and is likely to be present at nanomolar levels over extended periods in the synaptic space of many forebrain areas. This might be linked to an evolved generalized toning activity through a number of other peptide receptors that use C-terminally amidated agonists (with LHRH and orexin receptors and GIR as examples). However, the Y1 and Y2 receptors (which constitute the bulk of Y receptors active in the neural matrix) possess subnanomolar affinities that, at saturating NPY levels, could produce excessive signaling, as well as receptor losses via repeated endocytosis. The related Y4 receptor shows an even higher agonist affinity, and faces the same problem in visceral and neural locations accessible to pancreatic polypeptide (PP). An examination of agonist peptide interaction with Y receptors shows that Y1 and Y4 receptors in particular (as located on either the intact cells, or on particulates derived from various cell types) develop a blockade dependent on ligand concentration, with the blocking ranks of [NPY]>>[peptide YY] (PYY) for the Y1, and [human PP]>>>[PYY-related Y4 agonist] for the Y4 receptor. This blockade is also echoed in a concentration-related reduction in biological activity of primary agonists (NPY and PP), resembling a partial agonism, and is influenced especially by the allosteric interactivity of agonists. With the Y2 receptor, the blocking by agonists is less pronounced, but the signaling by NPY-related peptides is apparently less than with PYY-related agonists. The extended occupancy and self-attenuation of primary agonist activity at Y receptors could represent an evolutionary solution contributing to a balancing of metabolic signaling, agonist clearance and receptor conservation.

Oligomerization of neuropeptide Y (NPY) Y2 receptors in CHO cells depends on functional pertussis toxin-sensitive G-proteins.

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as approximately 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi alpha subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi alpha subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.

Parallel inactivation of Y2 receptor and G-proteins in CHO cells by pertussis toxin.

The Y(2) receptor for neuropeptide Y (NPY) interacts with pertussis toxin (PTX)-sensitive G-proteins, but little is known about interdependence of their levels and functions. We found that PTX reduces Y(2) receptors expressed in CHO cells in parallel to inactivation of Gi G-proteins, to loss of inhibition by Y(2) agonists of forskolin-stimulated adenylyl cyclase, and to decrease in the binding of GTP-gamma-S. These losses were attenuated by the endosome alkalinizer ammonium chloride. Affinity of the Y(2) receptor was not changed by PTX treatment. Prolonged treatment induced a large decrease of Y(2) receptor immunoreactivity (more than 70% in 48 h). The Gi(3) alpha-subunit immunoreactivity decreased slowly (about 46% in 48 h). There was a significant increase in Gq alpha immunoreactivity and in fraction of Y(2) binding sensitive to a Gq-selective antagonist. Possibly linked to that, the surface Y(2) sites and the internalization of the Y(2) receptor were less than 40% reduced. However, the abundant masked Y(2) sites were eliminated by the toxin, and could be mainly coupled to PTX-sensitive G-proteins.

Internalization of cloned pancreatic polypeptide receptors is accelerated by all types of Y4 agonists.

Internalization of cloned rat or human Y4 receptors expressed in Chinese hamster ovary (CHO) cells increased with concentration of all types of Y4 agonists, including human and rat pancreatic polypeptides, the Y1 receptor group co-agonists possessing C-terminal TRPRY.NH2 pentapeptide, and a C-terminally amidated dimeric nonapeptide related to neuropeptide Y, GR231118. These peptides also inhibited forskolin-stimulated adenylyl cyclase activity in Y4 receptor-expressing cells, and stimulated the binding of 35S-labeled GTP-gamma-S to pertussis toxin-sensitive G-proteins in particulates from these cells. Peptide VD-11 (differing from GR231118 only by C-terminal oxymethylation) acted as a competitive antagonist in all of the above processes. Agonist-induced stimulation of the Y4 receptor internalization persisted in the presence of allosteric inhibitors of hPP binding, N5-substituted amilorides, which also were relatively little active in G-protein stimulation and cyclase inhibition by Y4 agonists. Acceleration of Y4 receptor internalization by agonists apparently is related to relaxation of allosteric constraints to ligand attachment and sequestration of the receptor-ligand complex.

Hypercalcemia in coccidioidomycosis.

