A.BSTRACT Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100 % of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 m~ ATP at 25°C, the reactivated sperm had an average frequency of 32 beats/see and progressed forward a distance of 2.4 him/beat; comparable figures for live sperm in seawater were 46 beats/see and 3 9/zm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16/~mole Pi/(min X mg protein), while sperm that had been rendered nonmotile by homogenizing had an activity of 0 045 ]~mole Pi/(min X mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72 % of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg ++, although some motility was also obtained with Mn ++ and Ca ++. The coupled ATPase activity had a Michaelis constant (Kin) of 0.15 raM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "Kin" of 0.2 raM.The flagella of sea urchdn sperm consist of a longitudinal fibrous axoneme enclosed within a membrane. The energy for motility is normally provided by adenosine triphosphate (ATP) diffusing down the flagellum from the mitochondria at the base of the sperm head (I, 9). In order to study the effects of varying chemical conditions on the properties of the motile apparatus, it is necessary to obtain preparations in which the selective permeability of the fiagellar membrane has been destroyed, so that the conditions can be controlled experimentally. Hoffmann-Berling (3) demonstrated that sperm in wlfich the membrane has been rendered permeable by extraction with glycerol would regain motility if transferred to a suitable medium containing exogenous ATP. Brokaw and Benedict have recently studied the properties of glycerinated sperm in some detail, and have shown that they possess adenosine triphosphatase (ATPase) activity, part of which is tightly coupled to their Inotihty (4, 5). However, the quantitative study of their properties has been handicapped by the fact that only 20-50 % of the g...

The motility of demembranated sea urchin sperm flagella and that of embryo cilia reactivated with 0.1 mM ATP are completely inhibited by 4 AM and 0.5 AM vanadium (V) [V(V), in vanadate], respectively. The Mg2+-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) of the latent form of dynein 1 is inhibited 50% by 0.5-1 AM V(V), while the Ca2+-activated ATPase activity is much less sensitive. The inhibition of flagellar beat frequency and of dynein 1 ATPase activity by V(V) appears not to be competitive with ATP. In agreement with other reports, the inhibition of (NaK)ATPase by V(V) shows a slow onset in the presence of ATP and is relatively rapid in its absence. With dynein, however, the inhibition occurs at a rapid rate whether or not ATP is present. Catechol at a concentration of 1 mM reverses the V(V) inhibition of reactivated sperm motility, dynein ATPase, and (NaK)ATPase. (5).In this paper we report that vanadium(V), V(V), is a potent inhibitor of dynein 1 and of the motility of reactivated sea urchin sperm flagella and embryo cilia. We have also examined the effect of V(V) on myosin and (Na,K)-ATPase. MATERIALS AND METHODSMaterials. Sodium metavanadate (NaVO3) and sodium orthovanadate (Na3VO4) were obtained from Fisher ScientificCo. Stock solutions of sodium metavanadate that had been recrystallized from methanol/water were prepared in 10 mM Tris.HCl buffer, pH 8.1. Identical results were obtained with sodium orthovanadate in 0.1 M NaOH. Cateohol, norepinephrine, ouabain, NADH, phosphoenolpyruvate, lactate dehydrogenase, and pyruvate kinase were obtained from Sigma. ATP was obtained from Boehringer Mannheim Corporation. Stock solutions of 0.25 M catechol and norepinephrine were prepared freshly in 1 mM HCl.Sperm and eggs were obtained from the sea urchin Tripneustes gratilla by injection with 0.5 M KCl.Reactivated Sperm and Cilia. Sea urchin sperm were demembranated with Triton X-100 and reactivated with 0.1 mM ATP as described previously (7,8). Cilia were obtained from sea urchin embryos grown in Ca2+_free artificial sea water, disrupted mechanically into individual blastomeres, and treated with demembranating solution (7) at pH 7.5. They were then transferred to reactivating solution, pH 7.5, containing 0.1 mM ATP.Preparation of ATPases. LAD-1 was extracted from freshly Before their Mg2+-and Ca2+-activated ATPase activities were compared, preparations of LAD-1 were dialyzed against 0.5 mM EDTA/7 mM 2-mercaptoethanol/5 mM Tris-HCl, pH 8.0, to remove all divalent cations. Activated dynein 1 was obtained by incubating LAD-1 with 0.1% (wt/vol) Triton X-100 for 10 min at room temperature (5). Myosin and actin were prepared from rabbit muscle by the procedures of Perry (10) 2220The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Sperm flagella of the sea urchin Tripneustes gratilla beat with asymmetrical bending waves after demembranation with Triton X-100 in the presence of EGTA and reactivation at pH 8.1 with 1 mM ATP in the presence of 2 mM MgSO4. Addition of 0.1--0.2 mM free Ca2+ to these reactivated sperm induces 70--95% of them to become quiescent. This quiescence can be reversed by reduction of the free Ca2% concentration with EGTA, or by dilution to reduce the MgATP2- concentration below 0.3 mM. The quiescent waveform is characterized by a sharp principal bend of approximately 5.6 rad in the proximal region of the flagellum, a slight reverse bend in the midregion that averages approximately 0.3 rad, and a principal bend of approximately 1.1 rad in the tip. The quiescent sperm are highly fragile mechanically, and disruption, including microtubule sliding, occurs spontaneously at a slow rate upon standing or immediately upon gentle agitation. Mild digestion by trypsin causes a gradual appearance of normal, symmetrical flagellar beating. Addition of increasing concentrations of vanadate to quiescent sperm causes a graded decrease in the proximal bend angle, with 50 micrometers vanadate reducing it to approximately 2.6 rad. In the presence of 0.1 mM free Ca2% and 10 micrometers vanadate, a characteristic, crescented stationary bend is induced in the demembranated sperm, without intermediate oscillatory beating, by the addition of either 0.1 or 1 mM ATP. In the absence of vanadate, these two concentrations of ATP produce asymmetric beating and quiescence, respectively. The results support the hypothesis that quiescence in live sperm is induced by an elevated concentration of intracellular Ca2%. In addition, they demonstrate that bending can occur in flagella in which oscillatory beating is inhibited and emphasize the close relationship between asymmetric beating and quiescence.

Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of cytoplasmic dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the dynein heavy chain genes identified in Drosophila, indicating that the radiation of the dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in cytoplasmic dynein have been more constrained than those in the axonemal dyneins.

Clinical, morphologic, and cytogenetic features were examined in a group of 68 children with myelodysplasia (MDS) referred to a single institution between 1971–1991. The morphologic French-American-British (FAB) system of classification proved of limited value in this group of patients because 50% of the cases were categorized as chronic myelomonocytic leukemia and three patients with eosinophilia and MDS were unclassifiable. Cytogenetic analysis was performed in 63 cases and clonal abnormalities were detected in 55%; the most common chromosome involved was number 7. Modification of the FAB system to incorporate additional diagnostic features such as pretreatment fetal hemoglobin (Hb F) and cytogenetics allowed incorporation of the categories of juvenile chronic myeloid leukemia (JCML) and infantile monosomy 7 syndrome (IMo7). The resulting groups of patients had highly significant differences in survival (P = .00009). The overall 5-year survival for the patients was 31.9% (95% CI 21.7 to 44.1) and factors influencing prognosis included: modified FAB type, platelet count, Hb F level, and cytogenetic complexity. We developed a scoring system (“FPC”) where each of the following findings at diagnosis scored one point: HbF greater than 10%, platelets < or = 40 x 10(9)/L, and complex karyotypic changes (two or more clonal structural/numerical abnormalities), which produced groups with highly significant differences, patients with a score of 0 having a 5-year survival of 61.6% (CI 33% to 84%), whereas those with a score of two or three all died within 4 years of diagnosis. The revised classification and scoring system may prove helpful in making treatment choices in pediatric MDS and now needs to be tested prospectively in large scale population-based studies.