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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 261:llO-114 (1992) RAPID COMMUNICATION Oxidative Metabolism in Thermogenic Tissues of the Swordfish and Mako Shark J.S. BALLANTYNE, M.E. CHAMBERLIN, AND T.D. SINGER Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG 2 W1 (J.S.B., T.D.S.); Department of Zoological and Biomedical Sciences and College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701 (M.E.C.) Mitochondria isolated from the heater organ of the swordfish Xiphias gladius and ABSTRACT the warm lateral red muscle of the mako shark Isurus oxyrinchus display respiratory control, indicating that uncoupling of oxidative phosphorylation is not important in heat production. The mitochondria of the swordfish heater organ resemble those of mammalian brown adipose tissue in their substrate preferences, oxidizing pyruvate plus malate, palmitoyl carnitine, and a-glycerophosphate at high rates. The high hexokinase levels in the heater organ indicate exogenous glucose is a n important energy source. The high activity of cytosolic a-glycerophosphate dehydrogenase and the high rates of mitochondrial oxidation of a-glycerophosphate indicate that cytosolic redox is balanced via the a-glycerophosphate cycle. Calcium stimulates mitochondrial a-glycerophosphate oxidation, and it is proposed that calcium released from the sarcoplasmic reticulum may act as the signal coupling enhanced rates of substrate oxidation with calcium transport. Based on mitochondrial respiration rates, heat generation by the intact heater organ was calculated. Complete oxidation of glucose produces heat at a greater rate than oxidation of lipids. Nevertheless, oxidation of either substrate could produce sufficient heat to maintain tissue temperature 10°C above ambient a t 20°C. The mitochondria from the red muscle of the mako shark oxidize pyruvate plus malate, glutamine, glutamate, proline, and P-hydroxybutyrate a t high rates. Palmitoyl carnitine was not oxidized. The respiration rates are similar to those described for mitochondria isolated from “cold” muscle of other elasmobranchs. Therefore, it does not appear that mako shark red muscle mitochondria are specialized for thermogenesis. Several large pelagic fishes maintain some tissues at temperatures above those of the surrounding water (Carey and Teal, ’69;Carey et al., ’81;Carey, ’82; Block, ’85).In swordfish (family Xiphiidae), one of the eye muscles, the superior rectus, has become modified specifically for heat production to warm the eye and brain (Carey, ’82).Ultrastructural studies of billfish (family Istiophoridae) indicate that a portion of the superior rectus muscle (“brainheater organ”; Block, ’86) is modified for heat production and contains abundant mitochondria, sarcoplasmic reticula (SR), and T tubules. The contractile elements are largely absent. Whereas the billfish heater organ appears to be specially modified for thennogenesis, other fish muscles retain their contractile function and conserve the heat by countercurrent heat exchangers. In lamnid sharks, heat generated by skeletal red muscle contraction and associated metabolism is retained by countercurrent exchangers (Carey et al., ’81). The metabolic characteristics of mitochondria found in these two types of “warmer-than-ambient” 0 1992 WILEY-LISS,INC. fish tissues have not been described. Such studies require freshly caught fish and depend on biochemical facilities being available at sea. Recently, we had the opportunity to establish facilities at sea for the biochemical analyses of fresh tissues of the swordfish Xiphias gladius and the shortfin mako shark Isurus oxyrinchus. Our goal was to characterize the metabolism of the swordfish heater organ and mako shark warm red muscle using isolated mitochondria and enzyme measurements. MATERIALS AND METHODS Animals As part of a Canadian Department of Fisheries and Oceans survey, swordfish and mako sharks were obtained by long-lining off Corsair Canyon near Georges Bank (66“W 41.5”N)between September 6, 1990, and