Two patients with disseminated coccidioidomycosis and hypercalcemia are presented. One patient studied showed normal levels of 25-hydroxyvitamin D with depressed levels of 1 alpha,25-dihydroxyvitamin D. The serum calcitonin level was appropriate for the level of serum calcium, and the serum parathyroid hormone level was suppressed with elevation of the nephrogenous cAMP level. Intestinal absorption of calcium was elevated at 63 percent. Hypercalcemia and hypercalciuria persisted despite a 300 mg calcium diet. An osteotropic substance similar to the humoral hypercalcemia of malignancy is postulated.

Upregulation of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors in estrogen-induced hypertrophy of the anterior pituitary gland in the Fischer-344 rat.

Implants of diethylstilbestrol inducing anterior pituitary prolactinomas in female Fischer-344 rats produced a considerable elevation of high-affinity binding of either rat or human pancreatic polypeptide (hPP). No comparable upregulation of high-affinity binding sites was noted for the ligand [125I](Leu31,Pro34)hPYY (LP-PYY) (masked by 2 nM hPP to force selectivity for the Y1 sites), or of the Y2-selective ligand [125I]hPYY(3-36). The Y5-like sites displayed the rank order of potency of hPP = rPP, hNPY, LP-PYY > pPYY(1-36) > hNPY(2-36) > hPYY(3-36) >> aPP, similar to the previously described rabbit kidney or hypothalamic Y5-like receptors. The PP binding in the anterior pituitary was not sensitive to the Y1-selective non-peptidic antagonist BIBP-3226. The PP binding was highly sensitive to alkali metal cations, and to a N5-substituted amiloride antagonist of sodium transport, but not to a C2-guanido substituted amiloride antagonist of sodium channels. The binding was also sensitive to phospholipase C antagonist U-73122, and to alkylating alpha-adrenergic agonist chloroethylclonidine. Lactotrophs isolated in Percoll gradients after enzymic dispersion of the anterior pituitary gland retained the high-affinity PP binding. Following removal of estrogen implants, the hPP binding sites decreased to very low levels within 3 days, in parallel to the decrease of plasma prolactin.

AIDS-related bronchogenic carcinoma: fact or fiction?

OBJECTIVE: Case reviews and retrospective analyses have raised the possibility of an increased frequency of primary lung carcinoma in HIV- and AIDS-infected patients. Conclusions have often been controversial and conflicting. We conducted a population-based epidemiologic study to assess the incidence of lung neoplasms in an HIV/AIDS cohort. MATERIALS AND METHODS: The Texas Department of Health generated descriptive data on lung neoplasms in HIV-AIDS patients whose conditions were diagnosed between 1990 and 1995. The cancer registry matched against all patients whose conditions were diagnosed during the same time interval. Relative risk was measured through standardized incidence ratios of lung neoplasms in the HIV-AIDS population as compared with that of the US population. RESULTS: The HIV-AIDS data file included 26,181 cases. A total of 76 lung cancer cases were identified, of which 36 (47.4%) were primary lung cancers. All major histologies were represented. The observed (36)/expected (5.6) ratio (standard incidence ratio) for primary lung cancer compared to the US population was 6.5 (4.5 to 8.9, 95% confidence interval). CONCLUSIONS: Our data indicate a 6.5-fold increased incidence of primary lung cancer in HIV- and AIDS-infected patients. We present the results of our study, a review of the work of other investigators, and address a potentially even greater public health problem in the HIV/AIDS population than previously realized.

Blockade of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors by agonist peptides is prevented by modulators of sodium transport. Implications for receptor signaling and regulation.

Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y4 or Y5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y4 receptors are also selectively blocked by human or rat pancreatic polypeptide (PP) and the blocking peptides are not dissociated by high concentrations of alkali chlorides (which restore most of the binding of subtype-selective agonists to Y1 and Y2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y1-selective agonists (IC50 300-400 pM). The cloned Y(4) receptors from three species are much less sensitive to NPY or PYY. The sensitivity of both the PP/NPY sites and the Y(4) sites to Y2-selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y1 antagonist ((Cys31,NVal34NPY27-36))2, which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na+ transport, and by RFamide NRNFLRF.NH2, but not by Ca2+ channel blockers, or by inhibitors of K+ transport. Protection of both PP/NPY and Y4 sites against blockade by human or rat pancreatic polypeptide is also afforded by short N-terminally truncated NPY-related peptides. The above results are consistent with a stringent and selective activity regulation for rabbit PP/NPY receptor(s) that may serve to differentiate agonists and constrain signaling, and could involve transporter-like interactants.

