The membrane systems of the cardiac muscle cell of Euchaeta norvegica Boeck (Crustacea,Copepoda)

Summary The membrane systems of the cardiac muscle cell of the copepod Euchaeta norvegica Boeck are described. The heart wall, which is between 0.12 and 1.36 m thick, consists of an epicardium and a single layer of muscle cells. Invaginations of the sarcolemma forming transverse tubules have been found at all levels of the sarcomere with the exception of the H-band level. The longitudinal tubules of the same system are closely associated with the sarcoplasmic reticulum to form interior couplings at the A-I level of the sarcomere. Triadic couplings at the Z band level were not seen in E. norvegica, but peripheral couplings were demonstrated. Nexuses were found in the intercalated discs.     

成年大鼠视皮层(17区)神经元间的染色耦合--离体脑片研究

在离体脑片上,用生物胞素（biocytin）对成年大鼠视皮层神经元进行胞内注射。在标记成功的49例中,21例（42．9％）有染色耦合现象,耦合细胞的个数从2个到5个不等,平均2．8±1．1个。其中,18例为锥体-锥体神经元之间的耦合,2例为锥体-非锥体神经元之间的耦合,1例为非锥体神经元之间的耦合。在耦合细胞中,只有5例的胞体紧靠在一起,其它细胞的胞体之间都有一定的距离,其中有1例达到了635μm。大多数情况下（19／21例）,耦合细胞的胞体位于同一层次内,只有2例胞体位于不同层次。在Ⅱ／Ⅲ、Ⅳ和Ⅴ层内都观察到染色耦合神经元,但以Ⅱ／Ⅲ层内发生染色耦合的比率为最高。比较了染色耦合与非耦合神经元的某些电生理特性,没有观察到在它们之间有明显的差别。     

Molecular Dynamics Study of Naturally Existing Cavity Couplings in Proteins

Couplings between protein sub-structures are a common property of protein dynamics. Some of these couplings are especially interesting since they relate to function and its regulation. In this article we have studied the case of cavity couplings because cavities can host functional sites, allosteric sites, and are the locus of interactions with the cell milieu. We have divided this problem into two parts. In the first part, we have explored the presence of cavity couplings in the natural dynamics of 75 proteins, using 20 ns molecular dynamics simulations. For each of these proteins, we have obtained two trajectories around their native state. After applying a stringent filtering procedure, we found significant cavity correlations in 60% of the proteins. We analyze and discuss the structure origins of these correlations, including neighbourhood, cavity distance, etc. In the second part of our study, we have used longer simulations (≥100ns) from the MoDEL project, to obtain a broader view of cavity couplings, particularly about their dependence on time. Using moving window computations we explored the fluctuations of cavity couplings along time, finding that these couplings could fluctuate substantially during the trajectory, reaching in several cases correlations above 0.25/0.5. In summary, we describe the structural origin and the variations with time of cavity couplings. We complete our work with a brief discussion of the biological implications of these results.     

Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.     

Study of phospholipid structure by 1H, 13C, and 31P dipolar couplings from two-dimensional NMR.

Various motionally averaged 31P-1H, 13C-1H, 1H-1H, and 31P-13C dipolar couplings were measured for natural-abundance and unoriented phosphocholine in the L alpha phase. The couplings were obtained and assigned by a variety of advanced and partly novel two-dimensional solid-state NMR experiments. Whereas 31P-1H and 31P-13C dipolar couplings provide long-range structural constraints, geminal 1H-1H couplings and the signs of 13C-1H couplings are important new elements in a segmental order-tensor analysis of the lipid headgroup and glycerol backbone. The implications of these measured dipolar couplings for the conformational exchange of the lipid headgroup and the bending of the headgroup from the glycerol backbone are discussed. These dipolar couplings are also analyzed semiquantitatively in terms of the segmental order tensor.     

Synthesis of new, base-modified PNA monomers

A number of N-Boc-protected peptide nucleic acids (PNA) monomers containing 5-aryl- and 5-alkynyl-uracil bases have been synthesized using different palladium-catalyzed cross-coupling reactions. Starting from the base-unprotected 5-iodo-uracil PNA monomer, only the Stille couplings were accomplished successfully, while Suzuki couplings with boronic acids containing the same aryl groups failed. During Sonogashira couplings with terminal alkynes, significant amounts of unrequired furano[2,3-d]pyrimidine by-products were formed. Protection of the lactam function by p-methoxybenzylation prevented the opportunity for intramolecular cyclization as well as formation of a negative charge on the 4-O atom, making it possible to reach almost quantitative yields at Sonogashira couplings and acceptable conversions in Suzuki reactions.     