September 11, 1990. Lines were set Received March 25,1991;revision accepted July 2,1991. THERMOGENIC TISSUES OF SWORDFISH AND MAKO SHARK at night and retrieved the following morning. Only live fish were used for the reported studies. A 111 D C Mitochondriaf isolation Mitochondria were isolated and characterized on board ship. Brain heater organs were dissected from swordfish heads, and lateral red muscle was excised from a region near the backbone of the mako shark. The tissue was minced, placed in cold isolation medium, and homogenized with three passes of a loosely fitting pestle of a Potter-Elvehjem homogenizer. The medium for isolating mitochondria from swordfish brain heater muscle consisted of 250 mM sucrose, 1mM EGTA (ethylene glycol bis-(@-aminoethyl ether) N,N,N',N'-tetraacetic acid), 30 mM HEPES (N-[2-hydroxyethyl]piperazine-Nr-[2-ethanesulfonic acid]),and 1%BSA (bovine serum albumin, Sigma A-7030, essentially fatty acid free). Mako muscle mitochondria were isolated using a medium composed of 500 mM sucrose, 150 mM KC1, 20 mM HEPES, 10 mM Na2 EDTA (ethylenediFig. 1. Typical traces of respiration of mitochondria isoaminetetraacetic acid), 5 mM MgC12,and 0.1% BSA. lated from the mako shark red muscle and swordfish heater The pH of both solutions was adjusted to 7.2 at 20°C. organ. ADP (400 nM) was added prior to the addition of substrate (GLN, 12.5 mM glutamine; a-GP, 1 mM a-glycerophosThe homogenates were centrifuged at 918g for 10 phate). A: Mako shark mitochondria (0.79 mg proteidml). B: minutes. The resulting supernatants were removed Swordfish heater organ mitochondria (0.38 mg protein/ml). C: and centrifuged at 9660g for 10 minutes. The pel- Swordfish heater organ mitochondria (0.31 mg protein/ml). D: lets were removed and resuspended in isolation Swordfish heater organ mitochondria (0.31 mg proteiniml); 1 medium and again centrifuged at 9,660g for 10 min- mM CaCl, added prior to the addition of a-glycerophosphate. utes. The final pellets were resuspended in isolaEnzyme measurements tion medium t o yield a final protein concentration of 3-17 mg/ml. Maximal enzyme activities were measured in samples of brain heater tissue that had been froMitochondria1 respiration zen in liquid nitrogen and stored at - 80°C. These Oxygen consumption was measured using a assays were performed at the University of Guelph Clark-type electrode in temperature-controlled using a Varian DMS UV-visible spectrophotome(20°C) 1ml cells. One volume of mitochondrial sus- ter (Varian Canada Inc., Georgetown, Ontario). pension was added t o 9 volumes of a reaction Reaction rates for all enzymes were determined at medium consisting of 150 mM KCl, 30 mM HEPES, 20°C in 50 mM imidazole buffer, pH 7.2. Lactate 10 mM KH2P04,and 1%BSA (swordfish heater dehydrogenase and hexokinase were measured acorgan) or 400 mM urea, 200 mM TMAO (trimeth- cording to the method of Singer and Ballantyne ylamine oxide), 150 mM KC1, 50 mM sucrose, 30 ('89). Glycerol kinase, a-glycerophosphate dehydromM HEPES, 1%BSA, and 10 mM KH2P04,pH 7.2 genase, and pyruvate kinase were assayed as des(mako red muscle). The pH of the reaction media cribed by Vijayan et al. ('91) except that 0.2 mM was adjusted to 7.2 at 20°C. State 3 respiration NADH was used in the pyruvate kinase assay. (defined by Chance and Williams, ,551,which occurs RESULTS in the presence of saturating ADP concentrations (0.4mM), was used to assess maximal rates of subThe methods described above yield mitochondria strate oxidation. displaying tightly coupled respiration (see Fig. lA,B). The highest respiratory control ratios (the Protein determination ratio of the respiratory rate in the presence of ADP Protein contents of mitochondrial and tissue t o that after ADP has been phosphorylated) were preparations were determined with a microassay of obtained when swordfish (9.76 3.03, n = 4) and Bradford ('76) using appropriate blanks and BSA mako(ll.21 k 1.39,n = 3)wereoxidizingglutamine. Heater organ mitochondria oxidize a wide variety as a standard. * J.S. BALLANTYNE ET AL. 112 TABLE 1. Oxidation of substrates by mitochondria isolated from the heater organ of the swordfish Xiphias gladius and warm red muscle of the mako shark Isurus oxyrinchus' Substrate Pyruvate (10 mM) + malate (0.1 mM) a-Glycerophosphate (5mM) + CaCl, (1mM) Succinate (5 mM) Glutamate (10 mM) Glutamine (12.5 mM) Proline (50 mM) Octanoyl carnitine (40pM) + malate (0.1mM) Palmitoyl carnitine (40pM) + malate (0.1 mM) Acetoacetate (0.3 mM) p-Hydroxybutyrate (25 mM swordfish); (10 mM mako) Endogenous State 3 respiration rate (nmoles 0,iminlmg mitochondrial motein) Swordfish heater Mako red muscle 151.28 ? 