Excitation-contraction coupling and extracellular calcium transients in rabbit atrium: reconstruction of basic cellular mechanisms

Interactions of electrogenic sodium-calcium exchange, calcium channel and sarcoplasmic reticulum in the mammalian heart have been explored by simulation of extracellular calcium transients measured with tetramethylmurexide in rabbit atrium. The approach has been to use the simplest possible formulations of these mechanisms, which together with a minimum number of additional mechanisms allow reconstruction of action potentials, intracellular calcium transients and extracellular calcium transients. A 3:1 sodium-calcium exchange stoichiometry is assumed. Calcium-channel inactivation is assumed to take place by a voltage-dependent mechanism, which is accelerated by a rise in intracellular calcium; intracellular calcium release becomes a major physiological regulator of calcium influx via calcium channels. A calcium release mechanism is assumed, which is both calcium- and voltage-sensitive, and which undergoes prolonged inactivation. 200 microM cytosolic calcium buffer is assumed. For most simulations only instantaneous potassium conductances are simulated so as to study the other mechanisms independently of time- and calcium-dependent outward current. Thus, the model reconstructs extracellular calcium transients and typical action-potential configuration changes during steady-state and non-steady-state stimulation from the mechanisms directly involved in trans-sarcolemmal calcium movements. The model predicts relatively small trans-sarcolemmal calcium movements during regular stimulation (ca. 2 mumol kg-1 fresh mass per excitation); calcium current is fully activated within 2 ms of excitation, inactivation is substantially complete within 30 ms, and sodium-calcium exchange significantly resists repolarization from approximately -30 mV. Net calcium movements many times larger are possible during non-steady-state stimulation. Long action potentials at premature excitations or after inhibition of calcium release can be supported almost exclusively by calcium current (net calcium influx 5-30 mumol kg-1 fresh mass); action potentials during potentiated post-stimulatory contractions can be supported almost exclusively by sodium-calcium exchange (net calcium efflux 4-20 mumol kg-1 fresh mass). Large calcium movements between the extracellular space and the sarcoplasmic reticulum can take place through the cytosol with virtually no contractile activation. The simulations provide integrated explanations of electrical activity, contractile function and trans-sarcolemmal calcium movements, which were outside the explanatory range of previous models.     

Pathogenesis of passive Heymann glomerulonephritis: chlorpromazine inhibits antibody-mediated redistribution of cell surface antigens and prevents development of the disease

Two hypotheses were tested: first, that in LEW rats the interaction of sheep (or rabbit) anti-brush border antibodies with antigens (Heymann antigens) expressed on the plasma membrane of glomerular visceral epithelial cells is characterized by initial redistribution of immune complexes on the cell surface and by subsequent shedding of immune complexes in the subepithelial part of the capillary wall; and secondly, that this interaction is inhibited by chlorpromazine, a drug that displaces calcium ions from binding sites linking the plasma membrane to the cytoskeleton, and which blocks the redistribution of IgG on the surface of B lymphocytes exposed to anti-IgG antibodies. The studies were performed in vitro on cultured LEW glomerular epithelial cells and in vivo in LEW rats. In cultured glomerular epithelial cells exposed at 37 degrees C to anti-brush border IgG, chlorpromazine prevented, in a dose-dependent manner, the redistribution ("capping") of Heymann antigens and the fixation of complement. The renal glomeruli of chlorpromazine-treated LEW rats examined 6 and 48 hr after transfer of anti-brush border antibodies had punctate and, later, punctate and diffuse deposits of sheep (or rabbit) IgG on glomerular epithelial cells, but not similar deposits of rat C3. Moreover, granular subepithelial deposits of sheep (or rabbit) IgG and rat C3, characteristic of passive Heymann glomerulonephritis, did not develop, although deposits of sheep IgG were detected by immunoelectron microscopy on the microvilli of glomerular epithelial cells. Comparative studies on rats with similar reductions in glomerular filtration rates, produced by high doses of chlorpromazine or with renal artery stenosis, showed that the findings were not the consequence of insufficient delivery of antibody to glomerular epithelial cells. The results are consistent with the interpretation that Heymann glomerulonephritis is induced by mechanisms of redistribution of cell surface antigens comparable to those that govern the interaction of surface antigens (or receptors) with appropriate ligands in B lymphocytes and other classical in vitro systems.     

Structure-function relation in hemoglobin Köln (β98 val → met)

Hemoglobin Köln, an unstable hemoglobin resulting from the substitution of normal valine by methionine at FG 5 (β98) is the most commonly encountered unstable hemoglobin. In Hb Köln from a hitherto undetected family, we confirmed earlier observations of heme depletion and high oxygen affinity, with the absence of co-operative interactions. In an effort to elucidate the basis of the altered oxygen equilibria, sedimentation velocity and the kinetics of the reactions of the abnormal hemoglobin with ligands were studied. The results of ultracentrifuge studies indicated that at pH 7 hemoglobin Köln, in the liganded as well as in the deoxy form, existed largely as dimers. The ratio of optical densities at 540 nm and 280 nm indicate that the abnormal β chains were heme depleted. Hb Köln reacted with

approximately 20 times faster than did hemoglobin A (Hb A). However, the corresponding rate constants for O₂ dissociation

are similar for Hb Köln and Hb A. For Hb Köln the two rate constants, l′ and k show little pH or concentration dependence. Thus, the high oxygen affinity of Hb Köln (P_1/2 = 0.2 mm Hg at 10 °C, pH 6.8) arises in part from a larger combination rate constant for the reaction with oxygen. Addition of a 20-fold excess of 2,3-diphosphoglyceric acid did not affect the kinetics of CO-combination. However, in the presence of a sixfold excess of heme, the fast monophasic CO-combination reaction was replaced by a biphasic one. The rate constant of the slow phase was approximately the same as the corresponding rate constant for Hb A. The fast and slow phases were presumably due to the reaction of CO with Hb Köln dimers (α^hβ^o heme-saturated tetramers, respectively. The results of the preeent study are explained in terms of weakened α?β and heme-globin contacts in the mutant.     

A tissue-culture model of cartilage breakdown in rheumatoid arthritis. Quantitative aspects of proteoglycan release

1. The destruction of articular cartilage in human rheumatoid and other arthritides is the result of diverse mechanical, inflammatory and local cellular factors. A tissue-culture model for studying cartilage-synovial interactions that may be involved in the final common pathway of joint destruction is described. 2. Matrix breakdown was studied in vitro by using bovine nasal-cartilage discs cultivated in contact with synovium. Synovia were obtained from human and animal sources. Human tissue came from patients with ;classical' rheumatoid arthritis, and animal tissue from rabbits with antigen-induced arthritis. 3. Cartilage discs increased their proteoglycan content 2-3-fold during 8 days in culture. Proteoglycan was also released into culture medium, approx. 70% arising from cartilage breakdown. 4. Synovial explants from human rheumatoid and rabbit antigen-induced arthritis produced equivalent stimulation of proteoglycan release. After an initial lag phase, the breakdown rate rose abruptly to a maximum, resulting in a 2-fold increase of proteoglycan accumulation in culture medium after 8-10 days. 5. High-molecular-weight products shed into culture media were characterized chromatographically and by differential enzymic digestion. Proteoglycan-chondroitin sulphate accounted for 90% of the released polyanion, and its partial degradation in the presence of synovial explants was consistent with limited proteolytic cleavage. 6. Rheumatoid synovium applied to dead cartilage increased the basal rate of proteoglycan release. Living cartilage was capable of more extensive autolysis, even in the absence of synovium. However, optimal proteoglycan release required the interaction of living synovium with live cartilage. These findings support the view that a significant component of cartilage breakdown may be chondrocyte-mediated.     

Inaccuracies in Measurement of Dried Milk Powders

Infant feeds made up from dried milk are known to be hazardous if the mixture is too concentrated. Tests with five commercial brands of dried milk showed that nurses, midwives, and mothers often made up feeds incorrectly; in most cases the amount of powder used was excessive.Fewer mistakes would be made if scoops were standardized or, better, if the milk was made up in small, accurately weighed packets.     

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematodePanagrellus silusiae

Summary The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematodePanagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous¹²⁵I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplification alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.Supported by the National Research Council of Canada     

Parasitism in the bathypelagic fish, Melanostigma pammelas

Three hundred and thirty-seven specimens of the zoarcid fish, Melanostigma pammelas, collected between December, 1950 and December, 1969 from bathypelagic waters off southern California, were examined for their parasites. Incidences of infection were: digentic Trematoda 61%, Myxosporida 39%, larval Nematoda 9%. Two adult trematodes are described: Fellodistomum melanostigmum nov. sp. from the intestine, and Aponurus pacificus nov. sp. from the stomach. This parasite record is markedly unlike the typical record reported for eastern Pacific mesopelagic fishes in which many larval nematodes and few, if any, adult trematodes were found. Our long-term objective is to explain parasite pattern differences in the deep ocean in terms of their biological and physical environments. Our initial purpose is to determine the numbers and kinds of parasites in M. pammelas.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

Further studies on the lipid metabolism of the normal and vitamin B12-deficient chick embryo

1. The triglyceride, cholesterol ester and total phospholipid fractions were isolated from the livers and yolk sacs of normal and vitamin B₁₂-deficient chick embryos after 13, 15, 17, 19 and 21 days of incubation, and the fatty acid compositions were determined. 2. At all stages of incubation, the concentration of cholesterol ester in the livers of the normal embryos were greater, and on days 15 and 17 the concentrations of triglyceride were considerably less, than the corresponding concentrations in the livers of the deficient embryos. 3. Between day 13 and day 21 of incubation the concentration of oleic acid in the liver triglycerides of the normal embryos increased, whereas the concentrations of palmitic acid and docosahexaenoic acid decreased. Vitamin B₁₂ deficiency resulted in higher concentrations of palmitic acid in the liver triglycerides on days 15, 17 and 19, higher concentrations of C₁₈ polyunsaturated acids on days 13 and 15 and lower concentrations of oleic acid on days 13, 15, 17 and 19. 4. At all stages of development, cholesterol oleate accounted for almost 80% of the total liver cholesterol esters in both normal and deficient embryos. 5. As development of the normal embryos progressed, the concentrations of palmitic acid and arachidonic acid in the liver phospholipid decreased, whereas the concentrations of stearic acid and docosahexaenoic acid increased. Vitamin B₁₂ deficiency resulted in markedly higher concentrations of stearic acid and palmitic acid and markedly lower concentrations of arachidonic acid and docosahexaenoic acid in the liver phospholipids. 6. Vitamin B₁₂ deficiency did not influence the fatty acid composition of the triglyceride, cholesterol ester and phospholipid fractions either in the yolks of fertile unincubated eggs or in the yolks obtained from eggs that had been incubated for 13, 15, 17, 19 and 21 days.