Ligand association with the rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y (NPY) receptors shows large subtype-related differences in sensitivity to chaotropic and alkylating agents.

The binding to rabbit kidney or hypothalamic particulates of the subtype-selective neuropeptide Y (NPY) receptor ligands [125I](Leu31,Pro34)hPYY (as Y1 site label at 2 nM human pancreatic polypeptide (hPP)), [125I]-hPYY(3-36) (Y2 label), and [125I]-hPP (Y5 label) displayed great differences in sensitivity to alkylators and chaotropic agents. Sensitivity to a nonionic chaotrope, urea, was much higher for the Y1 binding than for the Y5-like binding or the Y2 binding. The non-selective alkylator N-ethylmaleimide (NEM) and several alkylators selective for aminergic receptors were much more efficacious against the Y1 relative to the Y2 binding. Similar differences could be confirmed with the attachment of Y1 and Y2-selective tracers to CHO cells expressing the cloned guinea-pig Y1 or Y2 receptors. The Y5-like binding was quite insensitive to NEM, but sensitive to chloroethylclonidine (CEC) and prazobind, which were less potent at the Y1, and especially at the Y2 site. The unrestricted-access alkylator 2-aminoethyl methanethiosulfonate inhibited the binding to all subtypes, while the restricted-access agent 2-(trimethylammonium)ethylmethanethiosulfonate poorly inhibited the Y5-like binding, or the guanine nucleotide-insensitive Y2 binding. These results are compatible with an active conformation of the Y5-like site dependent on maintenance of a shared hydrophobic cavity. The Y2 sites resistant to guanosine polyphosphates and restricted-access alkylators were detected mainly in particulates slowly solubilized by cholate at 0-5 degrees C; these sites could be clustered.

Characterization of Y1, Y2 and Y5 subtypes of the neuropeptide Y (NPY) receptor in rabbit kidney. Sensitivity of ligand binding to guanine nucleotides and phospholipase C inhibitors.

The binding of two peptide YY/neuropeptide Y analogues selective for major subtypes of neuropeptide Y (NPY) receptors was compared in particulates from rabbit kidney cortex employing modulators of activity of G-proteins, phospholipase enzymes, and ion channels. The binding of (Leu31,Pro34)human peptide YY resembled the patterns observed previously for the brain tissue Y1 receptor, exhibiting a high sensitivity to monovalent cations, disulfide disruptors, guanosine polyphosphates and phospholipase C inhibitors. However, this binding was bimodal in response to human pancreatic polypeptide and to peptides selective for the Y2 subtype of the NPY receptor, displaying a large component pharmacologically similar to the brain Y5 receptor. This kidney Y5-like binding largely shared the sensitivity to monovalent cations, guanine nucleotides and phospholipase C inhibitors found for either the kidney or the brain Y1 receptor, and also was activated by Ca2+ ion. Both Y1- and Y5-like binding in the kidney displayed a uniformly low reactivity to a nonpeptidic Y1 antagonist, BIBP-3226, and to a receptor peptide mimetic, mastoparan analogue MAS-7. The kidney Y2 binding shared the low sensitivity to ionic environment observed for the brain Y2 subtype, and was only partially sensitive to guanine nucleotides or to MAS-7. The Y2 liganding had a sensitivity to phospholipase C inhibitors similar to the Y1/Y5 binding. This reactivity was retained in the fraction of the Y2 receptor persisting detergent solubilization in a high-affinity form, which, however, was activated rather than inhibited by G-protein agonists.

Characterization of rabbit kidney and brain pancreatic polypeptide-binding neuropeptide Y receptors: differences with Y1 and Y2 sites in sensitivity to amiloride derivatives affecting sodium transport.