61.80 (4) 34.34 t 12.53 (3) 167.03 t 91.16 (4) ND 106.66 t 49.50 (3) 21.74 t 3.25 (3) 167.16 t 48.10 (4) 45.94 t 13.81 (3) 148.81 t- 50.39 (4) 49.84 t 2.21 (3) 37.48 t 16.56 (4) 38.43 - 41.16 (2) 116.33 47.61 (4) ND TABLE 2. Maximal activities of selected enzymes in the brain heater organ of the swordfish' Enzyme Hexokinase Pyruvate kinase Lactate dehydrogenase glycerol kinase a-glycerophosphate dehydrogenase Activity 2.34 2 1.76(4) 116.42 t 23.69(4) 104.96 t 15.37(4) 0.08 t 0.02 (4) 10.83 t 2.99(4) 'Values given are means ? SEM. Units are micromoles substrate convertedlminigm. Methods are as described in Materials and Methods. Numbers of preparations are given in parentheses. Brown fat mitochondria are poorly coupled due to the presence of the regulated proton channel thermogenin. When the channel is activated, the pH * gradient across the inner mitochondrial membrane is dissipated, and oxidative metabolism proceeds at a high (uncoupled) rate resulting in substantial 143.70 t 49.80 (4) ND heat production (Nedergaard and Cannon, '90). Block and Carey ('87) indicated that thermogenin ND 21.00 - 26.94 (2) 60.32 t 27.34 (4) 46.53 t 3.92 (3) is not present in the heater organ of billfish and proposed that the mitochondria in this tissue are coupled and produce ATP. The findings of our study 18.41 t 8.97 (3) 3.72 ? 2.24(3) support their proposition, since mitochondria iso'Values given are means t SE. The number of mitochondrial prep- lated from the heater organ had high respiratory arations are in parentheses, and ND indicates not detected. Substrates control ratios and high rates of oxidation in vitro not oxidized: sarcosine (5 mM), p-alanine (5 mM), citrate (20 mM), are dependent on ADP. alanine (10 mM), glycine (10 mM), isocitrate (1 mM), betaine (10 mM), According to the model of Block and Carey ('87), aspartate (10 mM), citrulline ( 2 mM), and arginine (1 mM). mitochondrial respiration in the intact heater organ of substrates (Table11, and oxidationof a-glycerophos- is stimulated by cytosolic ADP generated by the phate was stimulated fourfold by the addition of Ca2+-ATPasein the SR. Calcium continuously leaks CaC12(Fig. lC,D). Although only octanoyl carnitine across the SR membrane, maintaining high rates and palmitoyl carnitine are listed in Table 1, the of ATP hydrolysis and high rates of oxidative metabheater organ also oxidized the carnitine esters of olism. Block and Carey ('87) focused on the hydrolstearic, decanoic, and hexanoic acids. In contrast ysis of ATP in heat production. Currently accepted to the heater organ mitochondria, the mitochondria estimates of the heat released upon hydrolysis of isolated from the red muscle of the shark did not ATP to ADP and Pi, under physiological conditions oxidize palmitoyl carnitine, although there was a of pH, Mg2+, and ionic strength, indicate that very slight oxidation of hexanoyl carnitine. The pre- about 50% of the energy available is released as ferred substrates for the shark mitochondria were heat (Alberty, '69). This is equivalent to about - 6.0 pyruvate plus malate, amino acids (glutamate, glu- kcal/mole. Far more heat is generated in the comtamine, and proline), and P-hydroxybutyrate (Table bustion of substrates for the synthesis of ATP. As1).The activities of hexokinase, a-glycerophosphate suming an enthalpy of combustion of - 2,398 kcall dehydrogenase,pyruvate kinase, and glycerol kinase mole for palmitate (Weast,'70) and 23 0 2 consumed and lactate dehydrogenase in the swordfish heater yielding 