New Cytotoxic Saponins from Lysimachia davurica Ledeb.

To investigate the saponins from whole plants of Lysimachia davurica Ledeb., two new saponins named davuricoside I （compound 1） and E （compound 2） were isolated. Their chemical structures were elucidated as 3β, 16α, 28, 29-tetrihydroxy-olean-12-en-3-O-β-D-glucopyranosyl-（1→2）-β-D-glucuronopyranoside （compound 1） and 3β, 16α, 29-trihydroxy-13, 28-epoxy-oleanane-3-O-β-D-glucopyranosyl-（1→2）-β-D-glucuronopyranoside （compound 2） on the basis of their one- and two-dimensional nuclear magnetic resonance and mass spectrometry data, and chemical methods. Compound 1 showed significant cytotoxic activity against human A2780 cells.     

Triterpenoid Saponins from Pulsatilla cernua （Thunb.） Bercht. et Opiz.

To investigate saponins from the roots of Pulsatilla cernua （Thunb.） Bercht. et Opiz., two new compounds together with five known trlterpenold saponins were isolated. The structures of the two new trlterpenoid saponins, named cernuasides A and B, were elucidated as 3-O-[β-D-xylopyranosyl（1-）2）]-[α-L-rhamnopyranosyl（1-）4）]-α-L- arablnopyranosyl hederagenin 28-O-β-D-glucopyranosyl ester （compound 1） and 3-O-[α-L-arabinopyranosyl（1→）3）]- [α-L-rhamnopyranosyl （1→）2）]-α-L-arabinopyranosyl hederagenin 28-O-β-D-glucopyranosyl ester （compound 2） by 1D, 2D-NMR techniques, ESIMS analysis, as well as chemical methods.     

Water-Soluble Constituents of Cudrania tricuspidata （Carr.） Bur.

In order to find new structural and biologically active compounds, the constituents of the bark of Cudrania tricuspidata （Carr.） Bur. were investigated and a new 6-p-hydroxybenzyltaxifolin glucoside, named tricusposide （compound 1）, together with 16 known compounds, was isolated by solvent partition, macroporous adsorption resin AB-8, silica gel, Sephadex LH-20 chromatography. Using spectroscopic methods, the structures of the compounds were elucidated as 6-p-hydroxybenzyl taxifolin-7-O-β-D-glucoside （compound 1）, dihydroquerctin-7-O-β-D-glucoside （compound 2）, dihydrokaempferol-3-O-β-D-glucoside （compound 3）, dihydroquercetin （compound 4）, peonoside （compound 5）, sphaerobioside （compound 6）, quercimeritrin （compound 7）, genistein （compound 8）, aromadendrin （compound 9）, kaempferol （compound 10）, genistin （compound 11）, 3,4-dihydroxystyryl alcohol （compound 12）, sucrose （compound 13）, 1,3,5,6-tetrahydroxyxanthone （compound 14）, gericudranin E （compound 15）, gericudranin C （compound 16）, and orobol （compound 17）. Compounds 2-6, 8, 9, 12-14, and 17 were isolated from this genus for the first time.     

A Cytotoxic Neolignan from Schisandra propinqua （Wall.） Baill.

In the course of our study of bioactive natural products from Schisandra plants, we isolated a neolignan from an EtOAc extract of the stems of Schisandra propinqua （Wall.） Baill. The structure of the new com- pound was determined to be 4, 4-di （4-hydroxy-3-methoxyphenly）-2, 3-dimethylbutanol （compound 1） on the basis of 1H- and 13C-NMR spectra and 2D NMR methods. Eight known compounds, compounds 2-9, were also isolated and identified, of which compounds 3, 4, 6 and 9 were isolated for the first time from this plant. In addition, compounds 1-4 were evaluated for cytotoxicity by an 3-（4,5-dimethyl-2 thiazoyl）-2,5-diphenyl-2H- tetrazolium bromide （MTT） assay. Compound 1 showed significant potential cytotoxic ability in the bioassay.     

A Novel Flavonoid Glucoside from Anoectochilus roxburghii （Wall,） Lindl.

