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Aims: Definition of sense and antisense microRNA matches in 3’utr. Background: Matches of mature microRNAs (m-miRs) in human 3’utr could be traced to mutations producing fragments of original m-miR sequences without physical separation. (The m-miR matches in 5’utr and cds should be by far fewer, but could follow similar patterns). Objective: To ascertain if the sense and antisense m-miR fragments in 3’utr occur at similar or different levels. Methods: Frequency of sense and antisense m-miR matches in 3'utr was examined in the range of 7-22 nucleotides. Results: The fragmentation occurs at gene level by mutation within one of the paired m-miRs, which upon transcription results in increased interactive capability for both former pre-micro (premir) RNA stem partners. The non-mutated stem partner can persist in 3’utr sequences, as is apparent from significant presence of miR-619-5p and miR-5096 and some conservation of 20 other simian- specific m-miR sequences. However, most of m-mir sequences in 3’utr are extensively fragmented, with low preservation of long matches. In flanks of individual m-miR embeds the mutated pre-mir positions are to a degree defined specifically. Conclusion: The m-mir matches of various sizes in 3’utr apparently reflect accumulation, on a phylogenetic time scale, of in-sequence point mutations. Across human 3’utr this fragmentation is significantly less for evolutionarily recent human m-miRs that originate in simians compared to human m-miRs first appearing in lower primates, and especially to human m-miRs introduced in nonprimates.

Research into the effectiveness of video as a tool to educate students about environmental issues and cause a change in their attitudes toward them in a classroom setting is limited. We sought to add to this sparse body of research. We created three videos that showcased a species in a different stage of protection under the Endangered Species Act. Each video focused on a different species and employed different strategies to deliver content to the students who viewed them. The videos were screened to students in six classes. Data was collected via preassessments and postassessments from 140 students and analyzed using Wilcoxen Matched-Pairs Signed Ranks test. Each video significantly increased student content understanding and one video, “The Champion Chub,” improved student attitudes toward the Endangered Species Act. The study provides additional support for the effectiveness of video content as an environmental education tool.

Background: Same-nucleotide repeats (iterons) are strongly expressed in many DNA regions and RNA classes. These repeats serve importantly in association of polynucleotides and proteins, but have not been characterized in miRNAs. Methods: Iterons and nucleotide strings were quantified in currently known human miRNAs, including some comparisons with miRNAs of other species. Results: Human 5p miRNAs have significantly more G iterons than other miRNA groups. The 3p miRNAs have an inverse excess of C iterons. The miRNAs lacking functional counter-stems (which we differentiate as 5n or 3n by position in pre-miRNAs) also have a large excess of G iterons. In 5p miRNAs G and C iterons have much higher density in the seed compared to the post-seed region. This difference is lower in 5n and 3n sequences, and much lower in 3p sequences. In all groups the contiguous GC strings constitute a larger part of sequences than the AU strings. A surplus of G or C iterons and of GC strings should enable a more stable association with the target mRNAs. Conclusion: From the available evidence, the G iteron- and GC-rich miRNAs should also interact more readily with miRNA-processing and similar proteins.

Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25–28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (

