摘要:

AbstractTwo monoclonal antibodies, GLU‐1 and A1.6, raised against γ‐L‐glutamyl‐L‐glutamic acid dipeptide (Glu‐Glu) and Ca2+‐dependent ATPase fromParamecium, respectively, recognized the dipeptide Glu‐Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross‐reaction of the antibodies to the α‐tubulin subunit. α‐Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU‐1 and A1.6 specifically recognize the detyrosinated from of α‐tubulin. When microtubule protein purified from brain was probed, not only α‐but also, to a lesser extent, β‐tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the γ position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class IIIβ isotype at amino acid position 437 to 439, it is suggested that GLU‐1 and A1.6 are able to recognize the glutamylated form of β‐tubulin. These results indicate that the C‐terminal Glu‐Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of α‐tubulin and the glutamyl side chain of β‐tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post‐translation

ISSN:0886-1544    

DOI:10.1002/cm.970300302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractWe had previously found no myosin heavy chain (MHC) changes in expression during pregnancy in human myometrium. In the present work, we compared the MHC pattern of expression in normal human myometrium, pregnant and non‐pregnant, to that in benign tumors of the uterine musculature and in cultured myometrial cells. We used a high‐resolution gel electrophoretic system and monoclonal antibodies directed against smooth muscle and nonmuscle MHCs. Smooth muscle MHCs (SM1, 204 kDa, and SM2, 200 kDa, MHCs) and a nonmuscle MHC of 196 kDa (NM MHC) were detected in pregnant and nonpregnant human myometrium. Pregnant myometrium was found to differ from nonpregnant myometrium by its slightly lower content in NM MHC, whereas the ration of SM1/SM2 was equivalent. In leiomyomas and in cultured cells grown from human myometrium explants, SM1, SM2, and NM MHCs were also expressed. In addition, a nonmuscle MHC of 198/200 kDa (SMemb MHC), which was present in a fetal human uterus but not in adult normal tissue, was observed in leiomyomas and in cultured cells. Expression of SM1 and SM2 MHCs was variable in the different leiomyomas studied. In cultured cells, SM1 and SM2 MHC content was low, but it was enhanced by suppression of serum after cell confluency. Present results confirm that pregnancy‐associated smooth muscle cell hypertrophy is not accompanied by major changes in MHCs. In contrast, cell culturing and cell hyperplasia leading to leiomyoma formation induce substantial modifications in MHCs, including the occurrence of a second type of nonmuscle MHC. © 1995 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970300303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractFetal rat brain (E18) expresses at least three c‐src‐like, membrane‐associated non‐receptor tyrosine kinases: c‐src, fyn, and lyn. c‐src and fyn are the most abundant and are highly enriched in a subcellular fraction of nerve growth cones (GCPs). To study the cytoskeletal association of these tyrosine kinases, Triton X‐100‐resistant fractions were prepared from GCPs. All three non‐receptor tyrosine kinases are associated with the cytoskeleton to a significant degree with the relative affinities: fyn>c‐src>lyn. The binding is sensitive to ionic strength and to phosphotyrosine, but not to phosphoserine or phosphothereonine. To investigate the regulation of this association we used phosphatese inhibitors to increase phosphotyrosine levels in GCPs. This resulted in the release of c‐src from the cytoskeleton. Under these conditions tyrosine phosphorylation was increased selectively in released c‐src and primarily on tyrosine 527. Cytoskeletally bound c‐src had a higher specific kinase activity than Triton X‐100‐soluble c‐src. These findings indicate that src family members interact in a regulated manner with the cytoskeleton in non‐transformed cells. This regulation is explained by a model in which c‐src binds to the cytoskeleton via its SH2 domain and is released when phosphorylated tyrosine‐527 binds to this domain intramolecularly, inhibiting ki

ISSN:0886-1544    

DOI:10.1002/cm.970300304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractHair bundles located on tentacles of sea anemones are morphodynamic mechanoreceptors employed to regulate discharge of nematocysts into swimming prey. Activation of chemoreceptors for N‐acetylated sugars is known to induce anemone hair bundles to elongate while shifting discharge to lower frequencies matching those produced by calmly swimming prey. In the continued presence of N‐acetylated sugars, activation of proline receptors is known to induce hair bundles to shorten while shifting nematocyst discharge to higher frequencies presumed to correspond to movements produced by wounded, struggling prey. In the present study, N‐acetylneuraminic acid (NANA) causes stereocilia to become more intensely fluorescent in confocal optical sections of phalloidin‐stained specimens, suggesting that receptors for N‐acetylated sugars initate processes to increase the density of F‐actin within stereocilia. Computer analysis of electron micrographs is consistent with this interpretation for large diameter stereocilia but not for small diameter stereocilia. In the continued presence of NANA, proline causes flurescence intensity of phalloidin to decrease to or below control levels. DNaseI uniformly stains large diameter stereocilia, suggesting that these stereocilia contain a pool of G‐actin. Fluorescence intensity of DNaseI in stereocilia is significantly less bright in specimens exposed to NANA alone than in specimens exposed to proline in the continued presence of NANA. It appears that whereas activated receptors for NANA induce G‐actin to polymerize in large diameter stereocilia, activated receptors for proline induce F‐actin to depolymerize, restoring G‐actin pools. ©

ISSN:0886-1544    

DOI:10.1002/cm.970300305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAs major determinants of cell shape and polarity, microtubules are required to have suitable rigidity. However, our knowledge of the mechanical properties of microtubules is far from satisfactory. We report here a new method of measuring the flexural rigidity of a single microtubule by direct buckling using the optical trapping technique. Microtubule buckling was induced by applying a small longitudinal compressing force through an optically trapped microsphere that was firmly attached to the microtubule. Three ways of estimating the flexural rigidity of a continuous slender rod, one from the observed critical load of buckling and two from deflected lengths and angles of bending, yielded values which agreed well when applied to the analysis of buckling microtubules. Unexpectedly, we found that the rigidity was not constant as expected but was dependent on microtubule length. This length dependency explains the discrepancies among reported values of microtubule flexural rigidity measured by different methods. Comparing microtubules of identical lengths, microtubules assembled with brain‐derived associated proteins (4 × 10−23Nm2at around 10 m̈m in length) were four times more rigid than those assembled from purified tubulin and stabilized with taxol (1 × 10−23Nm2). © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970300306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractDuring its motion inside host cells,Listeria monocytogenespromotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria‐column interface. To get a measure of filament organization in the column,Listeriagrown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in theListeria‐associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: (1) the surface of the bacteria, (2) the cytoplasm next to the bacteria, (3) the outer shell of the actin column, and (4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant ofListeriathat can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility ofListeria monocytogenesinside infected cells. © 1995 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970300307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970300301

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY