Analysis of the relationship between the rise-time and the amplitude of single-fibre potentials in human muscles

Using the core-conductor theory, a single fibre action potential (SFAP) can be expressed as the convolution of a biolectrical source and a weight function. In the Dimitrov–Dimitrova (D–D) SFAP convolutional model, the first temporal derivative of the intracellular action potential (IAP) is used as the source. The present work evaluates the relationship between the SFAP peak-to-peak amplitude (V_pp) and peak-to-peak interval (rise-time, RT) at different fibre-to-electrode distances using simulated signals obtained by the D–D model as well as real recordings. With a single fibre electrode, we recorded 63 sets of consecutive SFAPs from the m. tibialis anterior of four normal subjects. The needle was intentionally moved whilst recording each SFAP set. We used the observed changes in RT and V_pp within each SFAP set as a point of reference with which to evaluate how closely the relationship between RT and V_pp provided by the D–D model reflects real data. We found that half of the recorded SFAP sets had rise-times higher than those generated by the D–D model. We also showed the influence of the IAP spatial length on the sensitivity of RT and V_pp with radial distance. The study reveals some inaccuracies in simulated SFAPs whose origin might be related to the assumptions made in the core-conductor theory.     

Effects of changes in the shape of the intracellular action potential on the peak-to-peak ratio of single muscle fibre potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet feasible, and consequently, knowledge about certain IAP characteristics of these IAPs is still limited. The ratio between the amplitudes of the second and first phases (the so-called peak-to-peak ratio, PPR) of a single fibre action potential (SFAP) is known to be closely related to the IAP profile. The PPR of experimentally recorded SFAPs has been found to be largely independent of changes in the fibre-to-electrode (radial) distance. The main goal of this paper is to analyze the effect of changes in different aspects of the IAP spike on the relationship between PPR and radial distance. Based on this analysis, we hypothesize about the characteristics of IAPs obtained experimentally. It was found that the sensitivity of the SFAP PPR to changes in radial distance is essentially governed by the duration of the IAP spike. Assuming that, for mammals, the duration of the IAP rising phase lies within the range 0.2-0.4 ms, we tentatively suggest that the duration of the IAP spike should be over approximately 0.75 ms, with the shape of the spike strongly asymmetric. These IAP characteristics broadly coincide with those observed in mammal IAPs.     

The morphology of single muscle fibre potentials – Part I: Simulation study of the distortion introduced by the distant-interfering potentials

Some morphologic aspects of human single fibre action potentials (SFAPs) are not sufficiently well-known. This uncertainty especially concerns the declining negative phase and the final positive phase (third phase) of SFAPs, as these parts are significantly affected by distant electrical activity. The incomplete characterisation of the SFAP shape is also explained by the limited knowledge of human intracellular action potentials (IAPs). The objectives of this study are to assess the morphologic features of human SFAPs and to derive information about the characteristics of human IAPs. To achieve this, the study has been divided into two parts. The present paper, Part I, aims to analyse the changes in the SFAP time-course introduced by distant-interfering potentials and to evaluate how these changes depend on the spike duration of the corresponding IAP. It was found that, for fibre-to-electrode distances shorter than about 0.2 mm, SFAPs generated by short-spike IAPs have a declining negative phase with a steep approximately constant slope that is largely unaffected by the potentials from distant fibres. For the same distances, SFAPs resulting from wide-spike IAPs have a declining negative phase with a slow return towards the baseline that is highly sensitive to distant-interfering potentials. The third phase of an SFAP is considerably distorted by distant potentials irrespective of the spike duration of the IAP.     

