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Individual nuclear spins in diamond can be optically detected through hyperfine couplings with the electron spin of a single nitrogen-vacancy (NV) center; such nuclear spins have outstandingly long coherence times. Among the hyperfine couplings in the NV center, the nearest neighbor 13C nuclear spins have the largest coupling strength. Nearest neighbor 13C nuclear spins have the potential to perform fastest gate operations, providing highest fidelity in quantum computing. Herein, we report on the control of coherences in the NV center where all three nearest neighbor carbons are of the 13C isotope. Coherence among the three and four qubits are generated and analyzed at room temperature. [ABSTRACT FROM AUTHOR]

Expression of the 37-kDa laminin binding protein (37LBP/p40), a precursor of the 67-kDa laminin receptor, is well-correlated with the biological aggressiveness of cancer cells. To elucidate the direct role played by 37LBP/p40 in cancer cells, a murine lung cancer cell line T11, the 37LBP/p40 expression of which was remarkably diminished, was established by the introduction of the antisense 37LBP/p40-RNA using a retroviral vector. As a result, the population doubling time of T11 was prolonged (60 h) compared with that of P29, the non-transfected parental cell line (42 h), and TN2, a transfectant with vehicle only (40 h). In-vitro studies also showed that T11 cells adhered to immobilized laminin less firmly than P29 cells did. When 5 × 105 cells were subcutaneously inoculated into syngenic mice, the mean survival time of T11-recipients (77.0 ± 14.8 days) was also significantly prolonged compared with that for P29 (34.8 ± 5.5 days) and TN2 (36.7 ± 6.1 days) recipients (P < 0.001). The electron-microscopic view of the tumour tissue revealed that T11 cells were loosely apposed and their intercellular space was markedly widened. Some of the T11 cells sporadically degenerated with the infiltration of lymphocytes and neutrophils. These results suggest that the suppressed expression of 37LBP/p40 reduces the capability of lung cancer cell proliferation in vitro and tumour formation in vivo. © 1999 Cancer Research Campaign [ABSTRACT FROM AUTHOR]

This corrects the article DOI: 10.1038/ncomms4524.

In this Letter, we propose a counterintuitive use of a hybrid system where the coherence time of a quantum system can be significantly improved by coupling it with a system of a shorter coherence time. Coupling a two-level system with a single nitrogen-vacancy (NV^{-}) center, a dark state of the NV^{-} center naturally forms after the hybridization. We show that this dark state becomes robust against noise due to the coupling even when the coherence time of the two-level system is much shorter than that of the NV^{-} center. Our proposal opens a new way to use a quantum hybrid system for the realization of robust quantum information processing.

The hybridization of distinct quantum systems has opened new avenues to exploit the best properties of these individual systems. Superconducting circuits and electron spin ensembles are one such example. Strong coupling and the coherent transfer and storage of quantum information has been achieved with nitrogen vacancy centres in diamond. Recently, we have observed a remarkably sharp resonance (~1 MHz) at 2.878 GHz in the spectrum of flux qubit negatively charged nitrogen vacancy diamond hybrid quantum system under zero external magnetic field. This width is much narrower than that of both the flux qubit and spin ensemble. Here we show that this resonance is evidence of a collective dark state in the ensemble, which is coherently driven by the superposition of clockwise and counter-clockwise macroscopic persistent supercurrents flowing in the flux qubit. The collective dark state is a unique physical system and could provide a long-lived quantum memory.

We have built a hybrid system composed of a superconducting flux qubit (the processor) and an ensemble of nitrogen-vacancy centers in diamond (the memory) that can be directly coupled to one another, and demonstrated how information can be transferred from the flux qubit to the memory, stored, and subsequently retrieved. We have established the coherence properties of the memory and succeeded in creating an entangled state between the processor and memory, demonstrating how the entangled state's coherence is preserved. Our results are a significant step towards using an electron spin ensemble as a quantum memory for superconducting qubits.

Prior to the emergence of life, it is believed that only l-amino acids were selected for formation of protein and that d-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, the occurrence of proteins containing d-beta-aspartyl (Asp) residues in various tissues from elderly subjects has been reported recently. Here, we demonstrate the presence of a d-beta-Asp-containing protein in the cardiac muscle of heart, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscle of the stomach, small intestine and large intestine. Since the d-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. Formation of the d-beta-Asp residue in proteins may be related to stress.

