The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identified. Aggrecanase-1 is one of two ADAMTS cartilage-degrading enzymes purified from interleukin-1-stimulated bovine nasal cartilage (Tortorella, M. D., Burn, T. C., Pratta, M. A. , Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., and Arner, E.C. (1999) Science 284, 1664-1666; 2 Abbaszade, I., Liu, R. Q., Yang, F., Rosenfeld, S. A., Ross, O. H., Link, J. R., Ellis, D. M., Tortorella, M. D., Pratta, M. A., Hollis, J. M., Wynn, R., Duke, J. L., George, H. J., Hillman, M. C., Jr., Murphy, K., Wiswall, B. H., Copeland, R. A., Decicco, C. P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Burn, T. C. (1999) J. Biol. Chem. 274, 23443-23450). The aggrecan products generated by this enzyme are found in cartilage cultures stimulated with cytokines and in synovial fluid from patients with arthritis, suggesting that aggrecanase-1 may be important in diseases involving cartilage destruction. Here we demonstrate that the thrombospondin type-1 (TSP-1) motif located within the C terminus of aggrecanase-1 binds to the glycosaminoglycans of aggrecan. Data from several studies indicate that this binding of aggrecanase-1 to aggrecan through the TSP-1 motif is necessary for enzymatic cleavage of aggrecan. 1) A truncated form of aggrecanase-1 lacking the TSP-1 motif was not effective in cleaving aggrecan. 2) Several peptides representing different regions of the TSP-1 motif effectively blocked aggrecanase-1 cleavage of aggrecan by preventing the enzyme from binding to the substrate. 3) Aggrecanase-1 was not effective in cleaving glycosaminoglycan-free aggrecan. Taken together, these data suggest that the TSP-1 motif of aggrecanase-1 is critical for substrate recognition and cleavage.     

REVIEWS

Book reviewed in this article
Growth Regulators in Plant Science, Monograph 8. Ed. by M. B. J ackson , B. G rout and I. A. M ackenzie .
An Introduction to Plant Taxonomy. By C. J effrey .
Pest and Diseases of Tropical Crops , Vol. 1. By D. S. H ill and J. M. W aller .
Flora of Turkey , Vol. 7 ( Orobanchaceae to Ceratophyllaceae and Ruhiaceae ). Ed. by P. H. D avis , assisted by J. R. E dmondson , R. R. M ill and K. T an .
Plant Growth Curves. The Functional Approach to Plant Growth Analysis. By R oderick H unt .
Bryophyte Ecology. Ed. by A. J. E. S mith . Bryophyte Ecology. Ed. by A. J. E. S mith . Bryophyte Ecology. Ed. by A. J. E. S mith . Bryophyte Ecology. Ed. by A. J. E. S mith .
Forest Succession: concepts and application. Ed. by D. C. W est , M. H. S hugart and D. B otkin .     

REVIEWS

Molds, Yeasts and Actinomycetes. By C. E. S kinner , C. W. E mmons and H. M. T suchiya
Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve. By D. J. F inney . With a foreword by F. T attersfield
Leguminous Forage Plants. By D. H. R obinson
Cytologie Végétale et Cytologie Générale. Par P ierre D angeard
German-English Science Dictionary for Students in Chemistry, Physics, Biology, Agriculture and Related Sciences. By L. D e V ries     

Book reviews

Book reviewed in this article:
Turner, R.M., Bowers, J.E. and Burgess, T.L. (1995) Sonoran desert plants. An ecological atlas . Phoenix: The University of Arizona Press.
Mori, S.A. and Lepsch-Cunha, N. (1995) The Lecythidaceae of a Central Amazonian moist forest . Memoirs of the New York Botanical Garden Volume 75. New York Botanical Garden, New York.
Mill, P.J. and McQuaid, C.D. (eds) (1995) Development in hydrobiology. Advances in littorinid biology . Dordrecht: Kluwer Academic Publishers.
Nogrady, T., Pourriot, R. and Segers, H. (1995) Rotifera Volume 3: The Notommatidae and The Scaridiiae. Guides to the identification of the microinvertebrates of the continental waters of the world . Coordinating editor H.J.F. Dumont. Amsterdam: SPB Academic Publishing.
Chapman, G.P. & Thompson, M. (eds) (1994) Water and the quest for sustainable development in the Ganges Valley . Mansell, London.
Begon, M., Harper, J.L. and Townsend, C.R. (1996) Ecology. Individuals, populations and communities . 3rd ed. Oxford: Blackwell Science.
Revista Geografica de America Central No. 27 . 1993. Heredia, Costa Rica: University National. 224pp., figs, tables. Paperback: Price unknown.     