Eight compounds were isolated from the ethyl acetate- and n-butanol-soluble fractions of the ethanollc extract of the whole plant of Anoectochllus roxburghll（Wali.） Lindi. （Orchidaceee）. On the basis of spectroscopic methods, the structures of these compounds were elucidated as quercetin-7-O-β-D-[6＂-O-（trans-feruloyi）]- giucopyranosids （compound 1）, 8-C-p-HydroxybenzyiquerceUn （compound 2）, isorhamnetin-7-O-β-D- giucopyranoside （compound 3）, isorhamnetin-3-O-β-D-giucopyranoside （compound 4）, kaempferoi-3-O-β-D- giucopyranoside （compound 5）, kaempferoi-7-O-β-D-giucopyranoside （compound 6）, 5-hydroxy-3＇,4＇,7- trimethoxyfiavonoi-3-O-β-D-rutinoside （compound 7）, and isorhamnetin-3-O-β-D-rutinoside （compound 8）. Of the compounds isoisted, compound 1 was a new flavonoid giucoside and exhibited strong scavenging activity against the 1,1-diphenyi-2-picryihydrazyi free radical, whereas the ethanolic extract showed weak activity. Compounds 2-8 were obtained from this family for the first time.     

Chemical Constituents of Siegesbeckia orientalis L.

The aerial parts of Siegesbeckia have been used as the traditional Chinese medicine in the treatment of rheumatic arthritis, hypertension, malaria, neurasthenia, and snakebite. In order to find new and bioactive compounds, the chemical constituents of the aerial parts of S. orientalis L. were investigated and two new compounds, namely β-D-glucopyranosyl-ent-2-oxo-15,16-dihydroxy-pimar-8（14）-en-19-oic-late （compound 1 ） and [1 （10） E,4Z]-8β-angeloyloxy-9α-methoxy-6α, 15-dihydroxy-14-oxogermacra-1 （10）,4,11 （13）-trien-12-oic acid 12,6-1actone （compound 2）, as well as five known ent-pimarane diterpenes （compounds 3-7） were isolated. The structures of the two new compounds were identified by their physicochemical properties and spectral analysis, particularly oneand two-dimensional nuclear magnetic resonance spectral methods.     

Chemical Constituents of the Roots of Vernonia cumingiana Benth.

To search for new and bioactive constituents from traditional Chinese medicines, a new steroidal saponin, named vernonioside G （1）, was isolated from the roots of Vernonia cumingiana Benth. （Compositae）. The structure of vernonioside G was elucidated using spectral methods, particularly two-dimensional nuclear magnetic resonance analysis. Together with the new compound, eight known compounds were also isolated and identified from the roots of V. cumingiana, among which, VE-1 （2） and 24-methylenelanost-9（11）- en-3β-ol acetate （3） were assigned NMR data for the first time and compound 3 was obtained as a natural product from a plant for the first time.     

Iridoid Glycosides from Pedicularis dolichocymba Hand.-Mazz.

During investigation of the chemical constituents of the whole plant ethanol extract of Pedlcularis dollchocymba Hand.-Mazz. （Scrophularlaceae）, four new irldold glycosides, dolichocymbosides A （compound 1）, B （compound 2）, C （compound 3） and D （compound 4）, were Isolated. Their structures were determined based on spectral data Including 1D and 2D-nuclear magnetic resonance spectroscopy （^1H-^1H COSY, HSQC, HMBC, ROESY） and FAB-MS.     

Two New Isoprenylated Stilbenes from Artocarpus chama

Two new stllbenes with two Isoprenoid groups, namely artostllbenes A （compound 1） and B （compound 2）, were Isolated from the stems of Arfocarpus chama Buch.-Ham. by repeated column chromatography. The＆ structures were elucldated as （E）-4-[2-（7-meth-xy-2-2-d-methy-6-（3-methy-but-2-eny-）-2H-1-benz-pyran-5-y-）v-ny-]benzene-1- 2-dlol （compound 1） and （Z）-4-[2-（7-meth-xy-2-2-dimethy--6-（3-methy-but-2-eny-）-2H-1-benz-pyran-5-y-）v-ny-]ben- zene-l,2-dlol （compound 2） by spectroscopic methods, mainly by 1D-, 2D-NMR and MS spectra. Compounds 1 and 2 are two cls- and trans-lsomers and compound 2 is the flrst cis-stllbene isolated from Moraceous plants.     

Biotransformation of 4-Hydroxybenzen Derivatives by Hairy Root Cultures of Polygonum multiflorum Thunb.

