Acid dissociation constants of diphytanylglycerolphosphorylglycerol-methylphosphate, and diphytanylglycerolphosphorylglycerophosphate and its deoxy analog

Acid dissociation constants of 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-methylphosphate (PGP-Me), the major phospholipid in extreme halophiles (Halobacteriaceae), and of the demethylated 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-phosphate (PGP) and its deoxy analog 2,3-diphytanyl-sn-glycero-1-phosphoryl-1′-(1′,3′-propanediol-3′-phosphate) (dPGP), were calculated by an original mathematical procedure from potentiometric titration data in methanol/water (1:1, v/v) and found to be as follows: for PGP-Me (and presumably PGP), pK₁=3.00 and pK₂=3.61; for PGP, pK₃=11.12; and for dPGP, pK₁=2.72, pK₂=4.09, and pK₃=8.43. High-resolution ³¹P NMR spectra of intact PGP-Me in benzene/methanol or in aqueous dispersion showed two resonances corresponding to the two P-OH groups, each of which exhibited a chemical shift change in the pH range 2.0–4.5, corresponding to acid dissociation constants pK₁=pK₂=3.2; no further ionization (pK₃) was detected at higher pH values in the range 5–12. The present results show that PGP-Me titrates as a dibasic acid in the pH range 2–8, but above pH 8, it titrates as a tribasic acid, presumably PGP, formed by hydrolysis of the methyl group during the titration with KOH. Calculation of the concentrations of the ionic molecular species of PGP-Me, PGP and dPGP as a function of pH showed that the dianionic species predominate in the pH range 5–9, covering the optimal pH for growth of Halobacteriaceae. The results are consistent with the concept that the hydroxyl group of the central glycerol moiety in PGP-Me and PGP is involved in the formation of an intramolecular hydrogen-bonded cyclic structure of the polar headgroup, which imparts greater stability to the dianionic form of PGP-Me and PGP in the pH range 5–9 and facilitates lateral proton conduction by a process of diffusion along the membrane surface of halobacterial cells.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.     

Metal–salt co-tolerance and metal removal by indigenous cyanobacterial strains

Chromium and salt tolerance in five indigenous cyanobacterial strains isolated from contaminated sites was investigated along with their metal bioaccumulative potential. All the five species showed significantly better growth when the medium was spiked with salt or chromium. As compared to single metal or salt treatment, the binary metal–salt (MS) treatments had more favorable effect on cyanobacterial growth as indicated by significantly higher concentration of the primary photosynthetic pigment chlorophyll at M₂₀S₂₀₀₀ (9.9–25.3 μg/mL) as compared to that at M₀S₀ (4.0–12.3 μg/mL). Similarly biomass was much higher at M₂₀S₁₀₀₀ and M₂₀S₂₀₀₀ (41.8–86.2 mg/10 mL) as compared to that at control, M₀S₀ (21.5–36.3 mg/10 mL). Accessory pigments like carotenoids and phycobilinproteins too tended to increase significantly in response to both metal and salts in the two species of Lyngbya (L. putealis and L. ceylanica var. constricta) and Gloeocapsa. These species also showed greater potential of chromium bioaccumulation, which increased further as both salt and metal concentration increased. In the two species of Nostoc however, bioaccumulative potential improve at higher metal concentration, but not affected significantly by salt concentration.     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

Combination of u.v.-B. and ozone reduces pollen tube growth more than either stress alone

The rate of in vitro Nicotiana tabacum L. “Bel-W3” pollen tube growth was reduced 62 and 44%, respectively, when pollen tubes were exposed to 120 ppb ozone (O₃) for 3 hr or 300 μW/cm² ultraviolet-B (u.v.-B) radiation for 30 min. Petunia hybrida Vilm. “White Cascade” pollen tube growth was reduced 34 and 59%, respectively, upon exposure to O₃ or u.v.-B at the above doses. The combination of u.v.-B at 300 μW/cm² for 30 min, followed by O₃ at 120 ppb for 3 hr, reduced pollen tube growth by 79% for “Bel-W3” and 75% for “White Cascade”. The effect appeared to be additive, implying that different target areas may be affected by the two stressors. In the Northeast, plants are exposed to both u.v.-B and O₃ during the normal growing season. This may result in an unexpectedly higher stress on the reproductive system than had been previously suspected based on these two stressors acting individually.     

High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C₁₈ column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.     

Simultaneous determination of zidovudine and its monophosphate in mouse plasma and peripheral red blood cells by high-performance liquid chromatography

Novel prodrugs for the intracellular delivery of zidovudine monophosphate (AZTMP) have recently been designed. To investigate the bioconversion and pharmacokinetic profiles of these compounds, an analytical method for the simultaneous determination of zidovudine (AZT) and AZTMP in mouse plasma and peripheral red blood cells was developed. Mouse whole blood samples were treated with TBAHS, EDTA and NaH₂PO₄, and separated into plasma and red blood cell portions. Samples were processed by solid-phase extraction using Bond Elut C₁₈ cartridges. Chromatography was performed using an Hypersil ODS column and a mobile phase of 2.9% (v/v) acetonitrile and 97.1% (v/v) phosphate buffer, pH 7.50, with UV detection at 267 nm. The average extraction recoveries of AZTMP and AZT in plasma were approximately 85% and 97% over their linear ranges of 0.05–5 μg/ml and 0.125–25 μg/ml, respectively. Extraction recoveries of AZTMP and AZT from peripheral red blood cells averaged 56 and 69% over their linear ranges of 0.125–5 μg/ml and 0.125–25 μg/ml, respectively. The accuracy of the assay was 90–100%. The intra- and inter-day variations of the assay were less than 14%. The analytical method was found to be applicable, reliable and suitable for pharmacokinetic studies.     