Book reviews

Book Reviewed in this article Ecological Studies—Analysis and Synthesis, Volume 43. Mediterranean-Type Ecosystems—the Role of Nutrients Edited by F. J. Kruger, D. T. Mitchell and J. U. M. Jarvis Insect Ecology E. G. Mathews and R. L. Kitching Donald Thompson's Mammals and Fishes of Northern Australia Edited and annotated by Joan M. Dixon and Linda Huxley Grasses. A Guide to their Structure, Identification, Uses and Distribution in the British Isles. 3rd edition C. E. Hubbard, revised by J. C. E. Hubbard Soil Salinity Under Irrigation. Ecological Studies Number 51 Edited by I. Shainberg and J. Shalhevet Nothofagus cunninghamii (Southern Beech) Vegetation in Australia. Australian Flora and Fauna Series, Number 1 John R. Busby Sandy Beaches as Ecosystems. Developments in Hydrobiology 19 Edited by A. McLachlan and T. Erasmus, Dr W. Junk BV     

REVIEWS

Book reviewed in this article:
Die Fusarien. By H. W. W ollenweber and O. A. B einking .
Biological Processes in Tropical Soils. By A. S teven C okbet .
Problems in Soil Microbiology. By D. W ard C utler and Lettice M. Crump.
Limnology. By P. S. W elch .
Plant Viruses. By K enneth M. S mith .
Heredity and the Ascent of Man. By C. C. H urst .
A Text-book of Mycology. By E. A. B essey .
The Arachnida. By T. H. S avory .
Garden Science. By J ohn G rainger .
Plant Physiology. By M eieion T homas .     

REVIEWS

Stomatal Physiology. Ed. by P. G. J arvis and T. A. M ansfield
Evolution and Pollution. By A. D. B radwhaw and T. McNJ eilly
The Biology and Taxonomy of the Solanaceae. Ed. by J. G. H awkes , R. N. L ester and A. D. S kelding
Advances in Legume Science . Ed. by R. J. S ummerfield and A. H. B unting
The Shaping of Cambridge Botany . By S. M. W alters
Microbial Control of Pests and Plant Diseases 1970–1980. Ed. by H. D. B urges     

REVIEWS

Atlas of Mapped Distributions of Dominance and Modern Pollen Percentages for Important Tree Taxa of Eastern North America . By P. A. D elcourt , H. R. D elcourt and T. W ebb III.
New Manual of Bryology. Volume 1. Edited by R. M. S chuster .
The Ecology and Physiology of the Fungal Mycelium. Edited by D. H. J ennings and A. D. M. R ayner .
Isotopes and Radiation in Agricultural Sciences. Edited by M. E. L'A nnunziata and J. O. L egg .
The Physiology of Flowering Plants : their growth and development . Third edition. By H. E. S treet and H. Ö pik .
Reconstructing Quaternary Environments. By J. J. L owe and M. J. C. W alker .
European Mires. Edited by P. M oore .
Compartments in Algal Cells and Their Interaction. (Proceedings in Life Sciences Series.) Edited by W. W iessner , D. G. R obinson and R. C. S tarr .
Chloroplast Metabolism. By B arry H alliwell .
Flooding and Plant Growth. Ed. by T. T. K ozlowski .
Mode of Action of Antifungal Agents. Ed. by A. P. J. T rinci and J. F. R yley .
Disease Resistance in Plants. By J. E. V anderplank .     

Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4)

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe(342) which is cleaved by matrix metalloproteinases and the other between Glu(373)-Ala(374) that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(374) bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred preferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE(1771)-(1772)AGEG and at VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of approximately 20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.     

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.     

REVIEWS

Book reviewed in this article:
Plant Cells . By F. A. L. C lowes and B. E. J uniper .
The History of British Vegetation . By W inifred P ennington . Modern Biology: General Editor J. E. W ebb .
Flora Europaea , Vol. II. Rosaceae to Umbelliferae . Ed. by T. G. T utin , V. H. H eywood , N. A. B urges , D. M. M oore , D. H. V alentine , S. M. W alters and D. A. W ebb .
Desert Wild Flowers . By E dmund J aeger .
British Mosses and Liverworts . By E. V. W atson .
Anatomy of the Monocotyledons , Vol. III. Commelinales—Zingiberales . By P. B. T omlinson . General Editor C. R. M etcalfe .
Biological Science—The Web of Life . Supervising Editor, D. M organ .
Plant Chimeras . By W. N eilson -J ones .
The Potatoes of Argentine, Brazil, Paraguay and Uruguay, a Biosystematic Study . By J. G. H awkes and J. P. H jerting .
Towards an Understanding of the Mechanism of Heredity . By H. L. K. W hitehouse .
Viruses in Plant Hosts; Form, Distribution and Pathologic Effects . By K atherine E sau .
An Introduction to Industrial Mycology . By G eorge S mith .     