Identification procedure in a model of single fibre action potential – Part II: Global approach and experimental results

The present paper describes a global procedure for estimating all the synthesis parameters that generate a single fibre action potential (SFAP) in the Dimitrov–Dimitrova (D–D) convolutional model. We call this inverse problem Identification Procedure, and it is presented in two parts, this paper being the second. The procedure incorporates the candidate pair (CP) method developed in Part I, which provides the values of radial distance r and fibre diameter d of the simulated SFAP that best matches a potential under study. The CP-method required prior knowledge of all the excitation parameters. However, since the Identification Procedure makes no assumption about the excitation, multiple combinations of the synthesis parameters result in very similar SFAPs whose shape is close the signal under study. Analysis of the possible combinations reveals that r and d can be modelled as two jointly Gaussian random variables. The interest of the Identification Procedure is that, for a certain SFAP, it provides estimates of r and d, along with estimates of different parameters that determine the IAP waveform. Moreover, the procedure is able to determine the degree of error that accompanies the estimation of r and d.     

Identification Procedure in a model of single fibre action potential – Part I: Estimation of fibre diameter and radial distance

The Dimitrov–Dimitrova (D–D) model generates a single fibre action potential (SFAP) as the convolution of an excitation function and a filter impulse function. We propose a method to estimate the parameters involved in these functions from a SFAP waveform (inverse problem) and call it Identification Procedure. The Identification Procedure comprises two parts. The present paper, Part I, is centred on the estimation of the radial distance, r, and the fibre diameter, d, of a given SFAP. To this end we develop a technique which we call the candidate pair method (CP-method), and we test it on fibrillation potentials (FPs) as experimental data. We found that the D–D model cannot synthesize all the SFAP waveforms observed in the experimental recordings, but in the cases where it can, the CP-method then provides the values of r and d that are more likely to have synthesized the SFAP. Having a method that provides information about the fibre diameter straight from a SFAP waveform is very desirable as this parameter has clinical and physiological relevance. Moreover, the CP-method plays a major role in the Identification Procedure that is carried out in Part II.     

The morphology of single muscle fibre potentials – Part II: Experimental findings

Studies dealing with single fibre action potentials (SFAPs) have been more interested in obtaining quantitative data of certain parameters of the SFAP waveform than in the analysis of its morphologic features. The characterization of the SFAP morphology is highly valuable as it will allow to obtain information about in vivo intracellular action potentials (IAPs). However, the SFAP final portion is highly sensitive to distant electrical activity, as shown in Part I of this study. The present paper, Part II, is aimed at analysing the morphologies found in human SFAPs and deriving data of the associated IAPs. It was found that, for most SFAPs (97%), the declining negative phase starts decreasing steeply up to a certain point (slope-discontinuity point), from which it returns more slowly towards the baseline. This return may be further slowed down due to the contamination from distant potentials, but the slope-discontinuity point seems to be an intrinsic feature of human SFAPs. The third phase of SFAPs was, either absent (65%), or of rather small amplitude and prolonged duration. The slope-discontinuity point was apparent for the SFAPs of larger amplitude and vanished gradually as the SFAPs got smaller. Several lines of evidence strongly suggest that the spike duration of human IAPs should be about 1.0 ms.     

Stimulation of the lateral hypothalamus provokes the initiation of robust long-term potentiation of the thalamo-cortical input to the barrel field of the adult, freely moving rat

Long-term potentiation in the thalamo-cortical input to the somatosensory cortex barrel field has been reported to be inducible in vitro only during a narrow critical period of the first postnatal week. Here we explored whether this is due to inability of adult synapses to express LTP or lack of appropriate conditions for LTP induction in slice preparations. We recorded thalamo-cortical field potentials (FPs) from the barrel field of chronically prepared adult rats. In the first series, several parameters of conditioning tetanization of thalamus (T) have been tried. Statistically significant LTP of 135-150% relative to the baseline was observed only in rare cases (3/18) so that the mean changes were not statistically significant. In the second series, five trains of 100 Hz stimulation of T were paired with a "reinforcing" stimulation of the lateral hypothalamus (LH). In most cases (9/13) thalamo-cortical FPs were potentiated. The mean post-tetanic amplitude was 238 +/- 42% (+/- SEM) relative to the baseline (n = 13). The potentiation persisted for > > 1 hr and typically even further increased when tested 24-48 hr later. LTP magnitude strongly correlated with the initial paired-pulse ratio (PPR, coefficient of correlation r = 0.98) so that LTP magnitude was larger (333 +/- 107, n = 6) in cases with PPR > 1.3. The mean PPR tended to decrease after LTP (from 2.05 to 1.65). Altogether the results suggest that LTP is inducible in the thalamo-cortical input to the barrel field of normal adult rats. The dependence of LTP magnitude upon the initial PPR suggests that inputs with low initial release probability undergo larger LTP. Together with the tendency to a decrease in the PPR this suggests an involvement of presynaptic mechanisms in the maintenance of neocortical LTP.     

Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction

Recently, it has been demonstrated that mouse sperm contain a protein with properties similar to the inhibitory guanine nucleotide-binding regulatory protein, G_i (Kopf, G. S., Woolkalis, M. J., and Gerton, G. L. 1986. J. Biol. Chem., 261, 7327–7331). Since sperm-zona pellucida interaction represents a specialized form of intercellular communication and signal transduction we examined the role of the mouse sperm G_i-like protein in the zona pellucida-induced acrosome reaction using mechanically isolated, structurally intact zonae pellucidae. Sperm capacitated for 90 min in the presence of increasing concentrations of islet-activating protein (IAP) bind to the zona pellucida to a similar extent as control sperm incubated in the absence of this toxin. The zona pellucida-induced acrosome reaction, however, is inhibited in a concentration dependent manner by IAP, with half-maximal effects at 0.1-1.0 ng/ml IAP. IAP does not affect the ability of the sperm to become capacitated, but inhibits the cells from progressing into an intermediate stage prior to the completion of the acrosome reaction. When sperm are capacitated in the presence of 100 μM guanosine-5′-O-(3-thiotriphosphate) for 60 min prior to the addition of IAP during the final 30 min, the IAP-induced inhibition of the zona pellucida-induced acrosome reaction is abolished; capacitation in the presence of 100 μM guanosine-5′-O-(2-thiodiphosphate) does not abolish the inhibitory effects of IAP. The target of the IAP effect on intact sperm appears to be at the level of the G_i-like protein since IAP-catalyzed ³²P-ADP-ribosylation of the M_r = 41,000 substrate in detergent extracts of sperm is reduced when intact sperm are preincubated with IAP during capacitation. These data suggest that the mouse sperm G_i-like protein plays an intermediary role in the zona pellucida-induced acrosome reaction.     

Evaluation of the criteria to identify single-fibre potentials in human muscle fibres

The criterion normally used to identify a potential generated by a single muscle fibre (SFAP) is that it must have identical shape at consecutive discharges. Technical problems accompanying the recording of single-fibre electromyographic (SFEMG) potentials introduce certain variability in the shape of these potentials, thereby compromising the ability to detect pure SFAPs. This study aims to determine the conditions necessary for two fibres to generate a compound potential that fulfils the single-fibre criterion. This has been done by analysing the alterations in the waveform of compound spikes formed by the summation of two SFAPs whose relative weight in the composite potential can differ considerably. Several factors responsible for this shape variability, with a geometrical, physiological or accidental origin, have been included in our study. It has been shown that a distant interfering component will be hardly detected in the composite potential if it is smaller than approximately 15?% of the main component. For this interfering component to generate a notch in the rising phase of the compound potential, it must be greater than about 30?% of the main component. A compound potential will fulfil the single-fibre criterion if the time dispersion between the individual components is less than 80-120?μs. These results permit the estimation of the amplitude of interfering potentials so they could be useful in fibre density studies. The article also emphasises the inherent variability of SFEMG potentials and the impact of this variability on jitter estimation.     