Selection and preliminary characterization of β-glycosidases producer Patagonian wild yeasts

Four pre-selected indigenous yeast strains belonging to Candida guilliermondii (V₂ and V₅), Candida pulcherrima (V₆) and Kloeckera apiculata (V₉), were used as β-glucosidase (βGL) and β-xylosidase (βXL) sources. The optimization of yeast culture conditions was carried out and the effects of oenological parameters on β-glycosidase activities were evaluated. C. guilliermondii V₂ and C. pulcherrima V₆ strains were selected. These strains showed intracellular (C. pulcherrima V₆) and parietal (C. guilliermondii V₂) constitutive βGL and βXL. The enzymatic activities were active at pH, glucose, ethanol and SO₂ concentrations usually found in winemaking and they were able to release monoterpenols and alcohols from grape juice glycoside extracts. Additionally, these yeast strains were not able to produce volatile acidity and off flavour. Regional ecological relevance of these species was also discussed. Our results evidence that the selected C. guilliermondii V₂ and C. pulcherrima V₆ strains have interesting oenological characteristics and allow us to think in their potential application in winemaking.     

Intracellular acidification does not account for inhibition of Saccharomyces cerevisiae growth in the presence of ethanol

Abstract Intracellular acidification has been considered one of a number of mechanisms underlying the inhibition of growth and fermentation by ethanol in yeast. However, most of the studies on the effect of ethanol on yeast intracellular pH (pH_i) were carried out by using unadapted cells to which ethanol was added. In this paper we show that the pH_i of exponential cells of Saccharomyces cerevisiae IGC 3507 III grown in a medium with glucose and inhibitory concentrations of ethanol only decreased to values below those in unstressed cells (6.9) for concentrations equal to or above 7% (v/v). Only at these supracritical levels (7–10% (v/v)) was pH homeostasis in ethanol-adapted yeast affected. This is consistent with the significant increase of plasma membrane permeability and decrease of plasma membrane H⁺-ATPase in comparison with the corresponding values in unstressed cells. These deleterious effects were only observed with those high concentrations of toxin. These results indicate that intracellular acidification does not account for inhibition of yeast growth in the presence of ethanol. In fact, growth was inhibited by ethanol concentrations (3–6% (v/v)) that did not lead to the decrease of pH_i. Furthermore, even for supracritical concentrations, close to the maximal that allowed growth (10% (v/v)), the dedrease of pH_i was not important reaching, at the most, values of 6.5–6.6.     

Increasing progesterone secretion and 3β-hydroxysteroid dehydrogenase activity of human cumulus cells and granulosa-lutein cells concurrent with successful fertilization of the corresponding oocyte

In many studies it has been documented that the induction of multiple follicular growth in humans results in an asynchrony between the degree of cumulus mucification, oocyte meiotic maturation, fertilizability, and follicular cell progesterone (P₄) secretion. The present study was carried out on oocytes enclosed in fully mucified cumulus. Thus, oocyte fertilizability was correlated to human cumulus cell (hCC) and human granulosa-lutein (G-L) cell competence for P₄ secretion in culture. In the G-L cells, P₄ secretion and percentage of cells manifesting 3β-hydroxysteroid dehydrogenase (3β-HSD) activity increased concurrently with the period of culture. In the hCC, however, P₄ secretion decreased concurrently with elongation of the culture period, whereas the percentage of 3β-HSD-positive cells increased. In hCC corresponding to the fertilized oocytes, P₄ accumulation in culture medium was 1.9-fold (P < 0.001) and 1.6-fold (P < 0.02) higher on days 0–3 and 3–5 of culture, respectively, as compared to P₄ accumulation in hCC of unfertilized oocytes. Also, in hCC corresponding to the fertilized oocytes, the degree of 3β-HSD activity was found to be significantly higher shortly after aspiration and after either 3 or 5 days, compared to hCC of unfertilized oocytes. In the G-L cells pooled from all follicles yielding mature cumulus-oocyte complexes, P₄ accumulation and percentage of 3β-HSD-positive cells increased concurrently with the increase in percentage of fertilized eggs of each individual woman. These results indicate that in stimulated cycles, follicles yielding mature cumulus-oocyte complex, oocyte fertilizability, and G-L cell or hCC competence for P₄ secretion are correlated and synchronous.     

