Atrial granules in the dog heart after cardiac denervation

Summary Because the increase in sodium excretion during left atrial distension in conscious dogs is abolished after chronic cardiac denervation, we have investigated whether this is a result of the disappearance of specific atrial granules. Electron microscopy and light-microscopical and ultrastructural immunohistochemistry of canine atria show that atrial granules displaying immunoreactivity for cardiac hormones of the cardiodilatin/atrial natriuretic polypeptide (CDD/ANP) family are still present in denervated left and right atria, although reduced in quantity. It is concluded that the atrial-induced natriuresis is not only related to the existence of specific atrial granules. The functional link between atrial-induced natriuresis provoked by atrial distension and the release of atrial polypeptide hormones remains uncertain because the denervated heart can secrete CDD although the diuretic-natriuretic effect is altered.     

Functional Analysis of the Photosynthetic Apparatus of Prochlorothrix hollandica (Prochlorales), a Chlorophyll b Containing Procaryote

Light-shade adaptation of the chlorophyll a/b containing procaryote Prochlorothrix hollandica was studied in semicontinuous cultures adapted to 8, 80 and 200 μmole quanta per square meter per second. Chlorophyll a contents based on dry weight differed by a factor of 6 and chlorophyll b by a factor of 2.5 between the two extreme light conditions. Light utilization efficiencies determined from photosynthesis response curves were found to decrease in low light grown cultures due to lower light harvesting efficiencies; quantum requirements were constant at limiting and saturating irradiances for growth. At saturating growth irradiances, changes in light saturated oxygen evolution rate originated from changes in chlorophyll a antenna relative to the number of reaction centers II. At light-limiting conditions both the number of reaction centers II and the antenna size changed. The amount of chlorophyll b relative to reaction center II remained constant. As in cyanobacteria, the ratio of reaction center I to reaction center II was modulated during light-shade adaptation. On the other hand, time constants for photosynthetic electron transport (4 milliseconds) were low as observed in green algae and diatoms. The occurrence of state one to two and state two to one transitions is reported here. Another feature linking photosynthetic electron transport in P. hollandica to that in the eucaryotic photosynthetic apparatus was blockage of the state one to two transition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Although chlorophyll b was reported in association with photosystem I, the 630 nanometer light effect does not exclude that chlorophyll b is the photoreceptor for the state one to two transition.     

Inhibition and labeling of sodium, potassium ATPase by the dialdehyde derivative of ATP

Canine renal Na,K-ATPase was treated with ATP dialdehyde, "oxATP" (20 microM), as described by G. Ponzio, B. Rossi, and M. Lazdunski (1983, J. Biol. Chem. 258, 8201-8205). In this system, a by-product, formaldehyde, was the inactivator. We modified the system to minimize such inhibition and to speed up the reaction. oxATP itself inactivated the enzyme at a rate that was slow at first and later speeded up. We fitted a precursor-product model to the data. Labeling with [3H]oxATP indicated about three sites per alpha beta protomer at complete inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled enzyme showed radioactivity in many components, in the alpha and beta subunits and in small molecules at the tracker dye region. ATP (20 mM) prevented all labeling and inactivation. Ponzio et al. concluded that oxATP labels covalently an ATP binding site. Our experiments did not support this conclusion. Ouabain did not affect labeling. Sodium stimulated both inhibition and labeling more than potassium did, indicating a high-affinity ATP binding site, if any. But nucleotide specificity for preventing or producing inhibition did not correspond to nucleotide specificity for binding of ATP to the native enzyme. Blocking the ATP binding center with fluorescein isothiocyanate or fluorosulfonyl benzoyl adenosine had no effect on [3H]oxATP labeling. ATP also prevented [3H]oxATP labeling of bovine serum albumin or of integral-membrane proteins.     

Time-dependent 31P saturation transfer in the phosphoglucomutase reaction. Characterization of the spin system for the Cd(II) enzyme and evaluation of rate constants for the transfer process

Time-dependent 31P saturation-transfer studies were conducted with the Cd2+-activated form of muscle phosphoglucomutase to probe the origin of the 100-fold difference between its catalytic efficiency (in terms of kcat) and that of the more efficient Mg2+-activated enzyme. The present paper describes the equilibrium mixture of phosphoglucomutase and its substrate/product pair when the concentration of the Cd2+ enzyme approaches that of the substrate and how the nine-spin 31P NMR system provided by this mixture was treated. It shows that the presence of abortive complexes is not a significant factor in the reduced activity of the Cd2+ enzyme since the complex of the dephosphoenzyme and glucose 1,6-bisphosphate, which accounts for a large majority of the enzyme present at equilibrium, is catalytically competent. It also shows that rate constants for saturation transfer obtained at three different ratios of enzyme to free substrate are mutually compatible. These constants, which were measured at chemical equilibrium, can be used to provide a quantitative kinetic rationale for the reduced steady-state activity elicited by Cd2+ relative to Mg2+ [cf. Ray, W.J., Post, C.B., & Puvathingal, J.M. (1989) Biochemistry (following paper in this issue)]. They also provide minimal estimates of 350 and 150 s-1 for the rate constants describing (PO3-) transfer from the Cd2+ phosphoenzyme to the 6-position of bound glucose 1-phosphate and to the 1-position of bound glucose 6-phosphate, respectively. These minimal estimates are compared with analogous estimates for the Mg2+ and Li+ forms of the enzyme in the accompanying paper.     