138ATP with - 6.0 kcal/moleconserved per ATP, oxidation of palmitate with ATP synthesis reorgan are presented in Table 2. leases 65.5%of the heat of combustion. This amounts DISCUSSION to 1,571kcal/mole of palmitate. In contrast, hydrolWhereas all animal tissues produce heat, only two, ysis of the 138 ATP produced yields only 828 kcal. mammalian brown fat and fish brain heater organ, ATP hydrolysis, therefore, can only account for appear to be specifically designed for heat produc- 34.5%of the total heat produced. In the present study, calcium stimulated the oxition. Both tissues have abundant mitochondria indicative of a high metabolic rate, yet maintenance dation of a-glycerophosphate in swordfish brain of these high rates occurs by different mechanisms. heater organ. A similar phenomenon has been ob- THERMOGENIC TISSUES OF SWORDFISH AND MAKO SHARK served in another type of muscle, insect flight muscle (Sacktor, '76). Such activation of mitochondrial respiration may play a role in coupling SR calcium release to mitochondrial respiration. In swordfish brain heater organ, calcium leaking from the SR may stimulate mitochondrial respiration before cytosolic levels of ADP rise. A similar phenomenon has been observed in several cell types (Balaban, '90). The coupling between the Ca2+-ATPaseand mitochondria oxidative phosphorylation may be either ADP or cytosolic calcium. Elucidation of the mechanism of respiratory control in intact heater organ cells must await further experimentation. Oxidation rates of swordfish heater organ mitochondria exceed those reported for other fish even when differences in temperature are taken into account (Moyes et al., '89, '90; Chamberlin et al., '91). If high rates of substrate oxidation are to be used to warm the eye and brain, heat must be produced at a rate greater than it is lost to the environment. Using the mitochondrial respiration rates we have obtained (Table 11,it is now possible to estimate the rates of heat production of an intact heater organ. Swordfish heater organ contains 35 nmol cytochrome c/g tissue (Carey, '82) and animal mitochondria typically contain 0.45 nmol cytochrome c/mg mitochondrial protein (Tzagoloff,'82). Therefore, oxidation of palmitoyl carnitine in the isolated mitochondria can be converted to a respiration rate of 11,176.7 nmol 02/min/g tissue in the intact tissue. Since 23 O2 are consumed for every palmitate oxidized, palmitate oxidation would yield 1.16 cal/g tissue or 0.0806 Wlg tissue. A heater organ from a large swordfish may weigh 150 g (Carey, '82) and would produce 12.09 W. A similar calculation for glucose oxidation, based on mitochondrial pyruvate oxidation and the production of two pyruvate molecules for every glucose molecule, yields a heat production of 0.365 W/g or 54.7 W for 150 g tissue. The value we have calculated for pyruvate oxidation (0.365 Wlg) is similar t o rates of 0.5 W per gram reported for brown adipose tissue (see Rothwell and Stock, '85, for review). These calculated values include the heat released by ATP hydrolysis. If it is assumed that the thermal conductivity (Weast, '70) of the heater organ is the same as that of water, a 150 g sphere of tissue with a n average diffusion distance of 3.296 cm (radius of the sphere) would lose 2.336 W when faced with a 10°C temperature gradient (Carey, '82, reports temperature gradients of 10"-14"C for free ranging swordfish). Clearly, at 20"C, the oxidation of palmitate or glucose can yield sufficient heat t o maintain a 10°C temperature gradient between the environment and 113 a 150 g heater organ. It should be noted, however, that our calculations of heat production are based on state 3 rates of mitochondrial respiration, which undoubtedly exceed the rate in the intact heater organ. Although mitochondrial respiration rate in the intact heater organ has not been measured, mitochondria in aerobic mammalian cells operate at 25% of their maximal capacity (Harris et al., '81). If a similar situation occurs in heater organs, our calculations indicate that a 150 g heater organ could