Biological and biochemical characterization of a factor produced spontaneously by adherent cells of human immunodeficiency virus-infected patients inhibiting interleukin-2 receptor alpha chain (Tac) expression on normal T cells.

Adherent cells from human immunodeficiency virus (HIV)-infected subjects but not from normal blood donors, patients with Gram-positive or -negative bacteremia, active tuberculosis, toxoplasmosis, pulmonary aspergillosis, and cytomegalovirus infection produce spontaneously an activity which inhibits alpha chain of interleukin-2 (Tac) expression and interleukin 2 (IL-2) production by normal activated T cells and IL-2 production by these cells. A similar biologic activity was detected in culture supernatants of in vitro HIV-I-infected normal adherent and leukemic U937 cells. Tac-inhibitory activity is not cytotoxic and it could be detected in serum-free conditioned media. Recombinant granulocyte/macrophage colony-stimulating factor and phorbol myristate acetate stimulation of patients' and normal adherent cells did not enhance specifically the production of the Tac inhibitor. Biologically active conditioned media did not contain infectious virus as well as secreted p24, gp120 viral proteins; the biologic activity could not be abolished by anti-p24, anti-gp120, and anti-nef monoclonal antibodies or human purified polyclonal anti-HIV IgG. Gel filtration of conditioned media followed by anion exchange chromatography resulted in a 1,200-fold degree of purification and revealed that the biologically active molecule was cationic. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this fraction and gel elution of the proteins showed that the biologic activity was associated with a 29-kD protein which was distinct from alpha- or gamma-interferon, tumor necrosis factor-alpha, and prostaglandin E2. The above findings demonstrate the production of inhibitory factor(s) during HIV infection, which might be involved in the pathogenesis of the patients' immune defect.

Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

A functional analysis of p53 during early development of Xenopus laevis.

p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis.

Purification and identification of a CD25 expression inhibitory protein from cell-free supernatants of chronically HIV-infected promonocytic cells.

The CD25 (IL-2-R alpha) cell surface glycoprotein expressed transiently during T-cell activation is implicated in the high affinity IL-2 receptor. This paper shows that cell-free supernatants from chronically HIV-infected promonocytic cells spontaneously produce a soluble factor which inhibits CD25 expression on PHA-activated human PBMC. We purified the CD25 expression inhibitory activity by a factor 12,350, using XM50 ultrafiltration, Superose 12 molecular sieving chromatography and MonoQ anion-exchange chromatography. Then we associated this activity to one single spot (M(r) 29,000, pI 6.8) on an O'Farrell two-dimensional gel. Our data demonstrate that this protein (M(r) 29,000, pI 6.8) is released from HIV-infected promonocytic cells and suggest that this factor is a new monokine regulating the T-cell activation process.

The angle of the dorsoventral axis with respect to the anteroposterior axis in the Drosophila embryo is controlled by the distribution of gurken mRNA in the oocyte.

Like most organisms, Drosophila embryos develop in relation to two orthogonal axes, the anteroposterior and dorsoventral. These two axes are established by four independent systems of maternal information. Mutations in any system disrupt either the anteroposterior or the dorsoventral patterning of the embryo but never affect the orthogonal orientation of the axes relative to each other. Here we show by analyzing their embryonic fate map, that K10 embryos still possess a dorsoventral polarity. However, instead of forming a 90 degrees angle, the dorsoventral and the anteroposterior axes lie parallel to each other. This axis misorientation was partially corrected by decreasing the wild-type grk gene copy number such that the embryos issued from K10/K10; grk/+ females showed a variability in their fate map which could be interpreted as a progressive rotation of dorsoventral axis relative to the unmodified anteroposterior axis. This rotation is maximal in the K10 embryos where it reaches 90 degrees and results in the congruence of the two axes. The alteration of the embryonic fate map could be traced back to oogenesis where it was shown to correlate with the mislocalization of the grk transcripts.

Autocrine growth of leukemic cells.

Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. This paper describes the stepwise malignant progression of leukemic cells which has been demonstrated in many experimental models of autocrine leukemic growth. In contrast, autocrine growth has not been proven as a major physiopathological mechanism for the growth of leukemic cells in vivo in human myeloid and lymphocytic leukemias. Growth-factor independency of human leukemic cell lines may be due to clonal selection.