Highlights • Homoiterons like GGGGGGG stabilize ribosomal RNAs of thermophile prokaryotes. • In eukaryotes, homoiterons are much more abundant in RNA of the larger subunit (LSU). • The LSU repeats increase with phylogenetic rank to 28% entire RNA sequence in hominids. • In mammal LSU RNAs, these repeats constitute 45% of the massive expansion segments. • These repeats may help in anchoring of ribosomes and export of secretory proteins., Ribosomal RNAs in both prokaryotes and eukaryotes feature numerous repeats of three or more nucleotides with the same nucleobase (homoiterons). In prokaryotes these repeats are much more frequent in thermophile compared to mesophile or psychrophile species, and have similar frequency in both large RNAs. These features point to use of prokaryotic homoiterons in stabilization of both ribosomal subunits. The two large RNAs of eukaryotic cytoplasmic ribosomes have expanded to a different degree across the evolutionary ladder. The big RNA of the larger subunit (60S LSU) evolved expansion segments of up to 2400 nucleotides, and the smaller subunit (40S SSU) RNA acquired expansion segments of not more than 700 nucleotides. In the examined eukaryotes abundance of rRNA homoiterons generally follows size and nucleotide bias of the expansion segments, and increases with GC content and especially with phylogenetic rank. Both the nucleotide bias and frequency of homoiterons are much larger in metazoan and angiosperm LSU compared to the respective SSU RNAs. This is especially pronounced in the tetrapod vertebrates and seems to culminate in the hominid mammals. The stability of secondary structure in polyribonucleotides would significantly connect to GC content, and should also relate to G and C homoiteron content. RNA modeling points to considerable presence of homoiteron-rich double-stranded segments especially in vertebrate LSU RNAs, and homoiterons with four or more nucleotides in the vertebrate and angiosperm LSU RNAs are largely confined to the expansion segments. These features could mainly relate to protein export function and attachment of LSU to endoplasmic reticulum and other subcellular networks.

BACKGROUND Heptahelical G protein coupled receptors (GPCRs) support numerous sensory and metabolic functions and differ considerably in levels of expression. GPCR protein levels should link to regulation of GPCR mRNAs by microRNAs (miRs), which might significantly depend on numbers, size and GC content of the canonical antisense matches in mRNAs. These parameters of GPCR mRNAs have not been studied in detail. METHODS Canonical matching profiles of human GPCR mRNAs and miRs were examined using segments of 7-15 nucleotides in windows shifted by one position over the entire microRNA sequence. RESULTS Human GPCRs mRNAs within larger function-related groups have a quite homogenous matching with miRs. Both the GC content and the melting temperature (and hence also the binding energy) are appreciably higher in 5'utr compared to 3'utr matches of the same length. Increase in the GC content correlates significantly with length in the ubiquitous matches of 7-12 nucleotides. However, several GPCR groups strongly differ in overall match numbers and density. The untranslated regions of sensory receptor mRNAs, especially the olfactory and Taste-2 mRNAs, have the lowest match numbers and density and the fewest miR partners. The glucagon and frizzled families show the highest canonical matching. CONCLUSION Partnership of GPCR mRNAs and miRs could significantly relate to the type of function of the receptor proteins, with mRNAs of the sensory receptors having the lowest and those of metabotropic GPCRs the highest targeting. This could be of interest regarding GPCR regulation by exogenous miRs.

Background: Canonical hydrogen-bonding multi-nucleotide matches of microRNAs (miRs) with mRNAs are considered as important in mRNA regulation. MiR "seed" positions 2-8 are frequently viewed as mRNA partners, but there is ample evidence for use of other (and even non-contiguous) miR parts. No detailed information is available about canonical matching, and the GC content of the matches is rarely considered, although it should have a major regulatory potential. Methods: Sequences of 2586 human miRs and of 5'utr, cds and 3'utr in 18810 human mRNAs were examined for number and GC content of contiguous Watson-Crick antisense matches of six or more nucleotides (nt) in successive windows shifted by 1 nt. Results: Frequency of the antisense matches is within all sectors similar for segments of up to 10 nt starting at positions 1-10 of miR sequences, with decrease of 3.5 to 4-fold for each 1-nt increment. Adenine and uracil rich elements (ARE-like) are very frequent in cds and 3'utr. All mRNAs have matches of up to 10 nt, and most also those of 11-15 nt. The match density is largest in 5'utr, and the match number in cds. The 5'utr and cds matches average much higher GC content than those of 3'utr. The GC content of matches is above that for the whole sector in 5'utr and cds, but lower in 3'utr. Conclusion: Human mRNA matches across miR sequences constitute a positionally similar matrix of canonical hydrogen-bonding reactivity. This presents ample opportunities for contiguous binding independent of miR position. The ubiquitous 10 to 15-nt matches could serve as binding foci. Interaction of miRs with the abundant GC-rich 5'utr and cds counterparts could be important in the regulation of mRNA-ribosome interaction as well as in mRNA disposal. The lower density and GC content of a majority of 3'utr matches could mainly support a dynamic regulation by miRs.