113CD-1H spin-spin couplings in homonuclear 1H correlated spectroscopy of metallothionein. Identification of the cysteine 1H spin systems

Heteronuclear spin-spin couplings between 113Cd and C beta protons of the metal-bound cysteines were observed in phase-sensitive, double-quantum filtered, homonuclear two-dimensional correlated (COSY) 1H NMR spectra of 113Cd-metallothionein-2 from rabbit liver. Comparison of 113Cd- and 112Cd-metallothionein-2 spectra revealed that 19 1H spin systems show heteronuclear couplings to at least one 113Cd and were thus identified as 19 of the 20 cysteines in this protein. From a detailed analysis of the manifestations of heteronuclear couplings in the homonuclear 1H COSY spectra, two cysteines could be identified as 'bridging cysteines', with spin-spin couplings to two different 113Cd nuclei. The observed 113Cd-1H coupling constants vary between less than or equal to 5 Hz and 80 Hz.     

Structural dependencies of protein backbone 2JNC' couplings

Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated (2)J(NC') couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H-bond strengths derived from (h3)J(NC') couplings, provides useful insight into the overall energy profile of the protein backbone in solution.     

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (¹D_CH) and long-range H-H residual dipolar couplings (ⁿD_HH) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the ⁿD_HH in natural abundance based on a ¹³C resolved ¹H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the ¹D_CH and the ⁿD_HH residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 16 glycosidic linkages.     

Development of the sarcotubular system in fusion-arrested myoblasts.

We have compared the effect of two different procedures, equally effective in preventing muscle cell fusion in culture, on the development of the sarcotubular system in rat muscle cells. Whereas in myoblasts grown in low Ca++ medium the T system was poorly developed and diadic or triadic couplings between T tubules and sarcoplasmic reticulum were rare, in cytochalasin B-treated myoblasts the development of the sarcotubular system was comparable to that seen in myotubes of the same age. We conclude that (a) muscle cell fusion is not essential for the development of the sarcotubular system, and (b) procedures used to prevent cell fusion in vitro may affect directly muscle cell differentiation by a process independent of the fusion block.     

Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.     

Solvent-induced differentiation of protein backbone hydrogen bonds in calmodulin

In apo and holoCaM, almost half of the hydrogen bonds (H-bonds) at the protein backbone expected from the corresponding NMR or X-ray structures were not detected by h3JNC' couplings. The paucity of the h3JNC' couplings was considered in terms of dynamic features of these structures. We examined a set of seven proteins and found that protein-backbone H-bonds form two groups according to the h3JNC' couplings measured in solution. H-bonds that have h3JNC' couplings above the threshold of 0.2 Hz show distance/angle correlation among the H-bond geometrical parameters, and appear to be supported by the backbone dynamics in solution. The other H-bonds have no such correlation; they populate the water-exposed and flexible regions of proteins, including many of the CaM helices. The observed differentiation in a dynamical behavior of backbone H-bonds in apo and holoCaM appears to be related to protein functions.     

Nonlinear coupling is absent in acute myocardial patients but not healthy subjects

We investigated whether autonomic nervous system imbalance imposed by pharmacological blockades and associated with acute myocardial infarction (AMI) is manifested as modifications of the nonlinear interactions in heart rate variability signal using a statistically based bispectrum method. The statistically based bispectrum method is an ideal approach for identifying nonlinear couplings in a system and overcomes the previous limitation of determining in an ad hoc way the presence of such interactions. Using the improved bispectrum method, we found significant nonlinear interactions in healthy young subjects, which were abolished by the administration of atropine but were still present after propranolol administration. The complete decoupling of nonlinear interactions was obtained with double pharmacological blockades. Nonlinear couplings were found to be the strongest for healthy young subjects followed by degradation with old age and a complete absence of such couplings for the old age-matched AMI subjects. Our results suggest that the presence of nonlinear couplings is largely driven by the parasympathetic nervous system regulation and that the often-reported autonomic nervous system imbalance seen in AMI subjects is manifested as the absence of nonlinear interactions between the sympathetic and parasympathetic nervous regulations.     