The biotransformation of four 4-hydroxybenzen derivatives （1,4-benzenediol （compound 1）, 4-hydroxybenzaldehyde （compound 2）, 4-hydroxybenzyl alcohol （compound 3） and 4-hydroxybenzoic acid （compound 4）） by the hairy root cultures of Polygonum multiflorum Thunb. as a new biocatalyst was investigated. It was found that the substrates were transformed to their corresponding glucosides, 4-hydroxyphenyl β-D-glucopyranoside （arbutin, compound la）, 4-hydroxymethylphenyl β-D-glucopyranoside （gastrodin, compounds 2a, 3a） and 4-carboxyphenyl α-D- glucopyranoside （compound 4a）, respectively. In the meantime, the hairy roots of P. multiflorum were able to stereoselectively and regioselectively glucosylate phenolic hydroxyl groups of compounds 1-4, but the cultures could not glucosylate the aldehyde group of compound 2 or the benzyllc hydroxyl group of compound 3, and no glucosyl esterification of carboxyl groups of compound 4 was detected. On the other hand, the result also showed that the hairy roots of P. multiflorum were able to reduce the 4-hydroxybenzaldehyde to its corresponding alcohol. This is the first report that substrate 4 has been converted into its α-D-glucopyranoside by a plant biotransformα- tion system.     

Molecular modeling and design of regioselectively addressable functionalized templates with rigidified three‐dimensional structures

Extensive conformational analysis of a series of β‐alkyl substituted cyclopeptides—cyclo(Pro¹–Xaa²–Nle³–Ala⁴–Nle⁵–Pro⁶–Xaa⁷–Nle⁸–Ala⁹–Nle¹⁰) and cyclo[Pro¹–Xaa²–Nle³–(Cys⁴– Nle⁵–Pro⁶–Xaa⁷–Nle⁸–Cys⁹)–Nle¹⁰] as well as their corresponding unsubstituted core structures cyclo(Pro¹–Xaa²–Ala³–Ala⁴–Ala⁵–Pro⁶–Xaa⁷–Ala⁸–Ala⁹–Ala¹⁰) and cyclo(Pro¹–Xaa²–Ala³–Cys⁴– Ala⁵–Pro⁶–Xaa⁷–Ala⁸–Cys⁹–Ala¹⁰) has been performed employing both the ECEPP/2 and the MAB force fields (Xaa = Gly, L ‐Ala, D ‐Ala, Aib, and D ‐Pro). Results show that (a) possible three‐dimensional structures of the cyclo(Pro¹–Gly²–Lys³–Ala⁴–Lys⁵–Pro⁶–Gly⁷–Lys⁸–Ala⁹–Lys¹⁰) molecule are not limited to a single extended “rectangular” conformation with all Lys side chains oriented at the same side of the molecule; (b) conformational equilibrium in monocyclic analogues obtained by replacements of conformationally flexible Gly residues for L ‐Ala, D ‐Ala, Aib, or D ‐Pro is not significantly shifted towards the target “rectangular” conformational type; and (c) introduction of disulfide bridges between positions 4 and 9 is a very powerful way to stabilize the target conformations in the resulting bicyclic molecules. These findings form the basis for further design of rigidified regioselectively addressable functionalized templates with many application areas ranging from biostructural to diagnostic purposes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 361–372, 1999     

Immunostimulatory activities of specific bacterial secondary metabolite of Anoxybacillus flavithermus strain SX‐4 on carp,Cyprinus carpio

Aims: To determine the capacity of secondary metabolite of strain SX‐4, to enhance the nonspecific immunity and survival of carp (Cyprinus carpio), and to identify the constituents that are responsible. Methods and Results: A thermophilic strain SX‐4 that is able to produce immunostimulatory metabolite was isolated from sludge sample of hot spring and identified by comparison with 16S rRNA sequences (99% of homology) as Anoxybacillus flavithermus. Bioactivity‐guided fractionation of methanol extract from its cell‐free culture, one bacterial peptide with the capacity of improving the nonspecific immune responses and disease resistance (relative per cent survival = 66·67%) was obtained and the compound was characterized as cyclo‐(L‐Pro‐Gly) by IR, ESI‐MS, ¹H NMR and ¹³C NMR spectroscopic analyses. After intraperitoneal administration of this peptide, selected innate immune parameters including phagocytic activity, superoxide anion production, serum lysozyme activity and serum SOD activity, along with immune‐related genes expression (i.e. interleukin‐1β and inducible nitric oxide synthase), in the blood were found to be significantly increased. Conclusions: The bacterial peptide cyclo‐(L‐Pro‐Gly) significantly enhances nonspecific immunity and survival of carp. Significance and Impact of the Study: There is a possibility of using cyclo‐(L‐Pro‐Gly) as a better natural immunostimulant, which could have a promising role in aquaculture to prevent diseases and disease outbreaks.     

