Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

Comparison of the diastereoisomeric excess of uridine,inosine and adenosine cyanohydrins determined by HPLC-DAD and 1H NMR.

The separation of the diastereoisomers of the nucleoside derivatives of uridine, inosine and adenosine was performed by HPLC using chiral and no chiral columns, it was observed with the no chiral columns the resolution was good enough to determine diastereoisomeric excess. These methods were compared with ¹H NMR, and no significant differences were observed between the three techniques. Diastereoisomeric uridine (3a), inosine (3b) and adenosine (4c) cyanohydrins were resolved by ¹H nuclear magnetic resonance (¹H NMR), chiral normal phase-high-performance liquid chromatography-diode array detector (NP-HPLC-DAD) and reversed phase (RP-HPLC-DAD); these methods allowed the assesment of the percent diastereoisomeric excess (% de) of the nucleosidic cyanohydrins of 3a (4, 6 and 4), 3b (10, 8 and 6) and 4c (4, 4 and 4). To the best of our knowledge, there are no reports using analytical techniques for the separation of the epimers of 3a, 3b and 4c.     

Effects of geometric isomerism in dinuclear antitumor platinum complexes on their interactions with N-acetyl-L-methionine

In this study, the reactions of N-acetyl-L-methionine (AcMet) with [{trans-PtCl(NH₃)₂}₂-μ-H₂N(CH₂)₆NH₂](NO₃)₂ (BBR3005: 1,1/t,t 1) and its cis analog [{cis-PtCl(NH₃)₂}₂-μ-{H₂N(CH₂)₆NH₂}]Cl₂ (1,1/c,c 2) were analyzed to determine the rate and reaction profile of chloride substitution by methionine sulfur. The reactions were studied in PBS buffer at 37°C by a combination of multinuclear (¹⁹⁵Pt, {¹H-¹⁵N} HSQC) magnetic resonance (NMR) spectroscopy and electrospray ionization time of flight mass spectrometry (ESITOFMS). The diamine linker of the 1,1/t,t trans complex was released as a result of the trans influence of the coordinated sulfur atom, producing trans-[PtCl(AcMet)(NH₃)₂]⁺ (III) and trans-[Pt(AcMet)₂(NH₃)₂]²⁺ (IV). In contrast the cis geometry of the dinuclear compound maintained the diamine bridge intact and a number of novel dinuclear platinum compounds obtained by stepwise substitution of sulfur on both platinum centers were identified. These include (charges omitted for clarity): [{cis-PtCl(NH₃)₂}-μ-NH₂(CH₂)₆NH₂-{cis-Pt(AcMet)(NH₃)₂}] (V); [{cis-Pt(AcMet)(NH₃)₂}₂-μ-NH₂(CH₂)₆NH₂] (VI); [{cis-PtCl(NH₃)₂}-μ-NH₂(CH₂)₆NH₂-{PtCl(AcMet)NH₃] (VII); [{PtCl(AcMet)(NH₃)}₂-μ-NH₂(CH₂)₆NH₂] (VIII); [{trans-Pt(AcMet)₂(NH₃)}-μ-NH₂(CH₂)₆NH₂-{PtCl(AcMet)(NH₃)] (IX) and the fully substituted [{trans-Pt(AcMet)₂(NH₃)}₂-μ-{NH₂(CH₂)₆NH₂] (X). For both compounds the reactions with methionine were slower than those with glutathione (Inorg Chem 2003, 42:5498–5506). Further, the 1,1/c,c geometry resulted in slower reaction than the trans isomer, because of steric hindrance of the bridge, as observed previously in reactions with DNA and model nucleotides.     