Based on ubiquitous presence of large ionic motifs and clusters in proteins involved in gene transcription and protein synthesis, we analyzed the distribution of ionizable sidechains in a broad selection of proteins with regulatory, metabolic, structural and adhesive functions, in agonist, antagonist, toxin and antimicrobial peptides, and in self-excising inteins and intron-derived proteins and sequence constructs. All tested groups, regardless of taxa or sequence size, show considerable segregation of ionizable sidechains into same type charge (homoionic) tracts. These segments in most cases exceed half of the sequence length and comprise more than two-thirds of all ionizable sidechains. This distribution of ionic residues apparently reflects a fundamental advantage of sorted electrostatic contacts in association of sequence elements within and between polypeptides, as well as in interaction with polynucleotides. While large ionic densities are encountered in highly interactive proteins, the average ionic density in most sets does not change appreciably with size of the homoionic segments, which supports the segregation as a modular feature favoring association.

Attempts to maintain refuge populations of Devils Hole pupfish Cyprinodon diabolis in artificial tanks have achieved limited success. Previous studies have documented changes in morphological, behavioral, and genetic characteristics of refuge populations, which suggest that environmental conditions (and thus selective pressures) differ from those found in Devils Hole (DH). Physical, chemical, and biological characteristics were compared among the three Devils Hole pupfish refuges (Hoover Dam, School Springs, and Point of Rocks) and between each refuge and DH. In contrast to the thermally constant environment in DH (∼33°C), mean refuge temperatures were cooler and fluctuated on a weekly and seasonal basis. On two occasions, extreme temperature fluctuations lasting several weeks (due to water supply malfunctions) were recorded at the Hoover Dam (6°C decrease) and School Springs (22°C decrease) refuges. The physical design of the refuges precludes surface runoff from entering them; thick layers of a...

Non-specific binding of Y receptor agonists to intact CHO cells, and to CHO cell or rat brain particulates, is much greater for human neuropeptide Y (hNPY) compared to porcine peptide Y (pPYY), and especially relative to human pancreatic polypeptide (hPP). This binding of hNPY is reduced by alkali cations in preference to non-ionic chaotrope urea, while the much lower non-specific binding of pPYY is more sensitive to urea. The difference could mainly be due to the 10-16 stretch in 36-residue Y agonists (residues 8-14 in N-terminally clipped 34-peptides), located in the sector that contains all acidic residues of physiological Y agonists. Anionic pairs containing aspartate in the 10-16 zone could be principally responsible for non-specific attachments, but may also aid the receptor site binding. Two such pairs are found in hNPY, one in pPYY, and none in hPP. The hydroxyl amino acid residue at position 13 in mammalian PYY and PP molecules could lower conformational plasticity and the non-selective binding via intrachain hydrogen bonding. The acidity of this tract could also be important in agonist selectivity of the Y receptor subtypes. The differences point to an evolutionary reduction of promiscuous protein binding from NPY to PP, and should also be important for Y agonist selectivity within NPY receptor group, and correlate with partial agonism and out-of group cross-reactivity with other receptors.

The minimal size of the fourth intracellular domain of heptahelical G-protein coupling receptors (GPCRs) is close to 15 residues, and a juxtamembrane 15-residue segment is predicted as helical (Helix-8) in most of the receptors. Sequences of opsins, non-visual opsin-like (family A) GPCRs and Taste-2 receptors correspond with bovine rhodopsin at four positions in this tract. This is especially evident in monoamine receptors. In most GPCRs, the conserved juxtamembrane segment also has a large fraction of basic sidechains, and a considerable excess of cationic over anionic residues. The conservation is not dependent on the preferred G-protein alpha subunit or the overall length of the domain, indicating an additive speciation. In rod opsins and some A-GPCRs this segment has been shown to associate with the bilayer and to interact with G-proteins. The segment could also be involved in precoupling of receptors and transducers. These interactions could be helped by both the structural propensities and the high content of cationic sidechains.