maintain a 10°C gradient when oxidizing either glucose or fats. The larger surface area to volume ratios of the heater organs of small fish would have greater difficulty in maintaining a 10°C gradient, especially if they are oxidizing lipids. The high rates of heat production from glucose oxidation may help to explain the high activities of glycolytic enzymes in this tissue. The very high hexokinase levels detected in this tissue indicate that exogenous glucose supplies much of the carbohydrate oxidized in this tissue. This is supported by ultrastructural studies that indicate that there are no stores of glycogen or lipid in billfish heater organs (Block,'86). High rates of glycolysis require rapid regeneration of cytosolic NAD. Lactate dehydrogenase activity is relatively low in the heater organ and may have a minor role in balancing cytosolic redox. In contrast, a-glycerophosphate is rapidly oxidized by the mitochondria and cytosolic NAD-dependenta-glycerophosphatedehydrogenase is found in high activity. The a-glycerophosphate cycle balances redox in insect flight muscle (Sacktor, '76) and squid mantle muscle (Storeyand Hochachka, '76) and may play a similar role in the heater organ. In mammalian liver, the a-glycerophosphatecycle (Wernette et al., '81) is responsive to changing cytoplasmic calcium mediated by catecholamines. Catecholamine stimulation of brain heater function has been proposed (Block and Carey, '87); perhaps it affects the a-glycerophosphate cycle in this tissue as well. Although swordfish heater organ mitochondria are tightly coupled, they do resemble brown adipose tissue mitochondria with respect to their substrate preferences. Mitochondria from both tissues oxidize pyruvate plus malate, palmitoyl carnitine, and a-glycerophosphate at high rates (Nicholls et al., '72). Other metabolic similarities lie in the high activities of hexokinase and a-glycerophosphate dehydrogenase in both tissues (Flatmark and Pedersen,'7 5 ) .Whereas the role of a-glycerophosphate dehydrogenase in redox balance is discussed above, another role for this enzyme may involve metabolism of glycerol, a by-product of triglyceride metabolism. J.S. BALLANTYNE ET AL. 114 There is no evidence to suggest high levels of glycerol kinase in brown adipose tissue; indeed, glycerol is released by brown adipose tissue during thermogenesis (Hull and Hardman, '70). The swordfish heater organ also resembles brown adipose tissue in that it lacks significant levels of glycerol kinase. An unusual finding of the present study is the oxidation of ketone bodies in swordfish heater organ mitochondria. It is generally assumed that ketone body oxidation does not occur in teleost fishes (Zammit and Newsholme, '79; Beis et al., '80). Nevertheless, this enzyme has been measured in only a few teleost species. Swordfish are the most advanced teleosts examined to date for this enzyme. Unless this enzyme arose independently in the Xiphiidae, it seems likely that ketone body oxidation exists in more primitive teleosts. Assays for this enzyme in other teleost families seems warranted. The warm red muscle of the mako shark displays no special characteristics for thermogenesis. The mitochondrial substrate preferences as well as respiration rates are similar to those described for the red muscle of the dogfish (Moyeset al., '90; Chamberlin and Ballantyne, unpublished data), an elasmobranch with "cold' red muscles. The mitochondria are well-coupled,indicating that ADP produced during muscle activity stimulates oxidative metabolism and heat production. ACKNOWLEDGMENTS This research was funded through a Natural Sciences and Engineering Research Council of Canada (NSERC) operating grant to J.S.B. and by a NSERCIDepartment of Fisheries and Oceans joint subventions grant to J.S.B. Ship time was generously provided by the Department of Fisheries and Oceans. We gratefully acknowledge the enthusiastic assistance of the captain and crew of the E.E. Prince in obtaining fish. In addition, we also thank Jaime Alvarado for his assistance, Martin Gerrits for shore support, and