Total synthesis,structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A ( 1a ) was reported to inhibit nitric oxide production in LPS‐stimulated murine macrophage RAW 264.7 cells. In the current study, solid‐phase total synthesis of stylissatin A was performed by using a safety‐catch linker and yielded the peptide with a trans‐Phe⁷‐Pro⁶ linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d ‐allo‐Ile in place of l ‐Ile and a different site for macrocyclization. The resulting product (d ‐allo‐Ile²)‐stylissatin A ( 1c ) was also found to have the trans‐Phe⁷‐Pro⁶ peptide conformations like rotamer 1b . Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a , 1b , and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2–7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product ( 1a ), the rotamer ( 1b ), and (d ‐allo‐Ile²)‐stylissatin A ( 1c ) exhibited significant inhibition of NO^.. Further investigations were focused on 1b , which also inhibited proliferation of T‐cells and inflammatory cytokine IL‐2 production. The analogs 2–7 weakly inhibited NO^. production, but strongly inhibited IL‐2 cytokine production compared with synthetic peptide 1b . All analogs inhibited the proliferation of T‐cells, with analog 7 having the strongest effect. In the analogs, the Pro⁶ residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Le système protéolytique de Penicillium roqueforti: II - Purification et propriétés de la protéase acide

An extracellular protease has been isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by precipitation with ammonium sulfate, filtration on Bio-gel P 100 columns and chromatography on D.E.A.E.-cellulose columns The purified preparation was homogenous by gel filtration on Bio-gel P 60 and electrophoretical analysis at pH 9.0 and 5.0.The protease exhibited the properties of an acid protease: the optimum pH was 3.5 for casein or hemoglobin hydrolysis and for bovine trypsinogen activation; at 40°C, the enzyme is most stable in the range of pH 3.5 to 5.5; the optimum temperatures was 50°C.E.D.T.A., D.F.P. and sulfhydryl reagents induced no inhibition.The enzyme exhibited a milk clotting activity that was fifty times weaker that the activity of rennin. Its molecular weight, estimated by gel filtration was 33 400. Amino acid composition is: Lys₁₅, His₂, Arg₁, Trp₅, Asp₃₃, Thr₂₇, Glu₁₆, Pro₁₀, Gly₄₀, Ala₂₅, Cys₂, Val₂₁, Ile₂₀, Leu₂₁, Tyr₁₄, Phe₁₉.The properties of this protease were closely similar to that of P. janthinellum and Aspergillus oryzae.     

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1‐naphthylalanine or 2‐naphthylalanine

We designed five ascidiacyclamide analogues [cyclo(‐Xxx¹‐oxazoline²‐d ‐Val³‐thiazole⁴‐l ‐Ile⁵‐oxazoline⁶‐d ‐Val⁷‐thiazole⁸‐)] incorporating l ‐1‐naphthylalanine (l ‐1Nal), l ‐2‐naphthylalanine (l ‐2Nal), d ‐phenylalanine (d ‐Phe), d ‐1‐naphthylalanine (d ‐1Nal) or d ‐2‐naphthylalanine (d ‐2Nal) into the Xxx¹ position of the peptide. The conformations of these analogues were then examined using ¹H NMR, CD spectroscopy, and X‐ray diffraction. These analyses suggested that d ‐enantiomer‐incorporated ASCs [(d ‐Phe), (d ‐1Nal), and (d ‐2Nal)ASC] transformed from the folded to the open structure in solution more easily than l ‐enantiomer‐incorporated ASCs [(l ‐Phe), (l ‐1Nal), and (l ‐2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1‐naphthyl isomer induced a more stable open structure than the 2‐naphthyl isomer. In particular, [d ‐1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL‐60 cells. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Temperature-correlated force and structure development in elastomeric polypeptides: the Ile1 analog of the polypentapeptide of elastin

The Ile¹ analog of the polypentapeptide of elastin, (L · Ile¹-L · Pro²-Gly³-L · Val⁴-Gly⁵)_n, abbreviated as Ile¹-PPP, was synthesized with n > 100 to determine the effect of the increased hydrophobicity of the pentamer resulting from Val¹ replacement by Ile¹ on the previously characterized inverse temperature transition of the polypentapeptide of elastin (PPP). Ile¹-PPP, dissolved in water at 4°C, was found to aggregate, forming a viscoelastic coacervate on raising the temperature. The onset of aggregation was 8°C for Ile¹-PPP, as compared to 24°C for PPP. Characterization by CD demonstrated an increase in intramolecular order on raising the temperature from 8°C to 25°C, and demonstrated similar conformations for PPP and Ile¹-PPP before and after their respective transitions. The CD-characterized transition also occurred at a temperature some 15°C lower than that of PPP. By means of 20-Mrad γ-irradiation cross-linking of the Ile¹-PPP coacervate, an elastomeric matrix was formed with an elastic modulus, similar to that of 20-Mrad cross-linked PPP. The temperature dependence of elastomeric force of cross-linked Ile¹-PPP showed an abrupt increase from essentially zero at 8°C to three-quarters of full force at 10°C and essentially full force by 20–25°C. This development of elastomeric force for the more hydrophobic Ile¹-PPP matrix, which parallels the increase in intramolecular order characterized by the CD studies, also occurs at a temperature some 15°C lower than that for the PPP matrix. Thus, in these elastomeric polypeptides, development of elastomeric force is coupled to an inverse temperature transition, the temperature of which depends inversely on the hydrophobicity of the constituent pentamer. It appears that a series of elastomeric polypeptide biomaterials are possible in which the temperature over which elastomeric force develops can be varied.     

