[Effects of topographical condition and sampling number on the interpolation precision of forest litter carbon density].

The territory of Zhejiang Province, East China was grouped into 3 topographical units (plain-coastal area, hill-basin area, and mountain area) to investigate the effects of topographical condition and sampling number on the Kriging interpolation precision of forest litter carbon density in the Province. The forest litter carbon density in the 3 topographical units showed a medium spatial correlation pattern, with the semi-variance nugget/sill ratio value ranged from 28.3% to 72.4%. The Kriging interpolation precision was in the order of plain-coastal area > hill-basin area > mountain area, indicating that the Kriging interpolation precision decreased with the increase of terrain complexity degree. Within the same topographical units, the Kriging interpolation precision improved with increasing sampling number, being most obvious in the mountain area. Therefore, under complicated topographical conditions, greater sampling number was required to achieve a high precision of Kriging interpolation.

Effect of solute hydrogen bonding capacity on osmotic stability of lysosomes.

The effect of solute hydrogen bonding capacity on the osmotic stability of lysosomes was examined through measurement of free enzyme activity of lysosomes after their incubation in sucrose and poly(ethylene glycol) (PEG) (1500-6000 Da molecular mass) media. Free enzyme activity of the lysosomes was less in the PEG medium than that in the sucrose medium under the same hypotonic condition. The lysosomal enzyme latency loss decreased with increasing hydrogen bonding capacity of the solute. In addition, the lysosomes lost less latency at lower incubation temperature. The results indicate that solute hydrogen bonding capacity plays an important role in the osmotic protection of an incubation medium to lysosomes.

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons.

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K+ and H+, and the organelle stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-beta-cyclodextrin (MbetaCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K+/H+ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MbetaCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K+ and H+. Loss of this membrane sterol is critical for the organelle acidification and stability.

Loss of membrane cholesterol affects lysosomal osmotic stability.

Lysosomal destabilization is critical for the organelle and living cells. Using methyl-beta-cyclodextrin (M beta CD) to selectively deplete lysosomal membrane cholesterol, we investigated the effect of cholesterol on the organelle osmotic stability. The results show that loss of membrane cholesterol caused changes in the lysosomal osmotic properties. The lysosomes lost the ability to resist osmotic shock and became more sensitive to osmotic stress. As a result, the lysosomes lost membrane integrity rapidly. Microscope observation showed that the lysosomes were liable to swell in the hypotonic sucrose medium. It is presumably due to an enhancement of the lysosomal permeability to water caused by the loss of membrane cholesterol. The results indicate an important role of cholesterol in the maintenance of lysosomal stability.

Mechanism of lysophosphatidylcholine-induced lysosome destabilization.

Lysosomal destabilization is critical for the organelle and living cells. Phospholipase A(2 )(PLA(2)) was shown to be able to destabilize lysosomes under some conditions. By what mechanism the enzyme affects lysosomal stability is not fully studied. In this study, we investigated the effects of lysophosphatidylcholine (lysoPC), a PLA(2)-produced lipid metabolite, on lysosomal ion permeability, osmotic sensitivity and stability. By measuring lysosomal beta-hexosaminidase free activity, membrane potential, proton leakage and their enzyme latency loss in hypotonic sucrose medium, we established that lysoPC could increase the lysosomal permeability to both potassium ions and protons and enhance lysosomal osmotic sensitivity. These changes in lysosomal membrane properties promoted entry of potassium ions into lysosomes via K(+)/H(+) exchange. The resultant osmotic imbalance across the membranes led to losses of lysosomal integrity. The enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. These results suggest that lysoPC may play a key role in PLA(2)-induced lysosomal destabilization.

Guanosine 5'-[gamma-thio]triphosphate-mediated activation of cytosol phospholipase C caused lysosomal destabilization.

Lysosomal disintegration is critical for the organelle functions and cellular viability. In this study, we established that guanosine 5'-[gamma-thio]triphosphate (GTP-gamma-S)-activated cytosol of rat hepatocytes could increase lysosomal permeability to both potassium ions and protons and osmotically destabilize the lysosomes via K(+)/H(+) exchange. These results were obtained through measurements of lysosomal beta-hexosaminidase-free activity, membrane potential and intralysosomal pH. Assays of phospholipase C (PLC) activity show that cytosolic PLC was activated upon addition of GTP-gamma-S to the cytosol. The effects of cytosol on the lysosomes could be abolished by D609, an inhibitor of PLC, but not by the inhibitors of phospholipase A(2). The cytosol-treated lysosomes disintegrated markedly in hypotonic sucrose medium, reflecting that the lysosomal osmotic sensitivity increased. Microscopic observations showed that the lysosomes became more swollen in hypotonic sucrose medium. This indicates that the cytosol treatment induced osmotic shock to the lysosomes and an influx of water into the organelle.

Effects of phospholipase A2 on the lysosomal ion permeability and osmotic sensitivity.

In this study, we investigated the mechanism of PLA(2)-induced lysosomal destabilization. Through the measurements of lysosomal beta-hexosaminidase free activity, their membrane potential, the intra-lysosomal pH and the lysosomal latency loss in hypotonic sucrose medium, we established that PLA(2) could increase the lysosomal membrane permeability to both potassium ions and protons. The enzyme could also enhance the organelle osmotic sensitivity. The increases in the lysosomal ion permeability promoted influx of potassium ions into the lysosomes via K(+)/H(+) exchange. The resulted osmotic imbalance across the lysosomal membranes osmotically destabilized the lysosomes. In addition, the enhancement of the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in the osmotic stress. The results explain how PLA(2) destabilized the lysosomes.