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. For Supplement material, please see the online version of the article.

With human neuropeptide Y Y2 receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp35Ala mutation, and especially the change of Pro34Asp35 to Ala34Ala35, decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y2 receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro34Ala mutation does not significantly affect the embedding and cycling of the receptor.

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.

Eighteen years of field observations and five summer field experiments in a coastal California river suggest that hydrologic regimes influence algal blooms and the impacts of fish on algae, cyanobacteria, invertebrates, and small vertebrates. In this Mediterranean climate, rainy winters precede the biologically active summer low-flow season. Cladophora glomerata, the filamentous green alga that dominates primary producer biomass during summer, reaches peak biomass during late spring or early summer. Cladophora blooms are larger if floods during the preceding winter attained or exceeded “bankfull discharge” (sufficient to mobilize much of the river bed, estimated at 120 m3/s). In 9 out of 12 summers preceded by large bed-scouring floods, the average peak height of attached Cladophora turfs equaled or exceeded 50 cm. In five out of six years when flows remained below bankfull, Cladophora biomass peaked at lower levels. Flood effects on algae were partially mediated through impacts on consumers in food webs....

Treatment with pertussis toxin in addition to a stable inhibition of G(i)alpha subunits of G-proteins also strongly reduced human neuropeptide Y Y(1) receptors expressed in Chinese hamster ovary (CHO) cells. This was reflected in abolition of the inhibition by Y(1) agonists of forskolin-stimulated adenylyl cyclase in intact cells, and of Y(1) agonist stimulation of GTPgammaS binding to particulates from disrupted cells. The loss of both receptor and G(i)alpha subunit function was attenuated by ammonium chloride, an inhibitor of acid proteinases, pointing to a chaperoning co-protection of active pertussis toxin-sensitive Galpha subunits and Y(1) receptors. The surface complement of the Y(1) receptor was changed a little in conditions of approximately 85% decrease of the Y(1) population, but the rate of the Y(1) receptor-linked internalization of agonist peptides was reduced about 70%. The preserved receptor fraction consisted of monomers significantly coupled to G(q)alpha subunits. The persistent pertussis toxin-insensitive internalization of agonists with the Y(1) receptor may reflect a rescue or alternative switching that could be important for cell functioning in neuropeptide Y-rich environments. The results are compatible with a loss, due to G(i)alpha subunit inactivation by the toxin, of a large Y(1) receptor reserve constituted of oligomers associating with heterotrimeric G-proteins.

Same-nucleotide repeats (iterons) are strongly expressed in many DNA regions and RNA classes. These repeats serve importantly in association of polynucleotides and proteins, but have not been characterized in miRNAs.Iterons and nucleotide strings were quantified in currently known human miRNAs, including some comparisons with miRNAs of other species.Human 5p miRNAs have significantly more G iterons than other miRNA groups. The 3p miRNAs have an inverse excess of C iterons. The miRNAs lacking functional counter-stems (which we differentiate as 5n or 3n by position in pre-miRNAs) also have a large excess of G iterons. In 5p miRNAs G and C iterons have much higher density in the seed compared to the post-seed region. This difference is lower in 5n and 3n sequences, and much lower in 3p sequences. In all groups the contiguous GC strings constitute a larger part of sequences than the AU strings. A surplus of G or C iterons and of GC strings should enable a more stable association with the target mRNAs.From the available evidence, the G iteron- and GC-rich miRNAs should also interact more readily with miRNA-processing and similar proteins.