Structural and positional studies of the antimicrobial peptide brevinin‐1BYa in membrane‐mimetic environments

Brevinin‐1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow‐legged frog Rana boylii, shows broad‐spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin‐1BYa was investigated in various solution and membrane‐mimicking environments by proton nuclear magnetic resonance (¹H‐NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2‐trifluoroethanol (TFE‐d₃)‐H₂O solvent mixture, as well as in membrane‐mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix‐hinge‐helix motif, with the hinge located at the Gly¹³/Pro¹⁴ residues, and the two α‐helices extending from Pro³ to Phe¹² and from Pro¹⁴ to Thr²¹. Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5‐doxyl‐labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non–pore‐forming mechanism.     

Association between CYP1A1 polymorphism and colorectal cancer risk: a meta-analysis

Colorectal cancer is one of the most common forms of cancer and is the third leading cause of cancer-related death worldwide. Published data on the association between CYP1A1 (MspI and Ile ⁴⁶² Val) polymorphisms and colorectal cancer risk are inconclusive. To address these issues, we carried out a meta-analysis of available case–control study. Online electronic searches of PubMed were performed. We identified 17 studies (6,673 colorectal cancer patients and 8,102 control subjects) that examined the association between CYP1A1 (MspI and Ile ⁴⁶² Val) polymorphisms and risk of colorectal cancer. For CYP1A1 MspI polymorphism, we performed a meta-analysis from 13 studies including 5,468 cases and 6,492 controls. Overall, there was no statistically significant association between CYP1A1 MspI polymorphism and colorectal cancer susceptibility. In the subgroup analyses based on ethnicities, no statistically significant associations were observed in all genetic models. With respect to CYP1A1 Ile ⁴⁶² Val polymorphism, a total of 14 studies including 6,654 cases and 7,859 controls were involved in this meta-analysis. The CYP1A1 Ile ⁴⁶² Val polymorphism was associated with risk of colorectal cancer. Ethnic subgroup analyses revealed that significant associations were found in Asians and Caucasians. In summary, this meta-analysis suggests that CYP1A1 Ile ⁴⁶² Val polymorphism was a low-penetrance susceptibility gene in colorectal cancer development. On the contrary, CYP1A1 MspI polymorphism does not seem capable of modifying colorectal cancer risk.     

Reduction of blood clearance rate of [Val5] angiotensin II by [Sar1, ILE8] angiotensin II in sheep

The present study examines the effect of [Sar¹, Ile⁸] angiotensin II ([Sar¹, Ile⁸] ANG II) on the blood clearance rate of [Val⁵] angiotensin II ([Val⁵] ANG II) in conscious, sodium-replete sheep. Animals were infused simultaneously with [Val⁵] ANG II and [Sar¹, Ile⁸] ANG II at a rate of 42 nmol/h and 6 μmol/h respectively. Blood [Val⁵] ANG II was quantitatively determined with care taken in separating [Val⁵] ANG II from [Sar¹, Ile⁸] ANG II prior to radioimmunoassay. The blood clearance rate of [Val⁵] ANG II calculated from infusion rate/blood concentration was significantly different before and during [Sar¹, Ile⁸] ANG II infusion, being 141 ± 13 L/h (n = 12) and 95 ± 10 L/h (n = 12) respectively. Plasma renin concentration remained suppressed after the commencement of [Sar¹, Ile⁸] ANG II infusion. $\text{In-vitro}$ studies showed no significant decrease in the rate of degradation of [Val⁵] ANG II in blood in the presence of [Sar¹, Ile⁸] ANG II. Possible interpretation of this reduction of blood clearance rate of [Val⁵] ANG II by 45 ± 15 L/h (n = 6) was discussed.