Effect of simulated microgravity on human lymphocytes

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

Contractile characteristics of the triceps surae muscle in healthy males during 120-days head-down tilt (HDT) and countermeasures

To reveal mechanisms responsible for changes in muscle contractility during microgravity, it seems expedient to perform similar studies under microgravity or conditions simulating microgravity. Among standard methods for simulating microgravity, hypokinesia modelling support unloading (or rather its redistribution), and hypodynamia are employed. Absence of weight loading, decreased muscular effort characteristic of the Earth conditions due to counteracting gravity, results in a general muscle underloading and therefore in lowered activity of the proprioceptive input. This may be one of the reasons not only for a resetting of motor coordination and control, but also for a gradual development of a persistent change in the motor control system. The basis of countermeasures against negative consequences of microgravity (hypokinesia) is the correct choice of countermeasures. In this connection of specific interest is a study of the magnitude of change in skeletal muscle contractility in humans after a variety of countermeasures when functional activity is lowered by a long-term 120-days HDT which is an adequate simulation of physiological microgravity-induced effects.     

Books

Book reviewed in this article:
B eehler , B.M. 1991. A Naturalist in New Guinea
B erthold , P. 1990. Vogelzug: eine kurze. aktuelle Gesamtübersicht
B ezzel , E. & P rinzinger , R. 1990. Ornithologie
B ub , H. 1991. Bird Trapping and Bird Banding
B urger , J. (ed.). 1988
B urke , T., D olf , G., J effreys , A.J. & W olff , R. (eds) 1991. DNA Fingerprinting: approaches and applications
C roxall , J.P. (ed.). 1991. Seabird Status and Conservation: a supplement
E rard , C. 1990. Ecologie et Comportement de Gobe-mouches
G alati , R. 1991. Golden-crowned Kinglets, Treetop Nesters of the North Woods
J ohnsgard , P.A. 1988. North American Owls: biology and natural history
J ohnsgard , P.A. 1991. Bustards. Hemipodes, and Sandgrouse: birds of dry places
K uroda , N. 1990. The Jungle Crows of Tokyo
L ekagul , B. & R ound , P.D. 1991. Birds of Thailand
L loyd , C. T asker , M.L. & P artridge , K. 1991. The Status of Seabirds in Britain and Ireland
L örhl , H. 1991. Die Haubenmeise
L oye , J. E. & Z uk , M. (eds) 1991. Bird-Parasite Interactions: ecology, evolution and behaviour
M itchell , A. 1989. The Fragile South Pacific: an ecological odyssey
O lney , P.J.S. & E llis , P. (eds) 1990. International Zoo Yearbook 1989, Vol. 29
O wen , M. & B lack , J.M. 1990. Waterfowl Ecology
R oberts , T.J. 1991. The Birds of Pakistan. Vol. 1; Regional studies and non-passeriformes
S chonn S., S cherzinger W., E xo K-L. & I lle R. 1991. Der Steinkauz. Athene noctua. Pp. 235, 139 monochrome photographs, 28 tables & 2 colour plates
S ibley , C.C. & A hlquist , J.E. 1990. Phylogeny and Classification of Birds: a study in molecular evolution
S kutch , A.F. 1991. Life of the Pigeon
S troud , D. & G lue , D. 1991. Britain's Birds in 1989–90: The conservation and monitoring review
Y eatman -B ekthelot , D. 1991. Atlas des Oiseaux de France en Hiver.     

Metabolic transitions in rat jaw muscles during postnatal development.

The program of acquisition of adult metabolic phenotypes was studied in three jaw muscles in order to determine the link between muscle metabolism and functional development. During early postnatal stages, there were similar transitions in the masseter, anterior digastric, and internal pterygoid muscles with respect to fiber growth, fiber type composition, and whole muscle energy metabolism. Oxidative capacity, as judged by the activities of the enzymes succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and beta-hydroxyacyl CoA dehydrogenase (beta OAC), rose sharply after birth to reach near maximal levels by 3 weeks. The capacities for glycolytic metabolism represented by lactate dehydrogenase (LDH), and for high-energy phosphate metabolism represented by adenylokinase (AK) and creatine kinase (CK) activities, rose gradually, not reaching peak values until 6 weeks or later. Thus, the maturation of oxidative metabolism preceded that of glycolytic metabolism in the developing jaw muscles. This was documented for individual fibers in the masseter muscle. Differential metabolic maturation among the jaw muscles was evident beyond 3 weeks. All three jaw muscles attained their specific adult fiber-type profile by about 6 weeks. This maturation program differed from that of hindlimb muscles [Nemeth et al., J Neurosci 9:2336-2343, 1989] and diaphragm muscle [Kelly et al., J Neurosci 11:1231-1242, 1991], reflecting their differential energy demands for contractile performance.     