Reviews of publications

Book reviewed in this article:
Plant Science An introduction to World Crops, by J. Janick. R. W. Schery, F. W. Woods & V. W. Ruttan. W. H. Freeman
Life of the Vertebrates, by J. Z. Young, F.R.S
The Mammals of North America, by E. Raymond Hall. John Wiley
The Giant Panda, edited by Zhu Jing & Li Yangwen
Modern Ethology; The Science of Animal Behaviour, by S. A. Barnett
Environmental Physiology of Fishes, edited by M. A. Ali
British Planarians, Synopsis of the British Fauna No. 19, by I. R. Ball & T. B. Reynoldson
Development and Specialization of Skeletal Muscle, edited by D. F. Goldspink
Human Navigation and the Sixth Sense, by R. R. Baker
Mosses of Eastern North America, by H. A. Crum & L. E. Anderson     

Book reviews

Book reviewed in this article:
C hemical D isinfection in H ospitals (1993). Edited by G. A. J. Ayliffe, D. Coates and P. N. Hoffman. 2nd Edition.
E xploitation of M icroorganisms (1993). Edited by D.G. Jones.
P redictive M icrobiology : T heory and A pplication (1993). By T.A. McMeekin, J.N. Olley, T. Ross and D.A. Ratkowsky.
F undamental I mmunology (1993). Edited by W. E. Paul
T he H uman H erpesviruses (1993). Edited by B. Roizman, R.J. Whitley and C. Lopez.
G eneral M icrobiology (1993). By H.G. Schlegel. 7th Edition. Translated by M. Kogut.
R eproduction in F ungi : G enetical and P hysiological A spects (1993). By C.G. Elliott.     

The statistical analysis of the in vitro chromosome aberration assay using Chinese hamster ovary cells

The purpose of the in vitro chromosome aberration assay (ABS) is to determine whether the test compound is a clastogen, i.e. induces structural changes in chromosomes. Details of this assay can be found in Galloway et al. [S.M. Galloway, M. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kirkland, M. Takeshi, P. Mosesso, T. Sofuni, Mutation Res. 312 (1994) 241-261]. The standard design consists of a negative control and at least three positive dose groups. At each dose, a sample, say 200, of metaphase cells is examined microscopically and cells exhibiting at least one type of chromosome aberration are identified. Using Chinese hamster ovary cells, Margolin et al. [B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E. Zeiger, Environ. Mutagen. 8 (1986) 183-204] and Richardson et al. [C. Richardson, D.A. Williams, J.A. Allen, G. Amphlett, D.O. Chanter, B. Phillips, Analysis of data from in vitro cytogenetic assays, in: D.J. Kirkland (Ed.), Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, 1989, pp. 141-154] demonstrated that a binomial sampling model could be used to describe the proportion of cells with chromosome aberrations.Statisticians and toxicologists have also suggested evaluation criteria for the dose response pattern of ABS. Margolin et al. [B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E. Zeiger, Environ. Mutagen. 8 (1986) 183-204] suggested one use the Cochran-Armitage trend test. Sofuni et al. [T. Sofuni, A. Matsuoka, M. Sawada, M. Ishidate Jr, E. Zeiger, M.D. Shelby, Mutation Res. 241 (1990) 175-213] considered the dose response to be (strong) positive if it had two significant doses out of three dose groups and decided it was weakly positive if it had only one significant dose and there was a significant trend. The criterion of Galloway et al. for a positive response was a clear dose-related increase in cells with structural aberrations in one experiment or a reproducible single positive dose [S.M. Galloway, M. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kirkland, M. Takeshi, P. Mosesso, T. Sofuni, Mutation Res. 312 (1994) 241-261].We formulate the above three procedures in terms of a Cochran-Armitage trend test and a Dunnett type test. We then compare the performance of these three procedures in terms of a Monte Carlo simulation study. We then develop a software program from the chosen procedure for its ease of use by statisticians and toxicologists.     