Passive stiffness of hindlimb muscles in anurans with distinct locomotor specializations

Anurans (frogs and toads) have been shown to have relatively compliant skeletal muscles. Using a meta-analysis of published data we have found that muscle stiffness is negatively correlated with joint range of motion when examined across mammalian, anuran and bird species. Given this trend across a broad phylogenetic sample, we examined whether the relationship held true within anurans. We identified four species that differ in preferred locomotor mode and hence joint range of motion (Lithobates catesbeianus, Rhinella marina, Xenopus laevis and Kassina senegalensis) and hypothesized that smaller in vivo angles (more flexed) at the knee and ankle joint would be associated with more compliant extensor muscles. We measured passive muscle tension during cyclical stretching (20%) around L₀ (sarcomere lengths of 2.2 μm) in fiber bundles extracted from cruralis and plantaris muscles. We found no relationship between muscle stiffness and range of motion for either muscle–joint complex. There were no differences in the passive properties of the cruralis muscle among the four species, but the plantaris muscles of the Xenopus and Kassina were significantly stiffer than those of the other two species. Our results suggest that in anurans the stiffness of muscle fibers is a relatively minor contributor to stiffness at the level of joints and that variation in other anatomical properties including muscle–tendon architecture and joint mechanics as well as active control likely contribute more significantly to range of motion during locomotion.     

Design of near-infrared single-domain fluorescent protein GAF-FP based on bacterial phytochrome

Fluorescent proteins (FPs) are widely used as genetically encoded markers for quantitative and noninvasive study of biological processes. Development of biomarkers that are fluorescent in the near-infrared spectral range allows the tissues of animals to be studied at a deeper level because they are more permeable to the light of this wavelength range than that of visible range. Such properties as low molecular weight and monomeric state are important for widespread use of FPs. In this paper, we managed to obtain FP based on the chromophore-binding domain of bacterial phytochrome (BphP) from Rhodopseudomonas palustris (RpB-phP1), named GAF-FP, with a molecular weight of ~19 kDa, which is half that of other FP based on BphP and 1.4 times lower than that of commonly used GFP-like proteins, which are fluorescent in the near-infrared range. In contrast to most other near-infrared FPs, GAF-FP is a monomer, which has a high photostability, and its structure is stable to the incorporation of small peptide inserts. Moreover, GAF-FP is capable of covalent attachment of two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is contained in mammalian tissues. GAF-FP with attached BV as a chromophore (GAF-FP–BV) has the main absorption band with a maximum at 635 nm. The fluorescence maximum falls at 670 nm, whereby GAF-FP has a high ratio of the fluorescence signal to the background signal even if FP is localized at a depth of several mm below the tissue surface. Together with the near-infrared absorption band, GAF-FP–BV also has an absorption band in the violet region of the spectrum with a maximum at 378 nm. We used this property to design a chimeric protein consisting of modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We showed resonance energy transfer from the substrate, the excited state of which occurs when oxidized by luciferase, to the chromophore GAF-FP–BV in the designed fusion protein. In the absence of an energy acceptor, RLuc8 catalyzes the cleavage of the substrate with the emission of the light with a maximum at 400 nm. At the same time, the energy from the substrate is transferred to the FP chromophore and then emitted in the near-infrared range corresponding to the spectrum of GAF-FP fluorescence in the GAF-FP–RLuc8 chimeric protein. These results open the way for the development of new small near-infrared FPs based on various natural BphPs with a view to their widespread use in cell and molecular biology.     

Shedding Light on Microbial Predator–Prey Population Dynamics Using a Quantitative Bioluminescence Assay

This study assessed the dynamics of predation by Bdellovibrio bacteriovorus HD 100. Predation tests with two different bioluminescent strains of Escherichia coli, one expressing a heat-labile bacterial luciferase and the other a heat-stable form, showed near identical losses from both, indicating that protein expression and stability are not responsible for the “shutting-off” of the prey bioluminescence (BL). Furthermore, it was found that the loss in the prey BL was not proportional with the predator-to-prey ratio (PPR), with significantly greater losses seen as this value was increased. This suggests that other factors also play a role in lowering the prey BL. The loss in BL, however, was very consistent within nine independent experiments to the point that we were able to reliably estimate the predator numbers within only 1 h when present at a PPR of 6 or higher, Using a fluorescent prey, we found that premature lysis of the prey occurs at a significant level and was more prominent as the PPR ratio increased. Based upon the supernatant fluorescent signal, even a relatively low PPR of 10–20 led to approximately 5 % of the prey population being prematurely lysed within 1 h, while a PPR of 90 led to nearly 15 % lysis. Consequently, we developed a modified Lotka–Volterra predator–prey model that accounted for this lysis and is able to reliably estimate the prey and bdelloplast populations for a wide range of PPRs.     