Effects of arachidonic acid on the lysosomal ion permeability and osmotic stability.

In this study, we investigated the effects of arachidonic acid, a PLA2-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K+/H+ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.

Mechanism of cytosol phospholipase C and sphingomyelinase-induced lysosome destabilization.

Lysosomal disintegration may cause apoptosis, necrosis and some diseases. However, mechanisms for these events are still unclear. In this study, we measured lysosomal beta-hexosaminidase free activity, membrane potential and intralysosomal pH. The results revealed that the cytosolic extracts of rat hepatocytes could increase the lysosomal permeability to both potassium ions and protons, and osmotically destabilize lysosomes via K(+)/H(+) exchange. The effects of cytosol on lysosomes could be completely abolished by D609, which inhibited both phospholipase C and sphingomyelinase, and partly prevented by sphingomyelinase inhibitor Ara-AMP, but not by the inhibitors of PLA(2). Moreover, purified phospholipase C could destabilize the lysosomes while phospholipase A(2) and phospholipase D did not produce such effects. The cytosolic phospholipases hydrolyzed lysosomal membrane phospholipids by 50%, which could be prevented by D609. Disintegration of the cytosol-treated lysosomes biphasically depended on the cytosolic [Ca(2+)]. The cytosol did not disintegrate lysosomes below 100 nM or above 10 muM cytosolic [Ca(2+)], but markedly destabilized lysosomes at about 340 nM [Ca(2+)]. The results suggest that cytosolic phospholipase C and sphingomyelinase may be responsible for the alterations in lysosomal stability by increasing the ion permeability.

Influence of membrane physical state on lysosomal potassium ion permeability.

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.

Lysosome destabilization by cytosolic extracts, putative involvement of Ca(2+)/phospholipase C.

Lysosomal disintegration is a crucial event for living cells, but mechanisms for the event are still unclear. In this study, we established that the cytosolic extracts could enhance lysosomal osmotic sensitivity and osmotically destabilize the lysosomes. The cytosol also caused the lysosomes to become more swollen in the hypotonic sucrose medium. The results indicate that the cytosol induced an osmotic shock to the lysosomes and an influx of water into the organelle. Since the effects of cytosol on the lysosomes could be abolished by O-tricyclo[5.2.1.0(2,6)]dec-9-yl dithiocarbonate potassium salt (D609), a specific inhibitor of cytosolic phospholipase C (PLC), the PLC might play an important role in the lysosomal osmotic destabilization. The activity of cytosolic PLC and the extent of enzyme latency loss of the cytosol-treated lysosomes exhibited a similar biphasic dependence on the cytosolic Ca2+ concentration. In addition, the cytosol did not osmotically destabilize the lysosomes until the cytosolic calcium ions rose above 100 nM. It suggests that the destabilization effect of cytosol on the lysosomes is Ca(2+)-dependent.

Influence of the physical states of membrane surface area and center area on lysosomal proton permeability.

The physical state of the lysosomal membrane was modulated with the membrane fluidizers n-propanol and n-octanol and with the membrane rigidifiers cholesteryl hemisuccinate and cholesterol. Membrane fluidity was examined by the steady-state fluorescence anisotropy of 2-(9-anthroyloxy) palmitic acid and 16-(9-anthroyloxy) palmitic acid. Fluidizing the membranes at the surface and center areas increased the proton permeability coefficient by 92.8 and 18.0%, respectively. Rigidifying the membranes at the surface and center areas decreased the coefficient by 68.2 and 40.2%, respectively. Proton leakage of the lysosomes increased and decreased similar to the coefficient changes with the treatments. The results indicate that lysosomal proton permeability is affected by its membrane's physical state, and the physical state of the membrane surface area affects the proton permeability more markedly. The proton permeability coefficient of liposomes was similar to that of lysosomes, suggesting that efflux of lysosomal protons might occur through the lipid part of the bilayer but not transmembrane proteins.

Enhancement of lysosomal proton permeability induced by photooxidation of membrane thiol groups.

Effects of photooxidation of membrane thiol groups on lysosomal proton permeability were studied by measuring intralysosomal pH with fluorescein isothiocyanate-dextran and monitoring proton leakage with p-nitrophenol. Methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linking of the membrane proteins, which was established by the measurement of residual membrane thiol groups with 5,5'-dithio-bis(2-nitrobenzoic acid) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The cross-linking of proteins could be abolished by subsequent treatment of the photodamaged lysosomes with dithiothreitol, indicating that the proteins were linked via disulfide bonds. In addition, the photodamage of lysosomes raised the intralysosomal pH and caused leakage of the lysosomal protons, which could also be reversed by subsequent dithiothreitol treatment. This indicates that lysosomal proton permeability can be increased by photooxidation of the membrane thiol groups and recovered to the normal level by reduction of the groups.

Enhancement of lysosomal osmotic sensitivity induced by the photooxidation of membrane thiol groups.

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) and examining the lysosomal beta-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75000 to 125000), which was visualized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.