Surface-charge related actions of polylysine on thylakoid membranes

Polycation binding to the negatively charged surface of chloroplast thylakoid membranes is known to cause an inhibition of photosystem I activity. It also interferes with the cation-dependent rearrangement of chlorophyll proteins in the thylakoid membrane. It was shown that added anions prevented or reversed the inhibition of photosystem I by polylysine without decreasing its binding to the membranes. Anions also caused a change in the interaction of the chlorophyll proteins in polylysine-treated thylakoids as indicated by an increase in the relative fluorescence intensity from photosystem II. In both cases, the relative effectiveness of the anions tested depended on their valence; for example, the tetravalent species Fe(CN)₆^4t- was effective at a concentration at least 2 orders of magnitude lower than the divalent species SO₄^2?. These results suggest that anions act by screening the positive charge of the polylysine-coated membrane surface. Measurements of the response of the anionic fluorescent probe 1-anilinonapthalene-8-sulfonate to an addition of anions to polylysine-treated thylakoids supported this contention. It was concluded that the action of polylysine on photosystem I and on the chlorophyll proteins is mediated by changes of the electrical properties of the thylakoid membrane and may not involve a direct binding of the polycation to the affected membrane proteins.     

Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.     

Paleolimnology of the Peten Lake District,Guatemala

Long-term changes of sedimentary particle-size distribution in two tropical lowland lakes were compared with changes of human population sizes, estimated archaeologically, in the drainage basins. Mean particle size of silt and clay fractions (<64 µm) varied between 3 and 15 µm. High positive skewness and kurtosis of the distributions were associated with smaller particle sizes; hence small mean size resulted from greater influx of small particles while influx of larger particles was probably constant. An inverse correlation between mean particle size and human population size is interpreted to mean that disturbance-induced erosion results in delivery of very fine inorganic particles at higher rates. Within any one basin, particle-size stratigraphy is more precisely related to archaeological time periods than is pollen stratigraphy. An absolute chronology still eludes us, owing to the failure of ¹⁴C dating of calcareous, colluvial sediments, but our relative chronology is now more precise than before. If certain assumptions about past hydrologic relations can be met, particle-size analysis is a way of comparing the histories of geographically very different lakes, including lakes from tropical, temperate, and arctic regions.     

Intracellular or extracellular calcium can be used to trigger C5a-stimulated enzyme secretion from rabbit neutrophils

The ability of C5a to stimulate lysosomal enzyme release and 45Ca2+ efflux from rabbit neutrophils was studied. C5a stimulated beta-glucuronidase release from cytochalasin B-treated neutrophils either in the presence or absence of extracellular calcium. Depletion of cell calcium by pretreatment with the calcium ionophore A23187 blocked both the ability of C5a to elicit enzyme release in the absence of extracellular calcium and its ability to stimulate 45Ca2+ efflux. Both actions were dose-dependent over the same concentration range (10(-8)-10(-6) M ionophore A23187). In contrast, ionophore pretreatment had no effect on C5a-stimulated enzyme release in the presence of extracellular calcium. These results suggest that (a) release of cell calcium is required for enzyme secretion in the absence of extracellular calcium, and (b) C5a can trigger near-maximal enzyme release by using calcium from either of two sources: the extracellular space or an intracellular site.     

An enzymatic assay for acetate in spent bacterial culture supernatants

A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supernatants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et al. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 microM. The assay is cheaper than commercially available assays and is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assay to the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs.     

PHOTOSYNTHETIC QUANTUM EFFICIENCIES OF PHYTOPLANKTON FROM PERENNIALLY ICE COVERED ANTARCTIC LAKES1

A recently introduced approach far estimating the photosynthetic quantum efficiency (φ) of a freshwater or marine phytoplankton community has been applied for the first time to high latitude polar ecosystems, namely four lakes of southern Victoria Land, Antarctica. Values for φ at various depths ranged from 0.0022–0.1560 when calculated using a recommended mean extinction coefficient for phytoplankton (i.e. k?_c= 0.016). By contrast, φ ranged from 0.0037–0.0760 when calculated using an empirically estimated value for k?_c of 0.0328. If the recommended k?_c= 0.016 more closely approaches an accurate estimate, then the φ valves indicate that the phytoplankton convert light to organic carbon more efficiently than elsewhere. However, if the empirically derived k?_c= 0.0328 more closely approaches an accurate estimate, then the φ values indicate the phytoplankton trap light more efficiently than elsewhere. Although we have not resolved whether light conversion (φ) or light trapping are more efficient, the results show that the phytoplankton of these Antarctic lakes are well adapted to performing photosynthesis under extremely low light conditions.     

Ammonia Regulation of Intermediary Metabolism in Photosynthesizing and Respiring Chlorella pyrenoidosa: Comparative Effects of Methylamine

The natural ¹³C abundance (¹³C value) of the field-grown leguminousplants (soybean, kidney bean, pea, azuki bean, mung bean, peanutand cowpea) was investigated by mass spectrometry with a precisionbetter than %0.2 for ¹³C. Among organs of premature plants,the leaves had the most negative values, and the nodules generallyhad the least negative values, and other organs, fruits, stemsand roots, showed intermediate values. In the soybeans so farinvestigated, the grains of nodulating plants exhibited higher¹³C values than nonnodulating lines. The ¹³C values of the grainsvaried depending on the species: peanuts showed the most negativevalues. Possible causes underlying these variations are discussed. (Received March 2, 1983; Accepted May 27, 1983)     

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.

Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.

When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.

     

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.