Novel PIKK inhibitor antibody-drug conjugates: Synthesis and anti-tumor activity

Novel neolymphostin-based antibody-drug conjugate (ADC) precursors were synthesized either through amide couplings between both cleavable and non-cleavable linkers and neolymphostin derivatives, or through Cu(I)-catalyzed acetylene-azide click cycloadditon between non-cleavable linkers and neolymphostin acetal derivatives. These precursors were site-specifically conjugated to cysteine mutant trastuzumab-A114C to provide neolymphostin-based ADCs. Preliminary in vitro data indicated that the corresponding ADCs were active against HER2-expressing tumor cell lines, thus providing a proof-of-concept for using neolymphostin as ADC-based anticancer agents.     

Short-range, long-range and transition state interactions in the denatured state of ACBP from residual dipolar couplings

Residual dipolar couplings in the denatured state of bovine acyl-coenzyme A binding protein (ACBP) oriented in strained polyacrylamide gels have been shown to be a sensitive, sequence-specific probe for residual secondary structure. Results supporting this were obtained by comparing residual dipolar couplings under different denaturing conditions. The data were analyzed using the program molecular fragment replacement (MFR), which demonstrated alpha-helix propensity in four isolated stretches along the protein backbone, and these coincide with the location of native helices. This is in full agreement with earlier findings based on secondary chemical shift values. Furthermore, N-H residual dipolar couplings provided direct evidence for the existence of native-like hydrophobic interactions in the acid-denatured state of ACBP at pH 2.3. It was shown that replacement of the hydrophobic side-chain of residue Ile27 with alanine in helix A2 leads to large decreases of residual dipolar couplings in residues that form helix A4 in the native state. It is suggested that the Ile to Ala mutation changes the probability for the formation of long-range interactions, which are present in the acid-denatured state of the wild-type protein. These long-range interactions are similar to those proposed to form in the transition state of folding of ACBP. Therefore, the application of residual dipolar couplings in combination with a comparative mutation study has demonstrated the presence of precursors to the folding transition state under acid-unfolding conditions.     

Structure and disorder in the ribonuclease S-peptide probed by NMR residual dipolar couplings

NMR residual dipolar couplings for the S-peptide of ribonuclease A aligned in C8E5/n-octanol liquid crystals are consistent with the presence of a native-like alpha-helix structure undergoing dynamic fraying. Residues 3-13, which correspond to the first alpha-helix of ribonuclease A, show couplings that become more negative at low temperature and in the presence of salt, conditions which stabilize alpha-helical structure in the S-peptide. By contrast, dipolar couplings from the N and C termini of the peptide are close to zero and remain nearly invariant with changes in solution conditions. Torsion angle dynamics simulations using a gradient of dihedral restraint bounds that increase from the center to the ends of the peptide reproduce the experimentally observed sequence dependence of dipolar couplings. The magnitudes of residual dipolar couplings depend on the anisotropy of the solute. Native proteins often achieve nearly spherical shapes due to the hydrophobic effect. Embryonic partially folded structures such as the S-peptide alpha-helix have an intrinsically greater potential for anisotropy that can result in sizable residual dipolar couplings in the absence of long-range structure.     

A new method for the simultaneous measurement of magnitude and sign of 1DCH and 1DHH dipolar couplings in methylene groups

Heteronuclear dipolar couplings of the protein backbone have proven to have a big impact on the accuracy of protein NMR structures. H,H dipolar couplings might have the same impact on side chains. Here we present a method that combines both heteronuclear and homonuclear dipolar couplings to investigate the local conformation of methylene groups. A new pulse sequence (SPITZE-HSQC) is presented, that allows to measure the two C,H and the H,H dipolar couplings at the same time, using spin state selective transfers. The new method has been applied to the methylene groups of glycines in the protein ubiquitin. The C,H and the H,H dipolar couplings might have a key role in fast stereospecific assignment of protons in CH₂ groups.     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods.

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-Cl method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.     

Correlation of the sweetness of variants of the protein brazzein with patterns of hydrogen bonds detected by NMR spectroscopy

In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (trans-hydrogen bond couplings). We have applied this approach to screen a series of five single site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long range, three-dimensional data correlating connectivities among backbone 1HN, 15N, and 13C' atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three "sweet" variants exhibited the same pattern of hydrogen bonds, whereas all three "non-sweet" variants lacked one hydrogen bond at the middle of the alpha-helix, where it is kinked, and one hydrogen bond in the middle of beta-strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.