In absence of receptor cycling, human/rat neuropeptide Y was found to persistently occupy the guinea pig neuropeptide Y Y1 receptors expressed on the surface of Chinese hamster ovary (CHO) cells (IC 50 ∼ 8 nM); a lasting occupancy was also evident with active receptor cycling. A similar blockade was obtained with the human neuropeptide Y Y1 receptor (in CHO or SK-N-MC cells). Peptidic antagonists GR238118 (1229U91) and VD-11 blocked the Y1 receptor in the same molarity range. A neuropeptide Y-related Y1 agonist, (Leu 31 Pro 34 ) human neuropeptide Y, also strongly adhered to the Y1 site. Similar blockade-like occupancy by neuropeptide Y was found with particulates from Y1-expressing CHO cells, and with native neuropeptide Y Y1 receptors of rat synaptosomes. Peptide YY and a related Y1-selective agonist, (Leu 31 Pro 34 ) human peptide YY, showed a much less stable binding to the neuropeptide Y Y1 receptor with either the intact cells or particulates. The Y1 binding of neuropeptide Y was also less sensitive to chaotropic agents and guanine nucleotides than the binding of peptide YY, indicating a larger stability for association of neuropeptide Y with the receptor. Inhibition of forskolin-stimulated adenylyl cyclase showed a distinctly attenuating agonism for neuropeptide Y, with an activity similar to peptide YY below 1 nM, but considerably lower above 3 nM of the peptides. This activity was largely exerted via pertussis toxin-sensitive G-proteins of Y1-CHO cells. Our findings indicate that signaling by neuropeptide Y via its Y1 receptor could be self-restricting at higher levels of the peptide, in relation to a strong association of the agonist with the Y1 binding site.

The receptor-linked internalization of [125I] human neuropeptide Y (NPY) in Chinese hamster ovary (CHO) cells expressing the guinea-pig Y1 receptors or in human endometrial carcinoma-1B (Hec-1B) cells expressing the human Y5 receptor, as well as the receptor-linked internalization of human pancreatic polypeptide (hPP) receptor expressed in CHO cells, is selectively inhibited by low molarities of the Li+ cation. The Na+ and K+ cations decreased the receptor-linked internalization of agonist peptides only at high molar inputs, and largely in proportion to the reduction of cell surface binding of Y ligand peptides, dependent on ion concentration and the type of Y receptor examined. With particulates isolated from disrupted cells, there was no preferential inhibition by Li+ relative to Na+ in the binding of type-specific ligand peptides to Y receptors of any type. The observed difference could be connected to the known ability of Li+ to modify active conformations of signal transducers, which may also directly or indirectly affect the internalization motors. The decrease in the rate of Y receptor internalization by Li+ also points to a possible alteration of Y receptor signaling in vivo by lithium at acute therapeutically employed dose levels.

Unlike neuropeptide Y receptors, the pancreatic polypeptide Y4 receptors display considerable differences in sequence and ligand-binding affinity across mammalian species. This could produce different receptor turnover rates in the same cellular membrane environment. Comparing rat, human and guinea-pig Y4 receptors expressed in Chinese hamster ovary (CHO) cells (K(d) with human pancreatic polypeptide 14, 45 and 116 pM, respectively), we indeed found human pancreatic polypeptide internalization in the rank order of receptor affinities. A large fraction of the internalized human pancreatic polypeptide, similar across the Y4 species, was associated with secondary endosomes (density approximately 1.05 in Percoll gradients) and lysosomes (density approximately 1.11). For all Y4 receptors examined, this intake was potently and selectively inhibited by cholesterol-complexing polyene antibiotic filipin III and also by clathrin lattice formation inhibitor, phenylarsine oxide. Internalization differences found across Y4 receptor species to a degree compare with those observed for the cloned guinea-pig neuropeptide Y Y1 and human neuropeptide Y Y5 receptors and, generally, support ligand-binding affinities as important determinants of internalization for neuropeptide receptors.