Biosynthesis of anthraquinone derivatives in a Sesamum indicum hairy root culture

In order to investigate the intermediacy of 2-(4-methylpent-3-en-1-yl)anthraquinone (MPAQ), a possible intermediate for the biosynthesis of anthraquinone derivatives in sesame (Sesamum indicum), ²H-labeled MPAQ was administered to a hairy root culture of S. indicum. Efficient conversion of fed MPAQ to 2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone ((Z)-MPDEAQ) was observed. Furthermore, administration experiment with ²H-labeled 2-geranyl-1,4-naphthohydroquinone, another possible intermediate, showed that it was converted to MPAQ and (Z)-MPDEAQ. The results clearly demonstrated that these substrates are the actual precursors for the production of (Z)-MPDEAQ. In contrast, neither MPAQ nor 2-geranyl-1,4-naphthohydroquinone was converted to anthrasesamone B and 2,3-epoxyanthrasesamone B, other anthraquinone derivatives in the hairy roots, suggesting that these substrates may not be the common precursors in the biosynthesis of anthraquinone derivatives.     

NAD-299 ANTAGONISES 5-HT-STIMULATED AND SPIPERONE-INHIBITED [35S]GTPγS BINDING IN CLONED 5-HT1A RECEPTORS

ABSTRACT

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxy-tryptamine_1A (5-HT_1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-³H]-3,4-dihydro-2H-1-benzopyran-5-carboxamide ([³H]NAD-299) exhibited high affinity (K_d = 0.16?nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-³H]di-n-propylamino)tetralin ([³H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [³⁵S]GTPγS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [³⁵S]GTPγS binding 2.5-fold while spiperone and methiothepin inhibited [³⁵S]GTPγS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [³⁵S]GTPγS binding to basal levels. The K_iL/K_iH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (±)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [³⁵S] GTPγS (r = 0.97).     

Temporal patterns of inorganic nitrogen uptake by mature sugar maple (Acer saccharum Marsh.) and red spruce (Picea rubens Sarg.) trees using two common approaches

Background: Plant uptake of nitrogen influences many ecosystem processes, yet uptake by trees in northern forests of the United States has not been quantified throughout the growing season.

Aims: To measure NH₄ ⁺ and NO₃ ^? uptake by mature sugar maple (Acer saccharum) and red spruce (Picea rubens) trees during the early, mid and late growing season.

Methods: At Hubbard Brook Experimental Forest, New Hampshire, we used two approaches to measure nitrogen uptake capacity by mature trees: an in situ depletion method using intact roots and an ex situ ¹⁵N tracer method using excised roots.

Results: NH₄ ⁺ uptake was greater than NO₃ ^? for both methods and tree species (P < 0.05). NH₄ ⁺ uptake was lowest during the early growing season, while NO₃ ^? uptake was lowest during the late growing season. Measured rates of NH₄ ⁺ uptake were 2–3 orders of magnitude greater using the in situ depletion method compared with the ex situ ¹⁵N tracer method.

Conclusions: These results demonstrate seasonal differences in nitrogen uptake by two dominant tree species in a northern forest and show that the method employed can significantly impact measured rates of uptake, which could have implications for understanding the magnitude of plant nitrogen uptake and for cross-study comparisons of this process.     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.     

Regulation of biosynthesis of vermiculin and vermistatin inPenicillium vermiculatum

Biosynthesis of vermiculin (1) and vermistatin (2) inPenicillium vermiculatum can be controlled by the carbon and nitrogen sources. Glucose and sucrose affect the levels of the two metabolites; cornsteep liquor influences the quality the biosynthesis. The concentrations of Fe³⁺ and Cu²⁺ ions also affect the biosynthesis, the effect being dependent on the type of carbon source utilized. The compounds capable of electron transport generally stimulate the production of1 and2 but do not influence the biosynthesis qualitatively. Translated by Č. Novotny     

Complementarity in the use of nitrogen forms in a temperate broad-leaved mixed forest

Background: The complementary use of different forms of soil nitrogen (N) might lead to a higher productivity of mixed forests than monocultures, but convincing evidence for temperate mixed forests is scarce.

Aims: We searched for species differences in N uptake rates and the preference for NH₄⁺, NO₃^? or glycine among five temperate broad?leaved tree species (Acer pseudoplatanus, Carpinus betulus, Fagus sylvatica, Fraxinus excelsior, Tilia cordata) in a mature mixed stand.