Calculation of free-Mg2+ concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo

We have attempted to resolve the differences between the levels of free Mg2+ in muscle calculated by Wu et al. [Wu, S. T., Pieper, G. M., Salhany, J. M., & Eliot, R. S. (1981) Biochemistry 20, 7399-7403] (2.5 mM in guinea pig heart) and by Gupta and Moore [Gupta, R. K., & Moore, R. D. (1980) J. Biol. Chem. 255, 3987-3993] (0.6 mM in frog skeletal muscle) on the basis of substantially identical measurements by 31P NMR of the phosphate peaks in the spectrum of MgATP2-. The differences depend on the methods of calculation, including which reactions in which multiple equilibria are being considered. Biochemists and physical chemists customarily use different working definitions of the stability constant for MgATP2- in particular. Wu et al. used in their calculations, without reconciliation, methods involving three different operational definitions of the chelation equilibria involved. An algorithm for calculating Mg2+ and total ATP, which can be carried out with a hand calculator, is described here. With it, we calculated Mg2+ levels that agree with those determined by Gupta et al. [Gupta, R. K., Benkovic, J. L., & Rose, Z. B. (1978) J. Biol. Chem. 253, 6165-6171] with their in vitro systems. We therefore agree with the finding of Gupta and Moore that the Mg2+ level in skeletal and cardiac muscle is 0.6 mM.     

Isometric training of young rats — Effects upon hind limb muscles

The soleus, rectus femoris, and gastrocnemius muscles of young rats trained isometrically for 4 weeks were studied by light and electron microscopy.--The percentage of fast-twitch oxidative muscle fibers decreased at the cost of the fast-twitch glycolytic fibers in the rectus femoris muscle. The percentages of the slow-twitch oxidative fibers did not change significantly in any of the muscles studied. The changes in the areas of the muscle fibers were specific for the muscle and the fiber type and indicate geometrical rearrangements of the fibers in the trained muscles. The Z and M lines were broader in the soleus (containing about 85% slow-twitch oxidative fibers) than in the rectus femoris muscle (containing about 90% fast-twitch glycolytic fibers), while the sarcomere length and the pseudo-H zone were similar. The length of the myosin filaments appeared to be slightly shorter in the fast rectus femoris than in the slow soleus muscle.--The hypothesis on the temporal progress of muscle adaptation to training (Müller, 1974) was substantiated. Correlations between biochemical (Exner et al., 1973a) and histochemical parameters measuring the oxidative capacity were preserved during adaptation to training. The comparison of the histochemical results with the physiological data on similar animals (Exner et al., 1973a) suggests a complex relationship between the contraction time and the percentage of fast-twitch muscle fibers.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

Antibiotic-resistant Escherichia coli from wastewater before and after treatment in stabilization ponds in the arid region of Marrakech, Morocco

Book reviewed in this article:
M edical M icrobiology (1991). By G.F. Brooks, J.S. Butel & L.N. Ornston.
I mmunoassays in F ood and A griculture (1991). Edited by A. Paraf & G. Peltre.
P rinciples of B acteriology , V irology and I mmunity .
F oodborne P athogens : A n I llustrated T ext (1991). By A.H. Varnam & M.G. Evans.     

Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2.

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.     

Total epidermal cell walls of pea stems respond differently to auxin than does the outer epidermal wall alone

The effect of auxin on cell wall mass in the epidermis of third internodes of Pisum sativum L. cv. Alaska grown in dim red light was investigated using epidermal peels, to determine whether epidermal peels reflect the behavior of the outer epidermal cell wall. In contrast to the outer epidermal wall itself, where auxin caused thinning in proportion to growth (M.S. Bret-Harte et al, 1991, Planta 185, 462–471), auxin promoted an increase in wall mass in epidermal peels from treated internode segments in the absence of exogenously supplied sugar. The percentage gain in mass was smaller than the percentage elongation, however, so mass per unit length decreased in peels from auxin-treated segments. Epidermal peels from auxin-treated segments gained more wall mass than control peels even when adhering internal tissue at the basal end of the peel was removed. Epidermal peels also had a gross composition different from that of the outer wall alone (M.S. Bret-Harte and L.D. Talbott, 1993, Planta 190, 369–378). These discrepancies can be explained by the observation that the outer wall makes up only 30% of the mass of the epidermal peel. It appears that the inner walls of the epidermis, and walls of the outer layer of cortical cells that remain attached to the epidermis during peeling, nearly maintain their thickness by biosynthesis while the outer wall loses mass as previously described (Bret-Harte et al. 1991). These results indicate that epidermal peels may not be a good system for examining the biochemical and physiological properties of the outer epidermal cell wall.I would like to thank Dr. Peter M. Ray, of Stanford University, for the use of experimental facilities, helpful discussions, and technical and editorial assistance, Dr. Winslow R. Briggs, of the Carnegie Institute of Washington, for helpful discussions and for the use of experimental facilities, Dr. Paul B. Green, of Stanford University, for financial support, and Dr. Wendy K. Silk, of the Department of Land, Air, and Water Resources, University of California, Davis, for financial support. This work was supported by a National Science Foundation Graduate Fellowship, National Science Foundation grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk in the final writing.     