Book reviews

Book Reviewed in this article: Birds of Wallacea C. M. N. White and M. D. Bruce Snakes, Ecology and Evolutionary Biology Edited by R. A. Seigel, J. T. Collins and S. S. Novak The Biology of Perch and Related Fish J. F. Craig Corals of Australia and the Indo-Pacific J. E. N. Veron Conservation Biology. The Science of Scarcity and Diversity Edited by M. E. Soulé Plant Ecology Edited by Michael J. Crawley Origins and Development of Adaptation. CIBA Foundation Symposium 102 Edited by D. Evered and G. M. Collins.     

Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695

We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.     

Modeling the cellular basis of altered excitation-contraction coupling in heart failure

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle. Cardiovasc. Res. 30, 281-290; Philips, P.J., Gwathmey, J.K., Feldman, M.D., Schoen, F.J., Grossman, W., Morgan, J.P., 1990. Post-extrasystolic potentiation and the force-frequency relationships: differential augmentation of myocardial contractility in working myocardium from patients with end-stage heart failure. J. Mol. Cell. Cardiol. 22, 99-110; Pieske, B., Hasenfuss, G., Holubarsch, C., Schwinger, R., Bohm, M., Just, H., 1992. Alterations of the force-frequency relationship in the failing human heart depend on the underlying cardiac disease. Basic Res. Cardiol. 87, 213-221.]. Analyses of protein levels in these failing hearts reveal that the SR Ca-ATPase is down-regulated on average by 50% and that the Na/Ca exchanger is up-regulated on average by a factor of two. In this paper, we test the hypothesis that this altered pattern of expression of Ca handling proteins is sufficient to account for changes in excitation-contraction coupling properties measured experimentally at the cellular level. To do this, we present an integrated model of excitation-contraction coupling in the guinea pig ventricular cell. The model is used to determine the effects of SR Ca-ATPase down-regulation and Na/Ca exchanger up-regulation on action potential duration, Ca transient shape and amplitude, and isometric force. Model analyses demonstrate that changes in Ca handling proteins play a direct and critical role in prolongation of action potential duration, and in reduction of contractile force in heart failure.     

Normal fertilization occurs with eggs lacking the integrin alpha6beta1 and is CD9-dependent

Previous results, based on inhibition of fertilization by an anti-alpha6 integrin mAb (GoH3), suggest that the alpha6beta1 integrin on mouse eggs functions as the receptor for sperm (Almeida, E.A., A.P. Huovila, A.E. Sutherland, L.E. Stephens, P.G. Calarco, L. M. Shaw, A.M. Mercurio, A. Sonnenberg, P. Primakoff, D.G. Myles, and J.M. White. 1995. Cell. 81:1095-1104). Because the egg surface tetraspanin CD9 is essential for gamete fusion (Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Suzuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324) and CD9 is known to associate with integrins, recent models of gamete fusion have posited that egg CD9 acts in association with alpha6beta1 in fusion (Chen, M.S., K.S. Tung, S.A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P.W. Kincade, J.C. Herr, and J.M. White. 1999. Proc. Natl. Acad. Sci. USA. 96:11830-11835; Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Su- zuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324). Using eggs from cultured ovaries of mice lacking the alpha6 integrin subunit, we found that the fertilization rate, fertilization index, and sperm binding were not impaired compared with wild-type or heterozygous controls. Furthermore, a reexamination of antibody inhibition, using an assay that better simulates in vivo fertilization conditions, revealed no inhibition of fusion by the GoH3 mAb. We also found that an anti-CD9 mAb completely blocks sperm fusion with either wild-type eggs or eggs lacking alpha6beta1. Based on these results, we conclude that the alpha6beta1 integrin is not essential for sperm-egg fusion, and we suggest a new model in which CD9 acts by itself, or interacts with egg protein(s) other than alpha6beta1, to function in sperm-egg fusion.     

REVIEWS

Books reviewed in this article:
Plant Anatomy: Experiment and Interpretation , Part II, Organs. By E lizabeth G. C utter .
Genetics. By D. J. C ove .
The Structure and Life of Bryophytes. By E. V. W atson .
Underwater Science. Ed. by J. D. W oods and J. J. L ythgoe .
8ème Symposium Européen sur les Maladies à Virus des Arbres Fruitiers.
Evolution and Plants of the Past. By H. P. B anks .
Project Aqua. A Source Book of Inland Waters Proposed for Conservation. IBP Handbook No. 21. By H. L uther and J. R zóska .
Variation and Adaptation in Plant Species. By D. A. J ones and D. A. W ilkins .
Flora of Lesotho (Basutoland). By A my J acot G uillarmod .
Introduction to Biogeography. By B rian S eddon .
Trees: Structure and Function. By M. H. Z immerman and C. L. B rown ;
Flora of New Zealand , Volume II. By L. B. M oore and E. E dgar .