Methods: ¹⁵N tracer was added to the soil and its accumulation in fine root biomass was analysed after 10 min, 1 h and 1 d.

Results: The estimated root uptake rates of the species were in the range of 5–46 µg N g^?1 root h^?1 for NH₄⁺, 6–86 µg N g^?1 h^?1 for NO₃^? and 4–29 µg N g^?1 h^?1 for glycine during the first hour after tracer application. Carpinus, Tilia and Acer tended to prefer NH₄⁺ over NO₃^?, while Fraxinus showed equal preference for both N forms and Fagus seemed to prefer NO₃^?.

Conclusions: The five co-existing tree species differed in uptake rates and partly in their N form preference, but complementarity in the use of different N forms seems to be of minor importance in this forest because tree species appear to be rather flexible in their N form use.     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

Antifungal activity of a novel chromene dimer

The activity on Aspergillus spp. growth and on ochratoxin A production of two novel chromene dimers (3) was evaluated. The results of the bioassays indicate that the chromene dimer 3a inhibited mycelia growth by approximately 50% (EC₅₀) at 140.1 μmol L⁻¹ for A. niger, 384.2 μmol L⁻¹ for A. carbonarius, 69.1 μmol L⁻¹ for A. alliaceus and 559.1 μmol L⁻¹ for A. ochraceus. When applied at concentrations of 2 mmol L⁻¹, 3a totally inhibited the growth of all Aspergillus spp. tested. Furthermore, ochratoxin A production by A. alliaceus was reduced by about 94% with a 200 μmol L⁻¹ solution of this compound. A moderate inhibitory effect was observed for the analogous structure 3b on ochratoxin A production but not in mycelia growth. No inhibition was registered for compounds 2a and 2b, used as synthetic precursors of the dimeric species 3.     

The relative orientation of the fibronectin 6F11F2 module pair: A 15N NMR relaxation study

The structure of a pair of modules (⁶F1¹F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from ¹H-¹H NOE and ³ J _HHN data [Bocquier et al. (1999) Structure, 7, 1451–1460] and a weak module–module interface, comprising Leu19 and Leu28, in ⁶F1, and Tyr68 in ²F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of ¹⁵N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and ³ J _HHN data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where ¹⁵N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443–449].     

Ce4+-stimulated ion fluxes are responsible for apoptosis and taxol biosynthesis in suspension cultures of Taxus cells

Ion fluxes across the plasma membrane activated by 1 mM Ce⁴⁺, cell apoptosis and taxol biosynthesis in suspension cultures ofTaxus cuspidata were studied. The extracellular pH sharply decreased upon the addition of 1 mM Ce⁴⁺, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular Ca²⁺ concentration decreased within the first 3 h after the addition of Ce⁴⁺, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a Ca²⁺-channel blocker indicated that the dynamic changes in extracellular pH and the Ca²⁺ concentration resulted from the Ce⁴⁺-induced activation of H⁺ uptake and Ca²⁺ influx across the plasma membranevia ion channels. A pretreatment of the ion channel blocker initiated Ce⁴⁺-treated cells to undergo necrosis, and the prior addition of the Ca²⁺-channel blocker inhibited Ce⁴⁺-induced taxol biosynthesis and apoptosis. It is thus inferred that H⁺ uptake is necessary for cells to survive a Ce⁴⁺-caused acidic environment and is one of the mechanisms of Ce⁴-induced apoptosis. Furthermore, the Ca²⁺ influx across the plasma membrane mediated both the Ce⁴⁺-induced apoptosis and taxol biosynthesis.     

Biosynthesis of Resorcylic Acid Lactone (5S)-5-Hydroxylasiodiplodin in Lasiodiplodia theobromae

An administration study of ²H-labeled precursors showed that the 9-hydroxydecanoyl unit, the acyl intermediate of lasiodiplodin (1), was also the intermediate of (5S)-5-hydroxylasiodiplodin (2) in Lasiodiplodia theobromae. The incorporation of [O-methyl-²H₃]-lasiodiplodin (6) into 2 indicated that hydroxylation at C-5 occurred after cyclization.     