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.     

Malaria and ovalocytosis — molecular mimicry?

Two recently published reports have described findings which will have a profound impact on the understanding of molecular mechanisms of human resistance to malaria infection. In Melanesian ovalocytosis, a genetic polymorphism found in Papua New Guinea and parts of South East Asia, the red cells are highly resistant to invasion by various species of malaria parasite. The molecular nature of the defect in ovalocytic erythrocytes was not known. Recent reports by Liu et al., (Liu, S.-C., Zhai, S., Palek, J., Golan, D., Amato, D., Hassan, K., Nurse, G., Babona, D., Coetzer, T., Jarolim, P. Zaik, M. and Borwein, S. (1990) N. Engl. J. Med. 323, 1530–1538.) and Jones et al. (Jones, G.L., Edmundson, H.M., Wesche, D. and Saul, A. (1991) Biochim. Biophys. Acta 1096, 33–40.) have now identified the abnormality in the band 3 protein of ovalocytic red cell membranes. A major discovery in the Jones et al, study is the presence of an extended peptide at the N-terminus of ovalocyte band 3 protein. This novel 13 amino acid extended sequence is not found in the primary structure of normal band 3 protein and was suggested to be the cause of band 3 defect in ovalocytes. We have analyzed this extended sequence through Genbank using SWISS-PROT database and found that an almost identical sequence exists in a malaria parasite protein called RESA.     

Books

A lstrom , P., C olston , P. & L ewington , I. 1991, A Field Guide to the Rare Birds of Britain and Europe
A mos , E.J.R. 1991. A Guide to The Birds of Bermuda
B enito -E spinal .E. 1990. OiseauxdesPetites Antilles. Birds of the West Indies
D owner , A. & S utton , R. 1990. Birds of Jamaica: Field guide
B irkhead , T.R. & M øller , A.P. 1992. Sperm Competition in Birds: Evolutionary causes and consequences
B rooke , M. & B irkhead , T. (eds) 1991. The Cambridge Encyclopedia of Ornithology
F orshaw J. (ed.) 1991. Encyclopedia of Animals: Birds
D eeming , D.C. & F erguson , M.W.J, (eds) 1991. Egg Incubation: its effects on embryonic development in birds and reptiles
D owner , A. & S utton , R. 1990. Birds of Jamaica: Field guide
F ehr , H. 1991. Die Vögel in Norden des Kreises Aachen
F inlayson , M. & M oser . M. (EDS) 1991. Wetlands
F orshaw . J. (ED) 1991. Encyclopedia of Animals: Birds
H epper . P.G. (ed.) 1991. Kin Recognition
H eches R.N. (ed.) 1990. Behavioural Mechanisms of Food Selection
J obling , J.A. 1991. A Dictionary of Scientific Bird Names
J ohnsgard , P. 1991 Crane Music. A natural history of American cranes
L ohmann , M. & R utschke , E. 1991. Vogelparadiese: 170 Biotope in Deutschland. Band 3: Ost- und Mitteldeutschland
L ongmore , W. 1991. Honeyeaters and their Allies of Australia
M aclean . G. L. 1990. Ornithology for Africa
Pinowski. J. K avanagh , B.P. & G orski . W. (eds) 1991. Nestling Mortality of Granivorous Birds due to Microorganisms and Toxic Substances
S ibley . C.G. & M onroe . B.L., Jr. 1990. Distribution and Taxonomy of Birds of the World
V oisin , C, 1991. The Herons of Europe     

Malaria and ovalocytosis--molecular mimicry?

Two recently published reports have described findings which will have a profound impact on the understanding of molecular mechanisms of human resistance to malaria infection. In Melanesian ovalocytosis, a genetic polymorphism found in Papua New Guinea and parts of South East Asia, the red cells are highly resistant to invasion by various species of malaria parasite. The molecular nature of the defect in ovalocytic erythrocytes was not known. Recent reports by Liu et al. (Liu, S.-C., Zhai, S., Palek, J., Golan, D., Amato, D., Hassan, K., Nurse, G., Babona, D., Coetzer, T., Jarolim, P. Zaik, M. and Borwein, S. (1990) N. Engl. J. Med. 323, 1530-1538.) and Jones et al. (Jones, G.L., Edmundson, H.M., Wesche, D. and Saul, A. (1991) Biochim. Biophys. Acta 1096, 33-40.) have now identified the abnormality in the band 3 protein of ovalocytic red cell membranes. A major discovery in the Jones et al. study is the presence of an extended peptide at the N-terminus of ovalocyte band 3 protein. This novel 13 amino acid extended sequence is not found in the primary structure of normal band 3 protein and was suggested to be the cause of band 3 defect in ovalocytes. We have analyzed this extended sequence through Genbank using SWISS-PROT database and found that an almost identical sequence exists in a malaria parasite protein called RESA.     

Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.