Identification of an ATPase activity associated with the high molecular weight proteins of the human spermatozoon axonemes.

The high salt extract obtained from demembranated human spermatozoa contains high molecular weight proteins. These proteins are associated with an ATPase activity inhibited by sodium orthovanadate. In association with lower molecular weight proteins, they constitute a 20 S particle and are probably localized in the dynein arms (and in the radial spokes) of the human spermatozoon axonemes. Evidence is shown for a biochemical analogy between the dynein ATPases extracted from the invertebrate axonemes and the human dynein-like ATPase described in this study.

Elastic extension and jump of the flagellar nexin links: a theoretical mechanical cycle.

The functions of the nexin links of a flagellar axoneme have not been clearly demonstrated. Taking into account both the elastic (Hookean) characteristics and the possible jump of the nexin links, we calculated the sliding to bending conversion of a theoretical model in a tip-ward direction step by step, according to the essential principles proposed by the geometric clutch hypothesis [Lindemann, 1994: J Theoret Biol 168:175-189]: the activity of the dynein arms depends on the transverse forces induced by the axonemal curvature. In our calculations, however, the transverse forces that are involved in the regulation of the activities of the dynein arms were due to the extension of the nexin links located upstream of a given abscissa. This allowed us to define a bent segment as the axonemal portion at whose proximal and distal ends the nexin links were relaxed, and as fully extended as possible, respectively. The model creates an apparent disorder in the orientation of the nexin links as already observed [Bozkurt and Wooley, 1993: Cell Motil Cytoskeleton 24:109-118; Wooley, 1997: J Cell Sci 110:85-94]. We propose that the nexin links are involved in a mechanical cycle, whose 3 stages are (1) rapid extension, (2) slow relaxation, and (3) stand-by. The rapid extension is compatible with the mechanical interactions between the nexin links and the inner dynein arms with which they form the dynein regulatory complex. This was correlated with the oscillating properties of the nexin links along the axoneme that allow them to be one of the regulatory elements of the local ATPase activity of the dynein arms.

Axonemal activity relative to the 2D/3D-waveform conversion of the flagellum.

The waveform of the flagellum of the sea urchin spermatozoon is mainly planar, but its 3D-properties were evoked for dynamic reasons and described as helical. In 1975, the apparent twisting pattern of the sea urchin axoneme was described [Gibbons I. 1975. The molecular basis of flagellar motility in sea urchin spermatozoa. In: Inoué S, Stephens R, editors. Molecular and cellular movement. New York: Raven Press, p. 207-232.] and was considered to be one of the main elements involved in axonemal behaviour. Recently, planar, quasi-planar, and helical waveforms were observed when the flagellum of sea urchin sperm cells was submitted to an increase in viscosity. The quasi-planar conformation seemed to be due to the alternating torsion of the inter-bend segments [Woolley D, Vernon G. 2001. A study of helical and planar waves on sea urchin sperm flagella, with a theory of how they are generated. J. Exp. Biol. 204:1333-1345]. These three waveforms, which are due to a change in axonemal activity, are possibly used by the sperm cells to adapt their movement to variations in the physico-chemical characteristics of the medium (seawater) in which the cells normally swim. We constructed a simple model to describe qualitatively the central shear (between the axonemal doublets and the central pair) and the tangential shear (between the doublets themselves). In this model, the 3D-bending is resolved into components in two perpendicular planes and each of the nine planes of inter-doublet interaction defines a potential bending plane that is independently regulated. These shears were calculated for the three waveforms and their inter-conversion. This allowed us to propose that axoneme is resolved in successive modules delineated by abscissas where the sliding is always nil. We discuss these data concerning the axonemal machinery, and especially the alternating activity of opposite sides of (two) neutral surface(s) that seem(s) to be responsible for this inter-conversion, and for the possible twist of the axoneme during the beating.

Morphometric analysis of the cell outlines of normal and polyomavirus-transformed FR 3T3 fibroblasts.