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min⁻¹/g⁻¹ cells) were achieved at an O₂ delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO₂ and inactivation of catechol-2,3-dioxygenase at high dO₂ levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of M_r 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a K_m of 0.9 mg/ml, and V_max of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.     

Testing of the anabolic stanozolol in human hair by gas chromatography–negative ion chemical ionization mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of stanozolol in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride and the sonication in methanol of 100 mg of powdered hair for 2 h. After elimination of the solvent, the hair sample was solubilized in 1 ml 1 M NaOH, 15 min at 95°C, in the presence of 10 ng stanozolol-d₃ used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C₁₈) and a liquid–liquid (pentane) extraction. After evaporation of the final organic phase, the dry extract was derivatized using 40 μl MBHFA–TMSI (1000:20, v/v), incubated for 5 min at 80°C, followed by 10 μl of MBHFBA, incubated for 30 min at 80°C. The derivatized extract was analyzed by a Hewlett-Packard GC–MS system with a 5989 B Engine operating in the negative chemical ionization mode of detection. Linearity of the detector response was observed for stanozolol concentrations ranging from 5 to 200 pg/mg with a correlation coefficient of 0.998. The assay was capable of detecting 2 pg of stanozolol per mg of hair when approximately 100 mg hair material was processed, with a quantification limit set at 5 pg/mg. Intra-day precision was 5.9% at 50 pg/mg and 7.8% at 25 pg/mg with extraction recoveries of 79.8 and 75.1%, respectively. The analysis of a 3-cm long hair strand, obtained from a young bodybuilder (27 year old) assuming to be a regular user of Winstrol (stanozolol, 2 mg), revealed the presence of stanozolol at the concentration of 15 pg/mg.     

Bimodal respiration, water balance and acid-base regulation in a high-shore crab, Cyclograpsus lavauxi H. Milne Edwards

Cyclograpsus lavauxi H. Milne Edwards, 1853 occurs under boulders near the littoral fringe, where it is wetted only briefly, if at all, in each tide. Mean water content of sea-water equilibrated crabs was 62.3 % of body weight. On warm, windy days crabs on the shore lose > 17.5% of body water. Water loss for crabs of 1.6 g mean body weight was 0.29 % water content · h ⁻¹ · mm Hg saturation deficit ⁻¹, which is similar to that of species occupying positions lower on the shore. C. lavauxi is, however, able to tolerate comparatively greater losses of body water (up to 36%) compared with species from lower shore levels.

As the gills dry, the lamellae separate into regular clumps which may help to maintain gas exchange. Resting oxygen consumption (V_o2) after 3–5 h settlement was similar in water and air. During enforced activity, V_o2 increased by factors of 5.3 in water and 2.6 in air at a standardized body weight of 1.5 g, indicating an appreciable aerobic scope in both media. Loss of up to 16% of body water did not depress resting aerial V_o2 or aerobic scope.

In resting crabs there was no change in haemolymph pH after 24 h of either immersion or emersion, but haemolymph [Ca²⁺], [HCO⁻₃] + [CO²⁻₃], and calculated P_co2 all rose in air relative to aquatic values. These results suggest that cuticular or other endogenous CaCO₃ is mobilized to compensate the respiratory acidosis in air. The implications of such physiological properties for aquatic and aerial activity on the shore are discussed.     

Chromatographic separation of chlorthalidone enantiomers using β-cyclodextrins as chiral additives

Different β-cyclodextrins have been tested as chiral additives in the mobile phase for the chromatographic analysis of chlorthalidone enantiomers in a C₁₈ LiChrospher (125×4 mm I.D.) column. The effect on enantioresolution of different parameters was studied: composition of the mobile phase (percentage of organic solvent, type of buffer and pH), mobile phase flow-rate, and type and concentration of β-cyclodextrin. A 25:75 mixture of methanol and 0.1 M phosphate buffer, pH 4, containing 2% triethylamine (v/v), and 12.5 mM β-cyclodextrin, at a flow-rate of 0.8 ml/min, was found to be the best option for the resolution of chlorthalidone enantiomers. Under such conditions, linear calibration curves were obtained in the 0.5–20-μg/ml interval using UV detection at 230 nm. The limit of detection for both isomers was 50 ng/ml. The utility of the described assay has been tested by analyzing chlorthalidone in different pharmaceutical preparations. Examples of application to biological samples are also given.     

Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.     

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4–25-fold higher affinity for the Y₂ receptor than for the Y₁ receptor. The affinity of [Leu³¹,Pro³⁴]NPY is 7–60-fold higher for the Y₁ receptor when compared with the Y₂ subtype. Species selectivity within the Y₂ receptors is demonstrated by PYY(3–36), NPY(2–36), NPY(22–36), and NPY(26–36). It is shown that NPY(22–36) is species selective for the human Y₂ subtype (K_i of 0.3 nM) compared with the rabbit and rat Y₂ receptor (K_i of 2 and 10 nM, respectively). PYY(3–36) displays highest affinity for the human and rabbit Y₂ subtype (K_i of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y₂ receptor is shown by NPY(2–36) and PYY(3–36). In addition, PYY(3–36) and NPY(2–36) are not only subtype selective, but also species selective.