Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Affinity, capacity and oxygen sensitivity of two different mechanisms for bicarbonate utilization in Ulva lactuca L. (Chlorophyta)

Ulva lactuca, collected on the west coast of Sweden at the end of May, was able to utilize the HCO₃ ^? pool of seawater only through extracellular dehydration via carbonic anhydrase, followed by uptake of the CO₂ formed. A decrease in the CO₂ supply via this mechanism resulted in the gradual development of an additional method of HCO₃ ^? utilization, namely a direct uptake of HCO₃ ^? . Photosynthesis could then be supported by either a ‘HCO₃ ^? dehydration mechanism’ or a ‘HCO₃ ^? uptake mechanism’. Through selective inhibition of either of these mechanisms, the physiological properties of the other could be assessed. These properties suggest that the HCO₃ ^? uptake mechanism of U. lactuca is important under conditions when low concentrations of inorganic C, high pH and high external O₂ concentrations would limit photosynthesis supported by the HCO₃ ^? dehydration mechanism. Such conditions may occur during intense irradiation of the alga in rockpools or in shallow bays with low rates of water exchange. The results are discussed in relation to a possible coupling between mechanisms for inorganic C acquisition and cell structure (or even morphology) of green macroalgae. They also illustrate some necessary precautions when using Michaelis–Menten kinetics for estimations of V_max and K_1/2 values.     

Characteristics of arylsulfatase in Russian sturgeon (Acipenser gueldenstaedti) semen

Spermatozoa of sturgeons (Acipenseriformes), unlike teleosts, possess an acrosome. This paper provides data concerning biochemical characteristics of arylsulfatase (AS), an acrosomal enzyme, found in Russian sturgeon spermatozoa and seminal plasma. The enzymes were purified by a four-step procedure, using n-butanol extraction, ion-exchange chromatography repeated twice and gel filtration. High purity of our enzymes was confirmed by silver staining electrophoresis and an immunological experiment. Kinetic parameters indicated that the purified enzymes belong to arylsulfatase type A. Similarity of the seminal plasma arylsulfatase to the spermatozoan enzyme showed us that arylsulfatase from seminal plasma might originate from damaged spermatozoa. The possible physiological consequences of the presence of arylsulfatase in Russian sturgeon semen are discussed.     

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells

Summary Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.     

Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n = 3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean ± SEM; 0.51 ± 0.21, 0.35 ± 0.09, p < 0.05), (2) lower (p < 0.05) secretion of IGF-binding protein 4 (9.6 ± 1.2 vs. 17.4 ± 2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6 ± 1.9 vs. 39.1 ± 6.3 μIU/ml), (3) lower protein expression of IGF2 “clearance” receptor in cell lysate (0.28 ± 0.03 vs. 0.53 ± 0.02 OD U/mm², p < 0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.     

Intracellular GABA-Activated In→Out Permeation of Chloride Across the Deiters' Neuron Membrane: Modulation by Phosphorylating Activities

The modulation of intracellular GABA activated ³⁶Cl^– inout permeation across single Deiters' neuron membranes has been studied in a microchamber system. Addition of Mg²⁺/ATP on the membrane cytoplasmic side reduces strongly the GABA effect as does ATP alone. However, the greatest inhibition of the GABA effect is given by the addition of Mg²⁺ to the intracellular side buffer: a complete block of the stimulation by GABA of ³⁶Cl^– inout permeation. This is interpreted as due to the presence in this case of a constant concentration of exogenous Mg²⁺ acting together with endogenous ATP in the small cytoplasmic layer on the membrane inner side. The addition of ADP to Mg²⁺/ATP increases the inhibitory effect of the latter. This is presumably due to an extra increase of ATP, locally under the membrane, due to phosphorylation of ADP by endogenous phosphocreatine. Overall, the data confirm that phosphorylating conditions impair the intracellular GABA action on ³⁶Cl^– inout permeation.     

Blood plasma model predictions for the proposed bone-seeking radiopharmaceutical [Sn]Sn(IV)-N,N′,N′-trimethylenephosphonate-poly(ethyleneimine)

In an attempt to elucidate the in vivo stability of the prospective radiopharmaceutical [^117mSn]Sn(IV)-PEI-MP, where PEI-MP stands for N,N′,N′-trimethylenephosphonate-polyethyleneimine, glass electrode potentiometry was used to determine the stability constants of the Sn⁴⁺ ion as complexed with a variety of physiological amino acids. In addition, linear free energy relationship (LFER) correlation plots were used to extrapolate the constants of the major blood plasma ligands, based on data from Cu²⁺, Pb²⁺, and Zn²⁺. In so doing, a thermodynamic model of blood plasma was established for Sn⁴⁺ from which the complexation tendencies of Sn⁴⁺ were predicted in the event of the intravenous administration of such a drug. It was found that the Sn(IV)-PEI-MP could succumb to competition by the glutamine amino acid, which forms more stable complex(es), whilst the PEI-MP gets taken up largely by Ca²⁺. Also, this study shows the value of the in vitro experiments and modeling performed for radiopharmaceutical research and for attempts to reduce the number of animal experiments.