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D₂O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.     

Development and validation of candidate gene-specific markers for the major fertility restorer genes, <Emphasis Type="Italic">Rf4</Emphasis> and <Emphasis Type="Italic">Rf3</Emphasis> in rice

Two major nuclear genes, Rf3 and Rf4, are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9-782-(M,I) and PPR762 (for Rf4) and SF21 (for Rf3), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9-782-(M,I), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer (n = 120) and maintainer lines (n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762, displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9-782-(M,I) is indeed the candidate gene for Rf4. With respect to Rf3, a codominant marker, named RMS-SF21-5 developed targeting SF21, displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4-specific markers. Validation of these markers in a F₂ mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3, together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.     

Vibrissal inputs into the motor cortex of adult and developing rats

Field potentials (FP) and responses of single neurones to electrical stimulation of vibrissal pads have been recorded in motor cortex in the albino mature and developing rats. The FPs were characterized by 3-phasic shape and high stability in mature rats. The FPs evoked by contralateral stimuli have a range of onset latency of 4 to 24 ms (peak of distribution 8-11 ms); those to ipsilateral stimuli have a latency of 4 to 23 ms (peak of distribution 12-16 ms). Responses of single neurones were evoked with a latency of 9 to 20 ms. Usually, the FPs were evoked by both contralateral and ipsilateral stimulation, and in some tracks were effective only ipsilateral stimuli in the developing rats beginning from the 11th day of life. The FPs in such animals were less stable and more fatigable. During 2-4 weeks of life, FPs evoked by contralateral stimulation appeared with a latency of 15 to 46 ms; during the same period, a latency of single unit responses ranged between 20 to 33 ms. The FPs to ipsilateral stimuli appeared with a latency of 18 to 47 ms, a latency of single unit responses of 27 to 47 ms. The results indicate functional immaturity of vibrissal system up to the end of the first month of rat life.     

Influence of intrapartum antibiotic prophylaxis against group B Streptococcus on the early newborn gut composition and evaluation of the anti-Streptococcus activity of Bifidobacterium strains

Several factors are known to influence the early colonization of the gut in newborns. Among them, the use of antibiotics on the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has scarcely been investigated, although this practice is routinely used in group B Streptococcus (GBS)-positive women. This work is therefore aimed at verifying whether IAP can influence the main microbial groups of the newborn gut microbiota at an early stage of microbial establishment. Fifty-two newborns were recruited: 26 born by mothers negative to GBS (control group) and 26 by mothers positive to GBS and subjected to IAP with ampicillin (IAP group). Selected microbial groups (Lactobacillus spp., Bidobacterium spp., Bacteroides fragilis, Clostridium difficile, and Escherichia coli) were quantified with real-time PCR on DNA extracted from newborn feces. Further analysis was performed within the Bidobacterium genus by using DGGE after amplification with genus-specific primers. Results obtained showed a significant decrease of the bifidobacteria counts after antibiotic treatment of the mother. Bifidobacteria were found to be affected by IAP not only quantitatively but also qualitatively. In fact, IAP determined a decrement in the frequency of Bidobacterium breve, Bidobacterium bifidum, and Bidobacterium dentium with respect to the control group. Moreover, this study has preliminarily evaluated that some bifidobacterial strains, previously selected for use in infants, have antibacterial properties against GBS and are therefore potential candidates for being applied as probiotics for the prevention of GBS infections.     