Soft and turbid collagen gel disks were previously converted into strong and transparent gel membranes utilizing a concept for the vitrification of heat-denatured of proteins. This novel stable and transparent gel has been termed 'vitrigel'. By encompassing the collagen vitrigel membrane in a nylon frame, it can be easily handled with tweezers, and functions as an excellent scaffold for three-dimensional cell culture models, as cells can be cultured on both sides. Here, we investigated the molecular permeability of the collagen vitrigel membrane in a time course-dependent manner using glucose and serum proteins. Glucose penetrated through the collagen vitrigel membrane to the opposite side, and concentrations on each side were found to be equilibrated within 24 h. Serum proteins up to a molecular weight >100 kDa also gradually passed through the collagen vitrigel membrane. In addition, human microvascular endothelial cells (HMVECs) were cultured on one surface of the collagen vitrigel membrane with a nylon frame, and human dermal fibroblasts (HDFs) or HT-29 (a human colon carcinoma cell line) cells were cocultured on the opposite surface. Histomorphological observations revealed the formation of three-dimensional crosstalk models composed of HMVECs and HDFs or HMVECs and HT-29 cells. Resulting data suggest that the protein-permeable scaffold composed of the collagen vitrigel membrane is useful for the reconstruction and/or modeling of 'crosstalk' between two different cells types. Hereafter, such crosstalk models in vitro could be applied to research not only of paracrine factors, but also to epithelial- or endothelial-mesenchymal transitions., (2007 S. Karger AG, Basel)

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.

Although proteins are generally composed of l-alpha-amino acids, biologically uncommon D-beta-aspartic acid (Asp)-containing proteins have been reported in various tissues from elderly individuals. Our previous study indicated that the N/N1003A cell line, derived from rabbit lens, includes D-beta-Asp-containing proteins of approximately 50 kDa by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-beta-Asp-containing proteins. In this study, we identified the D-beta-Asp-containing proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. The results indicate that one of these 50 kDa proteins is an enolase showing homology with tau-crystallin. Other D-beta-Asp-containing proteins, which we have recently discovered include lamin A/C, cytoplasmic NADP+-dependent isocitrate dehydrogenase, fructose-bisphosphate aldolase A, aldose reductase, L-lactate dehydrogenase A or calponin H2, phosphoglycerate mutase 1, phosphatidylethanolamine-binding protein, alpha-B-crystallin, and peptidyl-prolyl cis-trans isomerase A (PPlase).

Three-dimensional reconstructed organoids in vitro are valuable for not only regenerative medicine but also drug development. However, the manipulation of conventional three-dimensional cultures is not simple. We describe a nylon membrane ring-embedded or a pressed silk sheet-embedded scaffold made of collagen "vitrigel" that can facilitate three-dimensional cultures for reconstructing an epithelial-mesenchymal model or a hard connective tissue model, respectively. Here we define vitrigel as a gel in a stable state produced by rehydration after the vitrification of a traditional hydrogel. The collagen vitrigel was successfully prepared in three steps involving a gelation process in which a cold and clear neutral salt solution containing type I collagen formed an opaque and soft gel by incubation at 37 degrees C, a vitrification process in which the gel becomes a rigid material like glass after sufficient drying out, and finally a rehydration process to convert the vitrified material into a thin and transparent gel membrane with enhanced gel strength. The framework-embedded collagen vitrigel scaffold that can be easily reversed by forceps was prepared by inserting a nylon ring or a silk sheet in the collagen solution prior to the gelation. The scaffold enabled culturing anchorage-dependent cells on both surfaces of the collagen vitrigel by the manipulation of two-dimensional cultures and consequently resulted in reconstructing a three-dimensional organoid. An intestinal epithelial-mesenchymal model was reconstructed by coculturing fibroblasts on the opposite side of monolayered Caco-2 cells on the nylon ring-embedded collagen vitrigel. Also, fibroblasts seeded on both surfaces of the silk sheet-embedded collagen vitrigel proliferated well and formed multilayers and some cells invaded into the vitrigel framed by the network of numerous strong silk filaments, suggesting a reconstruction of a hard connective tissue model. These data demonstrate that the collagen vitrigel is a valuable scaffold for tissue engineering.