Sites sensitive to human and rat pancreatic polypeptides (hPP and rPP) accounted for more than 30% of the specific binding of [125I](Leu31,Pro34) human peptide YY (LP-PYY) in particulates from rabbit kidney cortex, and about 10% of the specific binding in membranes from rabbit hypothalamus. The binding of [125I]hPP or [125I]rPP showed a high-affinity displacement with either hPP, rPP, LP-PYY, neuropeptide Y or peptide YY (Ki below 50 pM for all), while being quite insensitive to Y2-selective ligands. The PP binding had a high sensitivity to alkali cations and inhibitors of phospholipase C, very similar to that of LP-PYY binding 'masked' by excess cold hPP. However, as different from the Y1-like LP-PYY binding, but similar to the binding of the Y2-selective ligand [125I]human peptide YY(3-36) (hPYY(3-36)), the PP binding showed a low sensitivity to guanosine polyphosphates. The PP binding was much more sensitive to N5-substituted amiloride inhibitors of Na+ transport than the binding of LP-PYY, or that of hPYY(3-36). The inhibition of PP binding by N5-substituted amilorides was not enhanced by guanine nucleotides or by phospholipase C blockers. However, pairing of N5-substituted amilorides disproportionately increased the inhibition of hPP binding. Thus, in rabbit kidney or hypothalamus, the high-affinity PP-responding sites share some of the basic properties of the Y1 and Y2 sites, but are distinguished from both by a high sensitivity to compounds affecting sodium transport. These PP/NPY receptors could associate with membrane structures involved in the control of ion balance and osmotic responses.

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY, endosome-linked entry and fast receptor recycling.

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.

The effect of influenza C virus on the Purkinje cells of chick embryo cerebellum.

Intra-amniotic inoculation of influenza C virus resulted in observable and quantitatively measurable changes in the Purkinje cells of chick embryo cerebellum. Purkinje cells were visualized by the Golgi-Cox procedure and prepared for statistical and computer evaluation from camera lucida drawings. Four computer-generated measurements (the area of the dendritic arbor, the perimeter of the dendritic tree, and the height and width of the cell's arborization) and two manually counted measurements (total number of branches and the number of first order branches) were made. Analysis of Purkinje cells from influenza C virus-infected embryos showed disturbances in dendritic arborization patterns and misalignment in the arrangement of the cells in the Purkinje cell layer compared to control cells. Statistical evaluation of Purkinje cell arborization showed significant decreases in all measured parameters for the influenza C virus-infected members when compared with the members of the uninfected control group.

Differences in cation sensitivity of ligand binding to Y1 and Y2 subtype of neuropeptide Y receptor of rat brain.

The binding of selective ligands to the Y1 subtype of neuropeptide Y receptor in rat brain particulates was promoted by Ca2+ and also stimulated by Sr2+, but reversibly reduced by Ba2+, Mg2+, Mn2+, by the organic polycations neomycin and spermidine, and by chelating agents. The alkali monovalent cations inhibited the Ca(2+)-enabled Y1 subtype binding with some selectivity (Cs+ > or = NH4+ > Li+ > Na+, K+), with half-inhibition between 70-120 mM. The specific Y2 subtype binding was enhanced by all alkaline-earth divalent cations, Mn2+, neomycin and spermidine in the range of 0.1-10 mM, and by alkali cations at up to 100 mM, and also by Na+ salts of the chelators EGTA and EDTA. The large disparity in cation sensitivity indicates substantial differences in the structure of the binding sites of the Y1 and Y2 receptors, predictable from known distinct features of ligand epitopes and of primary structure of the receptors.

Novel variant of the P2X2 ATP receptor from the guinea pig organ of Corti.

ATP functions as a neurotransmitter and a neuromodulator in various tissues by acting on metabotropic (P2Y) and ionotropic (P2X) receptors. Evidence suggests that ATP activates P2X receptors on several cell types in the organ of Corti of guinea pig including outer hair cells (OHCs), Deiters' cells, Hensen's cells, pillar cells and inner hair cells (IHCs). Determining the sequence and structure of P2X receptors in guinea pig organ of Corti is important for understanding the function of ATP in the cochlea. We screened a guinea pig organ of Corti cDNA library for P2X2 ATP receptors using rat P2X2 cDNA as a probe. We sequenced three P2X2 variants which were found to be abundant in this library. One is a novel P2X2 isoform (P2X2-3) created by a retained intron coding for an additional 27 amino acids (81 bp) in the putative extracellular domain. We have also sequenced a variant (P2X2-2) that lacks both the 81-bp sequence and a 192-bp sequence in the 3' intracellular domain. A third variant (P2X2-1) contains the intracellular 192-bp sequence but not the extracellular 81-bp sequence found in P2X2-3. The multiple transcripts arise from alternative intron and exon splicing events. In situ hybridization with a probe common to the three variants localized P2X2 to many of the cells of the organ of Corti.