the inventor of duct tape for equipment security in stormy seas. LITERATURE CITED Alberty, R.A. (1969) Standard Gibbs free energy, enthalpy, and entropy changes as a function of pH and pMg for several reactions involving adenosine phosphates. J. Biol. Chem., 244: 3290-3302. Balaban, R.S. (1990) Regulation of oxidative phosphorylation in the mammalian cell. Am. J. Physiol., 258:C377-C389. Beis, A., V.A. Zammit, and E.A. Newsholme (1980) Activities of 3-hydroxybutyrate dehydrogenase, 3-oxoacid CoA-transferase and acetoacetyl-CoAthiolase in relation to ketone-body utilisation in muscles of vertebrates and invertebrates. Eur. J. Biochem., 104:209-215. Block, B.A. (1985) Warm brain and eye temperatures in sharks. J. Comp. Physiol., 156B:229-236. Block, B.A. (1986) Structure of the brain and eye heater tissue in marlins, sailfish and speartishes. J. Morphol., 190:169-189. Block, B.A., and Carey, F.G. (1987)Billfish brain and eye heater: A new look at nonshivering heat production. NIPS, 2:208-212. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248-254. Carey, F.G. (1982) A brain heater in the swordfish. Science, 216:1327-1329. Carey, F.G., and J.M. Teal (1969) Mako and porbeagle: Warmbodied sharks. Comp. Biochem. Physiol., 28:199-204. Carey, F.G., J.M. Teal, and J.W. Kanwisher (1981) The visceral temperature of mackerel sharks. Physiol. Zool., 54:334-344. Chamberlin, M.E., H.C. Glemet, and J.S. Ballantyne (1991) Glutamine metabolism in a holostean fish (Amia calua) and a teleost (Saluelinus namaycush). Am. J . Physiol., 260: R159-R166. Chance, B., and G.R. Williams (1955)A simple and rapid assay of oxidative phosphorylation. Nature, 175:1120-1121. Flatmark, T., and J.I. Pedersen (1975) Brown adipose tissue mitochondria. Biochim. Biophys. Acta, 416:53-103. Harris, S.I., R.S. Balaban, L. Barrett, and L.J. Mandel (1981) Mitochondria1 respiratory capacity and Na+- and K'-dependent adenosine triphosphatase mediated ion transport. J . Biol. Chem., 259:10319-10328, Hull, D., and M.J. Hardman (1970) Brown adipose tissue in newborn mammals. In: Brown Adipose Tissue. 0. Lindberg, ed., Elsevier, New York, p. 337. Moyes, C.D., L.T. Buck, P.W. Hochachka, and R.K. Suarez (1989) Oxidative properties of carp red and white muscle. J. Exp. Biol., 143:321-331. Moyes, C.D., L.T. Buck, and P.W. Hochachka (1990) Mitochondrial and peroxisomal fatty acid oxidation in elasmobranchs. Am. J . Physiol., 258:R756-R762. Nedergaard J., and B. Cannon (1990) Mammalian hibernation. Lond., 326B:669-686. Philos. Trans. R. SOC. Nicholls, D.G., H.J. Gray and 0.Lindberg (1972) Mitochondria from hamster brown-adipose tissue. Eur. J . Biochem. 31: 526-533. Rothwell, N.J., and M.J. Stock (1985) Biological distribution and significance of brown adipose tissue. Comp. Biochem. Physiol. 824:745-751. Sacktor, B. (1976) Biochemical adaptations for flight in the insect. Biochem. SOC.Symp. 41:111. Singer, T.D., and J.S. Ballantyne (1989) Absence of extrahepatic lipid oxidation in a freshwater elasmobranch, the dwarf stingray Potamotrygon magdalenae: Evidence from enzyme activities. J . Exp. Zool., 251:355-360. Storey,K.B., and P.W. Hochachka (1976)Alpha-glycerophosphate dehydrogenases role in the control of the cytoplasmic arm of the alpha-glycerophosphate cycle in squid muscle. Comp. Biochem. Physiol., 52B3169-173. Tzagoloff, A. (1982) Mitochondria.Plenum Press, New York. p 342. Vijayan, M.M., J.S. Ballantyne, and J.F. Leatherland (1991) Cortisol-induced changes in some aspects of the intermediary metabolism of brook charr (Saluelinus fontinalzs). Gen. Comp. Endocrinol. (in press). Weast, R.C. (1970) Handbook of Chemistry and Physics, 51st Ed., Chemical Rubber Publishing, Cleveland, OH. Wernette, M.E., R.S. Ochs, and H.A. Lardy (1981) Ca2+stimulation of rat liver mitochondrial glycerophosphate dehydrogenase. J. Biol. Chem., 256:12767-12771. Zammit, V.A., and E.A. Newsholme (1979)Activities of enzymes of fat and ketone body metabolism and effects of starvation on blood concentrations of glucose and fat fuels in teleost and elasmobranch fish. Biochem. J., 184:313-322.