Purpose: Although the presence of biologically uncommon D-beta-aspartate (D-beta-Asp) in lens protein is thought to be related to aging, we recently found this isomer in lens alphaA-crystallin from human newborns. The objective of this study was to examine whether D-beta-Asp occurs in protein from lens-derived cell lines., Methods: We examined the expression of D-beta-Asp-containing protein in the lens-derived cell lines alphaTN4-1 and N/N1003A, by western blot and immunoprecipitation analysis using a polyclonal antibody against Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr (peptide 3R), which corresponds to three repeats of positions 149-153 in human alphaA-crystallin. The anti-peptide 3R antibody, prepared in a previous study, is a useful tool for investigating D-beta-Asp-containing peptides., Results: Western immunoblot and immunoprecipitation analysis showed that a 50 kDa protein in N/N1003A cells was strongly immunoreactive with the anti-peptide 3R antibody. Antibodies against alphaA- and alphaB-crystallin also stained this protein. On the other hand, the alphaTN4-1 cell line only expressed proteins of about 20 kDa, which also reacted to antibodies against alphaA-crystallin and alphaB-crystallin., Conclusions: The results indicate that the N/N1003A cell line expressed a 50 kDa D-beta-Asp-containing protein, which may share a common amino acid sequence with alphaA- and alphaB-crystallin.

Biologically uncommon d-aspartyl residues have been reported in proteins of various elderly tissues. We prepared a polyclonal antibody against d-beta-Asp-containing peptide and examined its immunoreactivity in the skin. The antibody recognized integrated or disintegrated elastic fibers in the sun-exposed skin but not in the sun-protected skin of the elderly donors. Western blot analysis of the proteins isolated from sun-damaged skin demonstrated that the 50 kDa protein was immunoreactive with both antibodies for d-beta-Asp-containing peptide and elastin. Ultraviolet (UV) irradiation on normal skin caused the appearance of d-beta-Asp-containing peptide-immunoreactive fibers in the dermis. These results suggest that UV irradiation is closely related to the formation of d-beta-Asp in the elastic fibers of skin. We propose that the antibody could be a useful indicator for sun damage of the skin.

Purpose: We have found that two aspartyl (Asp-151 and Asp-58) residues of alphaA-crystallin are inverted and isomerized to the biologically uncommon D-beta-Asp residues during aging. In order to elucidate the correlation between the formation of the D-beta-Asp isomer and the environment surrounding the Asp in the protein, we performed a Raman spectroscopic study using two synthetic peptides: T6 peptide containing Asp-58, and T18 peptide, containing Asp-151, which correspond to the tryptic peptides of human alphaA-crystallin., Methods: Both T6 (Thr-Val-Leu-Asp(58)-Ser-Gly-Ile-Ser-Glu-Val-Arg) and T18 (Ile-Gln-Thr-Gly-Leu-Asp(151)-ala-thr-his-ala-Glu-Arg) peptides were synthesized with four optical isomers which have L-alpha-, D-alpha, L-beta and D-beta-aspartyl residues. These peptides were subjected to Raman measurement., Results: The Raman spectrum of the L-alpha-Asp T18 peptide measured as dry powder revealed that the secondary structure of this peptide is mainly anti-parallel beta-sheet. The main structure of the D-beta-Asp T18 peptide when in dry powder form was altered to an alpha-helix and/or random structure. The main structure of L-alpha-Asp T18 peptide when measured in aqueous solution also converted to an alpha-helix and/or random structure. The conversion of L-alpha-to D-beta-Asp in T6 peptides when in dry powder form revealed no alteration of secondary beta-sheeted structure., Conclusion: Raman spectroscopy clearly revealed a large conformational change in the secondary structure of T18 peptide caused by substitution of normal L-alpha-Asp to biologically uncommon Asp-isomers. This result indicates that the inversion of an amino acid in a protein greatly affects the secondary structure of the protein.