The cloned guinea-pig Y2 neuropeptide Y (NPY) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the Y2 receptors natively expressed in rat forebrain, are distributed in two populations. A smaller population that is readily accessed by agonist peptides on the surface of intact cells constitutes less than 30% of Y2 receptors detected in particulates after cell homogenization. A much larger fraction of cell surface Y2 sites can be activated by sulfhydryl modifiers. A fast and large activation of these masked or cryptic sites could be obtained with membrane-permeating, vicinal cysteine-bridging arsenical phenylarsine oxide. A lower activation is effected by N-ethylmaleimide, an alkylator that slowly penetrates lipid bilayers. The restricted-access alkylator, 2-[(trimethylammonium)ethyl]methanethiosulfonate, was not effective in unmasking these sites. Some of the hidden cell surface Y2 sites could be activated by polyene filipin III through complexing of membrane cholesterol. The results are consistent with the presence of a large Y2 reserve in a compartment that can be accessed by alteration of sulfhydryl balance or fluidity of the cell membrane, and by treatments that affect the anchoring and aggregation of membrane proteins.

Cloned rat, human and guinea-pig Y4 pancreatic polypeptide (PP) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the rabbit Y4-like PP receptor, show a selective sensitivity to Na+ over K+ ion in PP attachment, but little sensitivity to Na+ in dissociation of bound PP peptides. Agonist binding to Y4 receptors of intact CHO cells also shows much greater sensitivity to Na+ over K+, and a tenacious attachment of the bound agonist. Binding sensitivity to K+ is greatly enhanced upon receptor solubilization. Pancreatic polypeptide sites also show large sensitivity to modulators of Na+ transport such as N5-substituted amilorides and to RFamides, as different from Y1 or Y2 receptors. Thus, PP binding is modulated by cation-induced changes in site environment (with selectivity for Na+) and ultimately results in a blocking attachment. This would support receptor operation in the presence of ion gradients, as well as prolonged agonist-delimited signaling activity (which can include partial antagonism). Also, this could point to an evolutionary adaptation enabling small numbers of PP receptors to perform extensive metabolic tasks in response to low agonist signals.

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.

While the ribosome constitution is similar in all biota, there is a considerable increase in size of both ribosomal proteins (RPs) and RNAs in eukaryotes as compared to archaea and bacteria. This is pronounced in the large (60S) ribosomal subunit (LSU). In addition to enlargement (apparently maximized already in lower eukarya), the RP changes include increases in fraction, segregation and clustering of basic residues, and decrease in hydrophobicity. The acidic fraction is lower in eukaryote as compared to prokaryote RPs. In all eukaryote groups tested, the LSU RPs have significantly higher content of basic residues and homobasic segments than the SSU RPs. The vertebrate LSU RPs have much higher sequestration of basic residues than those of bacteria, archaea and even of the lower eukarya. The basic clusters are highly aligned in the vertebrate, but less in the lower eukarya, and only within families in archaea and bacteria. Increase in the basicity of RPs, besides helping transport to the nucleus, should promote stability of the assembled ribosome as well as the association with translocons and other intracellular matrix proteins. The size and GC nucleotide bias of the expansion segments of large LSU rRNAs also culminate in the vertebrate, and should support ribosome association with the endoplasmic reticulum and other intracellular networks. However, the expansion and nucleotide bias of eukaryote LSU rRNAs do not clearly correlate with changes in ionic parameters of LSU ribosomal proteins.

Guinea-pig neuropeptide Y1 and rat pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y1 and Y2, but not the Y4, receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y2 receptor expressed in Chinese hamster ovary cells was small at 37 degrees C, and essentially absent at or below 15 degrees C, possibly in connection to the large molecular size of the receptor-ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 degrees C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y1 and the Y4 receptors could be blocked, and that of the Y2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y2 subtype. The restoration of Y1 and Y4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y1 and Y4 receptors have much larger subcellular dynamics than the Y2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes.