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL^pro) and a papain-like protease (PL^pro) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1–9) and four coumarins (10–13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL^pro and PL^pro) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL^pro and PL^pro inhibitory activity with IC₅₀ values of 11.4 and 1.2?µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL^pro, whereas noncompetitive inhibition was observed with the SARS-CoV PL^pro.     

Enhanced production of ansamitocin P-3 by addition of Mg2+ in fermentation of Actinosynnema pretiosum

The effect of divalent metal ions (i.e., Mn²⁺, Mg²⁺, Zn²⁺, Cu²⁺, and Co²⁺) on the production of anticancer ansamitocin P-3 (AP-3) by submerged cultures of Actinosynnemapretiosum in medium containing agro-industrial residues was investigated, and Mg²⁺ was found to be the most effective. Under the optimal condition of Mg²⁺ addition, the maximal AP-3 production titer reached 85 mg/L, which was 3.0-fold that of the control. The activities of methylmalonyl-CoA carboxyltransferase (MCT) and methylmalonyl-CoA mutase (MCM) were enhanced. The content of two precursors, malonyl-CoA and methylmalonyl-CoA, was lower than that of control. This work demonstrates that Mg²⁺ addition is a simple and effective strategy for increasing AP-3 production through the regulation of enzyme activity and pools of precursors. The information obtained can be helpful to its efficient production on large scale.     

Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ Oc mutation site of purG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein. Project supported by the National Natural Science Foundation of China.     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

Synthesis and Antimicrobial Evaluation of Novel Pyrazolones and Pyrazolone Nucleosides

The synthesis of a novel series of 4-arylhydrazono-5-methyl-1,2-dihydropyrazol-3-ones 4a–h, and their N ²-alkyl and acyclo, glucopyranosyl, and ribofuranosyl derivatives is described. K₂CO₃ catalyzed alkylation of 4a–h with allyl bromide, propargyl bromide, 4-bromobutyl acetate, 2-acetoxyethoxymethyl bromide, and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide proceeded selectively at the N ²-position of the pyrazolinone ring. Glycosylation of 4a with 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose under Vorbruggen glycosylation conditions gave the corresponding N ²-4-arylhydrazonopyrazolone ribofuranoside 9a in good yield. Conventional deprotection of the acetyl protected nucleosides furnished the corresponding 4-arylhydrazonopyrazolone nucleosides in good yields. Selected numbers of the newly synthesized compounds were screened for antimicrobial activity. Compounds 4b, 12a, and 14d showed moderate activities against Aspergillus flavus, Penicillium sp., and Escherichia coli.     

The characterization of a monovalent cation-selective channel of mammalian cardiac muscle sarcoplasmic reticulum

Summary Rabbit cardiac muscle sarcoplasmic reticulum (SR) was isolated and separated into ryanodine-sensitive and-insensitive fractions (L.R. Jones and S.E. Cala,J. Biol. Chem. 256:11809–11818, 1981). Vesicles of cardiac SR were incorporated into planar phospholipid bilayers by fusion and the channel activity of the membrane studied under voltage-clamp conditions (C. Miller,J. Membrane Biol. 40: 1–23, 1978). Both fractions contain a monovalent cation-selective three-state channel. In the presence of 75mm K₂SO₄, the fully open state () conductance of this channel is 157.2±30 pS and the sub-state () conductance is 100.7±21 pS. Both open states display the same selectivity sequence for monovalent cations, i.e. K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺ and may be blocked by the skeletal muscle relaxants decamethonium and hexamethonium. Block occurs when the compounds are added to either side of the membrane. The properties of the cardiac SR cation channel are compared with those of the previously reported monovalent cation-selective channels of mammalian and amphibian skeletal muscle SR.