Purpose: Previous studies demonstrated that the Asp-151 residue of alphaA-crystallin from human eye lens is stereoinverted to the biologically uncommon D-isomer and isomerized to the beta-aspartyl residue (isoaspartate) with age. To detect the locality of the D-beta-Asp-containing peptide in aged human lens, we prepared a highly specific antibody against peptide Gly-Leu-D-beta-Asp-Ala-Thr which corresponds to positions 149-153 of human alphaA-crystallin using peptide Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta- Asp-Ala-Thr (designated peptide 3R) as an immunogen., Methods: Peptide 3R was synthesized with F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry and then the peptide was immunized in rabbits to generate antibody against peptide 3R. The antibody in rabbit serum was purified by affinity chromatography using peptide 3R and bovine alphaA-crystallin as ligands. The specificity and titer of antibody were checked by ELISA assay. We synthesized four kinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acid sequences 146-157 in human alphaA-crystallin) in which Asp-151 residues were normal L-alpha-Asp, abnormal D-alpha-Asp, L-beta-Asp, and D-beta-Asp, respectively. The specificity of antibody was confirmed by ELISA using these peptides and utilized in immunohistochemistry., Results: The antibody we prepared crossreacted specifically to D-beta-Asp-151-containing alphaA-crystallin. Immunohistochemical staining of human lens with the antibody demonstrated that D-beta-Asp-151-containing alphaA-crystallin was predominantly localized in the core of aged human lens., Conclusions: The peptide 3R antibody clearly recognized the presence of racemized and isomerized Asp-151 in both protein solution and lens tissue obtained from aged human lens.

The laminin binding protein of 37 kDa (37LBP) is regarded as a precursor protein of the high-affinity 67-kDa laminin receptor (67LR). Expression of 67LR/37LBP is well correlated with biological aggressiveness of cancer, particularly with invasive and metastatic potential. To investigate in detail the role of 37LBP in cancer cells, we synthesized recombinant 37LBP (r37LBP) as a fusion protein and generated an IgG-type polyclonal antibody P4G against r37LBP. Western blot analysis with P4G showed a single band of 67LR under both nonreducing and reducing conditions using cell extract of human fibrosarcoma cells HT1080. It was shown that P4G inhibited cell attachment to immobilized laminin in a dose-dependent manner. Further, the intravenous injection of HT1080 cells pretreated with P4G, compared with that of cells pretreated with normal rabbit serum, resulted in a reduced number of experimental metastases (3.3+/-5.1 vs. 58.0+/-38.0 nodules per mouse, respectively) (P<0.005). These results suggest that P4G inhibits the colonization and growth of HT1080 cells in the lungs of mice, and that the blocking of r37LBP with the specific antibody P4G may offer a potential strategy for preventing cancer metastasis.

A heparin-binding peptide was isolated from a proteolytic hydrolysate of bovine lactoferrin by affinity chromatography using an immobilized heparin column. Analysis of amino acid sequences at the N-terminus showed that this heparin-binding peptide is derived from the region beginning at the 17th amino acid residue of the bovine lactoferrin sequence. The molecular mass of this peptide was 3195.5 as measured by matrix-assisted laser desorption-time of flight mass spectrometry. This peptide is the same as the bactericidal peptide lactoferricin B. In an aqueous environment, this peptide displays mainly a beta-sheet structure and an unordered structure as assessed by measurements of circular dichroism spectra. When this peptide was mixed with heparin, a distinct spectral change was induced because of conformational alteration of the peptide. This spectral change was reversible. Analysis of data from peptide synthesis indicated that binding by the sequence Arg28-Met29-Lys30-Lys31 of bovine lactoferrin is significant and that there is a synergistic contribution from Lys18-Cys19-Arg20-Arg21, and Arg38-Arg39.