The length of a river food chain changed from year to year, shifting with the hydrologic regime. During drought years, grazers suppressed algae across a nutrient gradient, while predators were functionally unimportant. Following flood disturbance, predators suppressed grazers, releasing algae. These results suggest that hydrologic regime, rather than productivity, determines the functional length of this river food chain. Within years, algae and grazer biomass responded to an experimental productivity gradient in patterns predicted by simple trophic models that assume efficient energy transfer. Understanding differences among species within trophic levels, however, was crucial in delineating the controlling interactions.

FMRFamide and related peptides (RFamides) were found to inhibit the association binding of iodinated human pancreatic polypeptide ([125I]hPP) to Y5-like neuropeptide Y (NPY) receptor in rodent tissues. An allosteric regulation of the activity of the rodent kidney PP-sensitive neuropeptide Y (NPY) receptor by RFamides was indicated by potency decrease with particle concentration in the inhibition of the association binding of125I-labeled human pancreatic polypeptide (hPP) by RFamides at rabbit kidney membranes. The competition by C-terminal hexapeptide of hPP (LTRPRY.NH2) did not show such affinity change. The steady-state binding of hPP showed little sensitivity to any of the RFamides tested. The Y1-selective binding of [125I][Leu31,Pro34]hPYY (at 2 nM hPP) was much less sensitive to RFamides than the binding of [125I]hPP, albeit with some differences across tissue or cell types. The binding of Y2-selective agonist125I-labeled human peptide YY (3-36) was quite insensitive to RFamides. The presence of a unique component in the inhibition of hPP binding by RFamides was further indicated by a degree of antagonism with phospholipase C inhibitor U-73122, and by an only limited cooperation with a N5-amiloride compound, and with alkylator chloroethylclonidine. Change of the chirality of individual residues in the FMRFamide molecule produced a significant reduction of inhibitory potency only with D-Phe in the C-terminal position. Substitution of the (C-3) L-Met by L-Leu greatly increased the inhibitory potency of RFamides relative to otherwise identical congeners. RFamides could act both as ligands of membrane neighbors of the PP receptor, and as competitors of Y5-like NPY receptor epitopes that accommodate the C-terminal aspects of agonist peptides.Key words: Y1receptor, Y2receptor, Y5receptor, RFamide, allosteric interaction, hydrophobic pocket, amino acid chirality.

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.

A multitrophic model integrating the effects of flooding disturbance and food web interactions in rivers predicted that removing floods would cause increases of predator-resistant grazing insects, which would divert energy away from the food chain leading to predatory fish. Experimental manipulations of predator-resistant grazers and top predators, and large-scale comparisons of regulated and unregulated rivers, verified the model predictions. Thus, multitrophic models can successfully synthesize a variety of ecological processes, and conservation programs may benefit by taking a food web perspective instead of concentrating on a single species.

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

For many G-protein coupling receptors (GPCRs), the upkeep of receptor dimers could depend on association with functional Gi α subunits. This is known for Y1, Y2 and Y4 neuropeptide Y receptors [presented in the companion paper (Estes et al., Amino Acids, doi: 10.1007/s00726-010-0642-z , 2010)]. Interactions with transducers use mainly intracellular domains of the receptors. Intracellular loops 1 and 2 in GPCRs are short and lack extensive helicity that could support transducer anchoring. Interaction with G-proteins is known to use the juxtamembrane Helix 8 in the fourth intracellular domain, for which we document a helix-stabilizing n/(n + 4) pattern of large hydrophobic sidechains. Another intracellular helix located in the C-terminal portion of the third intracellular loop does not display a strong stabilizing pattern, and is found in many studies to serve dynamically in association and activation of transducers and effectors. We show that these tracts share features across metazoan phyla not only in opsins and opsin-like receptors (including the Y receptors), but also in Taste-2 and Frizzled receptors. Similarities of these helices across GPCR groups could have both phylogenetic and functional roots.