Photoprotective pigment as an adaptive strategy in the antarctic moss Ceratodon purpureus

Summary Variation in leaf pigmentation from green to ginger is observed for Ceratodon purpureus (Hedw.) Brid. in Antarctica. Electron microscopy of ginger and green leaves reveals less thylakoid stacking, a response to greater light exposure, in the ginger leaves. In extremely exposed sites C. purpureus has low chlorophyll a/b ratios which correlate with decreased 77K chlorophyll fluorescence, indicating damage to chlorophyll a. Pigment analysis of ginger moss shows that even when the chlorophyll a/b ratio has not decreased the pigment composition differs from green moss. The increase in anthocyanin and decrease in chlorophyll concentrations largely account for the visual change from green to ginger. The ratio of total carotenoid to chlorophyll varies from 0.35 in green moss to 0.55 in the ginger moss, with violaxanthin increased preferentially. Since these changes in pigmentation are consistent with photoprotection and they are linked to light dependent variations in chloroplast structure, it appears that photoprotective pigments are a useful adaptation for the bright Antarctic environment.     

Transient states of geosmin,pigments, carbohydrates and proteins in continuous cultures of Oscillatoria brevis induced by changes in nitrogen supply

Transitions in the growth limiting factor from light (I) to nitrogen (N) and vice versa caused changes in geosmin production, protein and carbohydrate content, and the synthesis of pigments such as chlorophyll a (Chl a), phycobiliproteins (PBPs), and -carotene of the cyanobacterium Oscillatoria brevis. Following IN transition the first 150h, the decrease in protein content was compensated for by an increase of carbohydrates, and thereby, a constant biomass level was maintained in this period. Thereafter, biimass dropped to 15% of its initial level. A decrease in geosmin and pigment content was observed during transition from IN-limited growth. However, geosmin increased relative to phytol (Chl a) and -carotene which may indicate that a lowered demand for phytol and -carotene during N-limited growth allows isoprenoid precursors to be directed to geosmin rather than to pigment synthesis. Synthesis of Chl a and -carotene at the expense of geosmin was suggested for the observed start of increase in geosmin production only at the time that Chl a and -carotene had reached their I-limited steady state. Transition from nitrogen to light limited growth caused an acceleration of metabolism shown by a rapid decrease in carbohydrate content accompanied by an increase in protein content. The growth rate of the organisms temporarily exceeded the dilution rate of the culture and the biomass level increased 6-fold. Due to the only modest changes in geosmin production (2-fold) compared to changes in biomass level (6-fold) during I-or N-limited growth, environmental factors seem to have limited effect on geosmin production.Abbreviations Chl a chlorophyll a - dry wt dry weight; - I-limited light-limited - N-limited nitrogen-limited - PBP phycobiliprotein This research was performed at the Department of Microbiology, University of Amsterdam, with finacial support provided by the Royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50.

Pseudorabies virus (PRV) glycoprotein gp50 is the homolog of herpes simplex virus (HSV) glycoprotein D. Several cell lines that constitutively synthesize gp50 were constructed. Vero cells, HeLa cells, and pig kidney (MVPK) cells that produce gp50 all gave reduced yields of PRV and HSV progeny viruses when compared with the parent cell line or the same cell line transfected to produce a different protein. The reduction in virus yield was greatest at low multiplicities of infection. The Vero and HeLa cells that produce gp50 showed an even greater reduction in HSV yield than in PRV yield. This phenomenon may be an example in a herpesvirus of the interference observed in retroviruses or cross-protection in plant virus systems.     

Analysis of flow and vascular resistance in a model of the circle of Willis

A very simple model of the flow in the circle of Willis is described in this paper. Disregarding pulsatility and vessel wall elasticity, fluxes in all segments of the circle of Willis and its afferent and efferent vessels are calculated by applying the Poiseuille-Hagen formula. Comparison with the fluxes calculated numerically from a more sophisticated mathematical model, including pulsatility, vessel wall elasticity and nonlinear effects, revealed only very slight differences. In short, fluxes in the afferent vessels and the segments of the circle of Willis are influenced by any change of resistance within the network, whereas the fluxes in the efferent segments are dominated by the efferent resistance distribution. However, a great advantage of the present simple model is that it offers the possibility of an analytical approach which yields both an easy sensitivity analysis of parameters and an insight into the mechanisms that govern the flow in a network like the circle of Willis. It can be concluded that these mechanisms are similar to the principles of the Wheatstone bridge, known from electrical circuit theory.     

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.