We investigated the expression and distribution of laminin in Lewis lung carcinoma LL2-Lu3 cells. The microscopic immunofluorescence study of the non-permeabilized cells and blotting assay after immunoprecipitation with anti-laminin antibodies of biotinylated cell surface proteins demonstrated that LL2-Lu3 cells retained laminin on their cell surfaces. This laminin was atypical in that it lacked A chain as revealed by the immunoblot analysis. The results of the reverse transcription polymerase chain reaction method indicated that LL2-Lu3 cells contained mRNA for B1 and B2 chains, but not A chain corresponding to those of typical laminin derived from murine Engelbreth-Holm-Swarm sarcoma. A precursor form of 67 kDa laminin receptor protein was also shown to exist on the surfaces of LL2-Lu3 cells. These findings suggest that the interaction between atypical laminin and the precursor form of the 67 kDa laminin receptor protein on the cell surfaces may function in regulating cell activities such as metastasis of LL2-Lu3 cells.

Some members of the integrin family recognize the RGD sequence which is common to cell adhesive proteins in a divalent cation-dependent manner. In the presence of Ca2+ and Mg2+, the fibronectin receptor of placenta recognizes the RGD sequence of fibronectin, but not that of vitronectin, while the vitronectin receptor of placenta recognizes the RGD sequence of vitronectin, but not that of fibronectin, although both receptors recognize the same RGD sequence. We have found by performing an enzyme-linked immunosorbent assay (ELISA) using receptor-specific monoclonal antibodies and by electrophoretic analysis that in the presence of Mn2+ a vitronectin receptor of placenta binds to an affinity column coupled with the cell-binding domain of fibronectin. By replacing divalent cations from Mn2+ to Ca2+ and Mg2+, the vitronectin receptor was completely eluted from the column. When the synthetic peptides GRGDSP and GRGESP were applied to the column as competitors, the Mn(2+)-dependent binding was inhibited by both peptides. These results suggest that Mn2+ elicits a binding activity of the placenta vitronectin receptor to the RGD site of fibronectin. The modulation of ligand specificity by Mn2+ will provide an important clue in the elucidation of the cause of individual ligand specificity of RGD-recognizing integrins.

Vitronectin is a 75 kilodalton (kDa) cell-adhesive glycoprotein found in animal blood and connective tissue, also termed serum spreading factor, S-protein, and epibolin. It promotes attachment and spreading of animal cells on tissue culture dishes, and it also binds to collagen. We established four mouse hybridoma lines producing monoclonal antibodies (M1, M2, M4 and M5) to human vitronectin. By immunoblotting, both epitopes recognized by M4 and M5 were suggested to exist in the amino terminal 5 kDa portion of vitronectin, and both M1 and M2 bound to the adjacent 35 kDa portion. Cell spreading on vitronectin-coated dishes was inhibited by M4 = M5 greater than M1, but not by M2. Collagen binding to vitronectin was inhibited by M2 greater than M4 = M5, but not by M1. These results indicate that the collagen-binding site is located near the cell-binding site in the amino terminal half of vitronectin. Independent inhibition of vitronectin binding to the cell and to collagen by these monoclonal antibodies will provide a potential tool to dissect the structure and function of vitronectin.

In order to establish an assay for the in vitro growth of myogenic cells, we investigated whether creatine kinase (CK) activity can be used as an index for the estimation of growth. The content of CK activity in a primary myogenic cell culture, almost all of which originated from myotubes, was shown to be linearly proportional to the content of proteins in the myotubes and increased concomitantly with myotube growth. CK activity can be easily and accurately assayed and used for routine assay of the cell growth. As an example of the application of CK activity for the bioassay of growth promoting substances, the relative potency (Pr) of turkey transferrin (Tf) to chick Tf in the promotion of chick myogenic cells was examined with a parallel line assay. The calculation limited that Pr = 0.363, ranging from 0.356 to 0.370, at 95% safety. The results demonstrated the usefulness of CK activity for estimating cell growth, as well as the value of a statistical method for the assay of a growth factor in vitro.