Characterization of antifungal metabolite phenazine from rice rhizosphere fluorescent pseudomonads (FPs) and their effect on sheath blight of rice

We have shown, the outcome of antifungal activity of phenazine derivatives which is produced by fluorescent pseudomonads (FPs) for the control of sheath blight of rice. A total of 50 fluorescent pseudomonads (FPs) were isolated from rice rhizosphere. Off which, 36 FPs exhibited antagonistic activity against Rhizoctonia solani, Macrophomina phaseolina, Fusarium oxysporum, Alternaria alternata and Sclerotium rolfsii up to 70–80% compared to control by dual culture method. BOX-PCR analyses of antagonistic isolates indicated that two phylogenetic group, where group I consisted of 28 isolates and eight isolates belongs to group II. Among 36 FPs, a total of 10 FPs revealed that the presence of phenazine derivatives on thin layer chromatography (TLC), which is coincided with that of authentic phenazine with R_f value 0.57. Similar to TLC analysis, antibiotic encoding gene phenazine-1-carboxamide (PCN) was detected in 10 FPs by PCR analysis with respective primer. Among, PCN detected isolates of FPs, a significant biocontrol potential possessing isolate designated as VSMKU1 and it was showed prominent antifungal activity against R. solani and other tested fungal pathogens. Hence, the isolate VSMKU1 was selected for further studies. The selected isolate VSMKU1 was identified as Pseudomonas aeruginosa by 16S rDNA sequence analysis. The antifungal metabolite phenazine like compound produced by VSMKU1 was confirmed by UV, FT-IR and HPLC analysis. The phenazine compound from VSMKU1 significantly arrest the growth of R. solani compared to carbendazim by well diffusion method. The detached leaf assay showed remarkable inhibition of lesion height 80 to 85% by the treatments of culture (VSMKU1), cell free culure filtrate and phenazine like compound compared to control and other treatments was observed in detached leaves of rice. These results emphasized that VSMKU1 isolate can be used as an alternative potential biocontrol agent against sheath blight of rice, instead of using commercial fungicide such as validamycin and carbendazim which cause environmental pollution and health hazards.     

Solid-Phase Synthesis and NMR Structural Studies of the Marine Antibacterial Cyclic Tetrapeptide: Cyclo[GSPE]

Twelve-membered head-to-tail cyclic tetrapeptides (CTPs) are rigid molecules found in nature and possess a diverse range of biological activities. A possible reason may be due to their ability to adopt rigid conformations in solution mimicking reverse-turns. Reverse-turns are common structural motifs which serve as molecular recognition sites in many protein-receptor interactions. In this paper, we describe the solid-phase synthesis of the antibacterial cyclic tetrapeptide cyclo[Gly-Ser-Pro-Glu] (cyclo[GSPE]), first isolated from the Ruegeria strain of marine bacteria by Mitova et al. (J Nat Prod 67:1178–1181, 2004). Our NMR experiments in H₂O:D₂O:DMSO (18:1:1) revealed that it possessed three conformations in an approximate ratio of 4:2:1 based on NMR amide peak intensities. 2D NMR studies and computer calculations revealed that the major conformer adopted a reverse-turn conformation and have ω torsion angles twisted by up to 2°, with two transoid amide bonds between Gly-Ser, Pro-Glu and two cisoid amide bonds between Ser-Pro, Glu-Gly in a cis–trans-cis–trans (ctct) pattern. This supports previous reports that majority of CTPs adopt a ctct pattern when dissolved in hydrogen-bond disrupting solvents (Che and Marshall in J Med Chem 49:111–124, 2006 and references cited therein). An ensemble of ten lowest-energy-minimised 3D structures generated using XPLOR-NIH software revealed that cyclo[GSPE] possessed a rigid backbone ring scaffold. The remaining two minor conformers were present in quantities too low for NMR structural studies.     

Ubiquitous distribution of fluorescent protein in muscles of four species and two subspecies of eel (genus <Emphasis Type="Italic">Anguilla</Emphasis>)

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4–100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490–496 and 527–530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.