A method is described for the analysis of cell shape, using an image analyzer connected to a computer to assess the cell outline. A series of parameters to assess the contribution of large cytoplasmic expansions to cell morphology and to cell spreading on a planar substratum were used to quantify the visual morphologic differences between normal (nontransformed; N.3T3) and polyomavirus-transformed (Py.3T3) Fisher rat 3T3 fibroblasts. The results show that the Py.3T3 fibroblasts are more spherical than are the N.3T3 fibroblasts and that the cytoplasmic expansions of the Py.3T3 fibroblasts are smaller than those of N.3T3, with the spreading of these two cell strains being different. These differences can be explained by the difference in cell-substratum affinity between these two cell strains.

Statistical analysis of morphometrically analyzed outlines of cells in culture. Comparison of populations of normal and polyomavirus-transformed FR 3T3 fibroblasts.

The correspondence analysis method was used to statistically characterize the morphologies of populations of normal (nontransformed; N.3T3) and polyomavirus-transformed (Py.3T3) Fisher rat 3T3 fibroblasts in culture, based on morphologic parameters calculated according to a previously described morphometric method. With this statistical method, each cell is considered as a vector in a space defined by an arrangement of the calculated morphologic parameters. The N.3T3 cells and the Py.3T3 have two distinct morphologic aspects in culture: they have either a smooth or a multipolar outline. The normal cell population contained twice as many cells with smooth outlines (46%) as did the transformed one (23%). Moreover, the cells with smooth outlines in the two strains could be classified into three homologous subpopulations that were present in significantly different proportions in the N.3T3 cells versus the Py.3T3 cells. In addition, morphologic differences were observed among the cells with multipolar outlines in these two strains, due to differences in the morphologies, size, number and distribution of the cytoplasmic expansions along the cell outline.

Microtubule tracks can be detected in mouse oocytes with an antibody directed against a calcium transporter.

In metaphase II-arrested mouse oocytes, most microtubules are found in the meiotic spindle, a structure that remains stable for hours despite microtubule instability. Microtubule organizing centres (MTOCs) are present at the poles of the spindle and in the cytoplasm, but the latter nucleate very few microtubules. This particular organization of the microtubule network enabled us to observe the unexpected behaviour of a protein that can associate with microtubules. We compared the distribution of a mitosis-activated calcium transport system with that of the microtubule network, by immunofluorescence, using two monoclonal antibodies, one directed against a component of the calcium transport system (7/13), and the other against the common tyrosinated form of alpha-tubulin (YL1/2). The 7/13 staining was associated with the spindle microtubules and with the kinetochore area. In addition, we observed many asters in the cytoplasm, around the cytoplasmic MTOCs. The majority of these asters were not stained with the antitubulin antibody. Moreover, these 7/13 asters either disappeared after nocodazole treatment or were enlarged after taxol treatment. Using a confocal microscope, we observed single fibres that were stained with both antibodies: the extremity furthest from the MTOC (corresponding to the + end of the microtubule) being detected by the 7/13 antibody only. All these observations suggest that the 7/13 antigen is associated with microtubule tracks that persist a few minutes after microtubule depolymerization. The possible role of these tracks in microtubule regrowth is discussed.

The vacuolar compartment is required for sulfur amino acid homeostasis in Saccharomyces cerevisiae.

In order to isolate new mutations impairing transcriptional regulation of sulfur metabolism in Saccharomyces cerevisiae, we used a potent genetic screen based on a gene fusion expressing XylE (from Pseudomonas putida) under the control of the promoter region of MET25. This selection yielded strains mutated in various different genes. We describe in this paper the properties of one of them, MET27. Mutation or disruption of MET27 leads to a methionine requirement and affects S-adenosylmethionine (AdoMet)-mediated transcriptional control of genes involved in sulfur metabolism. The cloning and sequencing of MET27 showed that it is identical to VPS33. Disruptions or mutations of gene VPS33 are well known to impair the biogenesis and inheritance of the vacuolar compartment. However, the methionine requirement of vps33 mutants has not been reported previously. We show here, moreover, that other vps mutants of class C (no apparent vacuoles) also require methionine for growth. Northern blotting experiments revealed that the met27-1 mutation delayed derepression of the transcription of genes involved in sulfur metabolism. By contrast, this delay was not observed in a met27 disrupted strain. Physiological and morphological analyses of met27-1 and met27 disrupted strains showed that these results could be explained by alterations in the ability of the vacuole to transport or store AdoMet, the physiological effector of the transcriptional regulation of sulfur metabolism.

Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II-arrested mouse oocytes.

The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 microM OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immunoblotting and [32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.

The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton and mitotic apparatus.

FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunophilin (HBI), was originally discovered in association with unliganded steroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bind cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of FKBP59-HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either 'soluble' proteins released from cells exposed to NP40 detergent, or proteins released from the cytoskeleton exposed to calcium ions (i.e. in microtubule depolymerizing conditions). Exposure of cells to 1 microM FK506 and rapamycin for 1 hour did not modify significantly the staining, although rapamycin treatment rendered the network stained by 173 clearly visible. Interestingly, during mitosis FKBP59-HBI segregated from the region of the chromosomes; it mainly localized with the mitotic apparatus (centrosome, spindle and interzone separating the chromosomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daughters cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Forskolin blocks the apical expression of dipeptidyl peptidase IV in Caco-2 cells and induces its retention in lamp-1-containing vesicles.

In a previous work, we showed that the differentiation-dependent expression of dipeptidyl peptidase IV (DPP IV) in Caco-2 cells, a human colon adenocarcinoma cell line, was controlled at the mRNA level (D. Darmoul et al. J. Biol. Chem., 1992, 267, 4824-4833). Whether post-translational events may contribute to the final control of DPP IV cell surface expression was explored here by studying the potential effect of forskolin (FK), a drug known to permanently stimulates adenylyl cyclase and to strongly perturbs glucose metabolism in fully differentiated Caco-2 cells. FK treatment reduces by about 50% the amount of active DPP IV present at the brush border membrane, whereas the total amount of active DPP IV remains unchanged. Biosynthesis and maturation of DPP IV were measured using [35S]methionine labeling and were shown to be essentially unaffected by FK treatment. Pulse-chase experiments demonstrate that up to 50% of the neosynthesized DPP IV do not appear at the apical membrane after FK treatment. To get further insight into this phenomenon, we have used confocal laser scanning microscopy. We demonstrate that the blockade of DPP IV transport is associated with the accumulation of this protein in intracellular vesicles. Double-staining experiments demonstrate that these vesicles are not labeled with a monoclonal antibody directed against the Golgi apparatus but display a strong staining with a polyclonal antibody raised against lamp-1, a lysosomal membrane protein. Using a newly developed image analysis procedure, we have been able to quantitate the relative distribution of lamp-1 and DPP IV labels in both control and forskolin-treated cells. We show that the overlap of the two labels dramatically increases in FK-treated Caco-2 cells. These results suggest that, beside the transcriptional level, post-translational events may be involved in the final control of the apical expression of a differentiation-dependent hydrolase.

Early defect in branching morphogenesis of the ureteric bud in induced nephron deficit.

Development of the metanephric kidney during embryogenesis can be altered both in vivo and in vitro by exposure to gentamicin, which may lead to oligonephronia. To study the role of the ureteric bud in nephron deficit genesis, we used metanephros organ cultures exposed to gentamicin as a model of impaired nephrogenesis. Ultrastructural localization of the antibiotic showed that by eight hours it was already present within the epithelial cells of the ureteric bud and in its growing ends, and also trapped in the adjacent blastema. Using confocal microscopy and image analysis, we devised a quantitative approach to analyze the branching pattern of the ureteric bud, and showed that by 24 hours of culture, despite no change of explants growth, gentamicin had significantly decreased the number of branching points. This effect involved the early branching events and was limited to end buds that had no nephron anlagen nearby. Our findings indicate that impaired branching morphogenesis of the ureteric bud is the likely event of gentamicin-induced nephron deficit.

Inhibition of flagellar beat frequency by a new anti-beta-tubulin antibody.

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

Effects of osmolality, morphology perturbations and intracellular nucleotide content during the movement of sea bass (Dicentrarchus labrax) spermatozoa.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.

Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair.

Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.