This report presents evidence suggesting the direct binding between tubulin and myosin: (1) coprecipitation of tubulin with myosin occurred at a low ionic strength at which no precipitation of tubulin by itself occurred; (2) the amount of tubulin coprecipitated was unchanged when the coprecipitate was washed thoroughly; (3) about 2 mol of tubulin dimer could bind per mol of myosin at the maximum under our experimental conditions. The binding of about 1 mol of tubulin dimer was influenced by the presence of F-actin, but that of the other 1 mol of tubulin dimer was uninfluenced. In the former binding, tubulin or actin which bound first to myosin was suggested to have a priority. With regard to the priority of the binding, a similar result was obtained from the experiments of tubulin interference in actin activation of myosin Mg2+-ATPase. The tubulin-myosin binding occurred moderately even at 0 degrees C and was not affected by Ca2+ (2 mM), colchicine (200 microM), or Mg-ATP (4 mM), reflecting that the ability of tubulin to bind to myosin was different from the ability of tubulin to form microtubules and that the nature of tubulin-myosin binding was different from that of F-actin-myosin binding. Besides tubulin-myosin interaction, a possible interaction between microtubule-associated proteins (MAPs) and actomyosin was suggested from the data that MAPs activated actomyosin MG2+-ATPase activity while purified tubulin inhibited the activity.

The specificity of transferrin (Tf) in its exertion of a growth-promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class-heterologous cells at higher concentrations and less so or completely ineffective on some class-homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity. To exert the growth-promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I-labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones. We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor.

Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronectin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effective for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the attachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronectin, but it did not cause any inhibition on the other proteins. The synthetic peptide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.

Localization of alpha-spectrin in chicken and monkey ventral horns has been studied by immunoperoxidase techniques at the electron microscopic level. For this purpose, an antiserum specific for chicken alpha-spectrin (240 kD subunit of spectrin) was prepared. The characteristics of the staining patterns of both chicken and monkey ventral horns were essentially identical. The reaction product for peroxidase was contained in the somata of large cells (presumably motor neurons), dendrites and axons. No specific staining was seen with either preimmune or blocked sera. The staining within the cell somata was primarily localized in cortical cytoplasm. Within dendrites and axons the immunocytochemical label was associated predominantly with the cortical cytoplasm and with microtubules. Staining was heavy over postsynaptic densities. Although presynaptic terminals showed weak staining as a whole, heavy staining was sometimes observed in areas adjacent to the presynaptic plasma membrane facing the postsynaptic density. These results indicate that spectrin distributes widely and functions in many biological activities in the nervous system.

Actomyosin Mg2+-ATPase activity was stimulated by a brain microtubule-associated protein (MAP) fraction. The stimulating activity of the MAP fraction was abolished by boiling and trypsin treatment, suggesting the presence of a protein factor. The factor stimulated actomyosin Mg2+-ATPase activity stoichiometrically by about four times in the optimum conditions (50--75 mM KCl, pH 6.6). The stimulating factor was coprecipitable with actomyosin and was found to be a pair of high-molecular-weight polypeptides (mol wts, 240,000 and 235,000). The polypeptides were not associated with microtubules or myosin, but with fibrous actin. In column chromatographies used for purifying the stimulating factor, the amount of polypeptides coincided with the stimulating activity. Increases in both specific activity and the amount of the paired polypeptides were nearly parallel in the process of the purification. A purified fraction (65% pure with respect to the paired polypeptides) showed a 56-fold increase of the specific stimulating activity as compared with the initial brain supernatant. The two peptides were similar but not identical with filamin and spectrin in terms of electrophoretic mobility. Hence, the pair of polypeptides was identified as an actin-binding protein newly found in brain.

Crude actomyosin fraction from porcine brain contained a large amount of high molecular weight actin-binding protein (BABP). The molar ratio of BABP to actin (BABP/actin) in the fraction was estimated to be 0.22. From this fraction, BABP and actin were solubilized with a molar ratio of 0.25, suggesting the existence of an interaction between BABP and brain actin. BABP was finally purified to 90% purity. The purified BABP was negatively stained and observed by electron microscopy; it appeared to be a slender, flexible, two-stranded molecule whose contour length was about 200 nm. The structure was very similar to those of fodrin and other high molecular weight actin-binding proteins such as filamin, spectrin, and ABP. Lattice cage-like structures composed of BABP molecules were occasionally observed at high BABP concentrations. The addition of BABP to actin filaments resulted in the appearance of many branching, filamentous bundles. The electron microscopic observations suggested that a single BABP molecule could crosslink actin filaments, that is, one BABP molecule has two actin binding sites.