Midge larvae (Diptera, Chironomidae) that weave filamentous algae into retreats of tufts, are dominant primary consumers in a river food web. In a previous study, densities of tuft—weaving midges increased in the presence of large fish. In the absence of large fish, midges decreased as densities of predatory invertebrates built up, and higher standing crops of algae were maintained. To examine the mechanisms underlying these dynamics, we compared the vulnerability of tuft—weaving midges (naked or in algal tufts) to fish and predatory invertebrates, in field and laboratory experiments. When midges were exposed for 1 h in the river to fish, 15 out of 15 midges in tufts survived, while 15 of 15 naked midges were consumed. Tufts afforded only partial protection to midges exposed to invertebrate predators, however. After 1 h, enhancement of survivorship by tufts was moderately significant for midges exposed to aeshnids, and insignificant for midges exposed to lestids and naucorids. We suggest that the vulnerab...

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.

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.

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.

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.

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.

Numerous rhodopsin-like G-protein coupling receptors induce or inhibit angiogenesis. The active human receptors include several chemokine receptors, apelin APJ receptor, neuropeptide Y Y2 receptor, Duffy antigen, and herpes virus-8 receptor. A common and striking feature of these receptors is the large fraction (up to 42%) of residues with anionic sidechains (Asp, Glu, and benzene anions Tyr, Trp, and Phe) in the N-terminal extracellular domain. These residues (which are frequently clustered) can assist the binding of ligand peptides, but should also support interactions that help tubular arraying of cells, e.g., via cationic bridges and/or hydrogen bonding with cell-connecting receptors such as integrins, or with proteins of the extracellular matrix.

Low concentrations of Li + reduce the rate of internalization of neuropeptide Y (NPY) Y1 receptors [M.S. Parker, S.L. Parker, J.K. Kane, Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions, Regul. Pept., 118 (2004) 67–74]. This Li + -induced decrease in Y1 receptor internalization could be alleviated by Y1 receptor agonists. As shown by fractionation on Percoll gradients, lithium treatment induces a concentration-related decrease of intermediate and higher endosomal densities that contain the internalized Y1 ligand–receptor complex. This indicates an inhibition of endosome processing and maturation. Internalization of human transferrin shows [Li + ] sensitivity similar to that of the Y1 receptor, and a similar Li + -induced decrease in endosomal processing. Lithium treatment thus decreases activity of the endosome system shared in the recycling endocytosis of the Y1 and transferrin receptors.

In human embryonic kidney-293 (HEK-293) cells, the cloned human neuropeptide Y Y 5 receptor saturably internalized agonists, with the rank order of neuropeptide Y-(19-23)-[Gly 1 ,Ser 3 ,Gln 4 ,Thr 6 ,Ala 31 ,Aib 32 ,Gln 34 ]human pancreatic polypeptide (neuropeptide Y-Aib-pancreatic polypeptide)>human neuropeptide Y>porcine peptide YY>[Pro 34 ]human peptide YY>[Leu 31 ,Pro 34 ]human peptide YY≫human peptide YY-(3-36). Human pancreatic polypeptide competed [ 125 I]neuropeptide Y binding and internalization in neuropeptide Y Y 5 receptor-expressing cells, but itself showed no internalization. The internalization was strongly dependent on temperature. The surface binding, and especially the internalization, of human neuropeptide Y were highly sensitive to the clathrin network inhibitor phenylarsine oxide, and to the cholesterol-complexing antibiotic filipin III. The internalized ligands were present in particles corresponding to secondary endosomes in Percoll gradients, but especially in particles banding with the acid hexosaminidase lysosomal marker. At any temperature tested, internalization of the neuropeptide Y Y 5 receptor driven by human neuropeptide Y in HEK-293 cells was much slower than the internalization of the neuropeptide Y Y 1 receptor reported in the same cells, or in Chinese hamster ovary (CHO) cells. The neuropeptide Y Y 5 receptor subtype could be the metabotropic receptor responding to protracted challenges by neuropeptide Y-like peptides, and its density could be little sensitive to concentration of extracellular agonists.