The effect of hematoporphyrin and light on human platelets. II. Uptake of hematoporphyrin

A linear relationship was demonstrated between the reciprocals of the concentration of free hematoporphyrin and the moles of hematoporphyrin taken up by the platelet in the dark. radiated platelets took up more hematoporphyrin than did controls; this increase in uptake was accounted for by the movement of the dye across the damaged membrane of the cell. platelets irradiated at 4°c remained impermeable to hematoporphyrin until warmed to 37°c. during the initial three to four minutes of exposure to light at 37°c, there was no additional uptake of hematoporphyrin by platelets in comparison to controls. between six to ten minutes irradiation, the uptake of hematoporphyrin increased linearly with the log time of irradiation. thereafter, no further uptake occurred. a further increase in uptake of dye was demonstrated by both control and irradiated platelets at a reduced ph. this study enables a correlation to be made between the effects of hematoporphyrin on the platelet and the uptake of this agent by the platelet.     

The effect of hematoporphyrin and light on human platelets. I. Morphologic, functional, and biochemical changes

The morphologic, functional, and biochemical changes produced by hematoporphyrin and light in human platelets have been characterized. by phase microscopy the cells appeared swollen and resembled signet rings; by electron microscopy they showed considerable loss of cytoplasm and their contour was smoother than normal. irradiated platelets were not aggregable by thrombin and calcium chloride, although they contained clottable protein, and were incapable of supporting clot retraction. a linear relationship was demonstrated between the per cent depletion of serotonin from irradiated platelets and the log dose of hematoporphyrin. the depletion of serotonin from these platelets was related lineraly to the log of time of exposure to light during the initial six minutes of exposure; but thereafter continued at a constant rate. the temperature of incubation influenced directly the rate of depletion of serotonin from irradiated platelets but did not influence the movement of serotonin into these platelets. atp was diminished considerably in irradiated platelets. these changes are attributable to damage to the membrane of the platelet by hematoporphyrin and light. These studies provide additional information about the blood platelet in terms of its response to photodynamic action.     

Aggregation and release reaction of washed platelets stimulated by lanthanum.

Ionized lanthanum caused clumping of washed platelets. This clumping response could be reversed by chelating agents but was not impaired by known inhibitors of platelets aggregation. Aggregation by lanthanum was not restricted to the unique clumping properties of platelets but occurred in fixed platelets and red cells and was most likely based on an electrostatic interaction.Lanthanum was able to stimulate as well as to inhibit serotonin release from platelets.At a concentration of 1 mM, lanthanum evoked a release of serotonin from washed platelets at 37°C. This release reaction was inhibited at 18°C or by prior treatment of platelets with neuraminidase or NEM.At a high concentration (10 mM), lanthanum did not stimulate the platelet release reaction but inhibited that induced by all stimuli investigated, presumably due to a fixation of membrane molecules.The release reaction promoted by thrombin or A 23187, but not that by collagen, was inhibited by a low concentration of lanthanum (0.1 mM). This inhibition is based on an interaction of lanthanum with the stimuli rather than with the platelet surface.     

Distribution of Acid Phosphatase During Autolysis in Bovine Liver

The percentage distribution of acid phosphatase between lysosomes and cytoplasm in bovine liver during autolysis at 37°C was investigated. The share of the cytoplasmic acid phosphatase activity of the total acid phosphatase activity in liver tissue increased during autolysis being before incubation 28–42 % and after 24 hrs.' incubation at 37°C 63–94 %. Microbiological contamination increased the proportion of cytoplasmic acid phosphatase. When bovine liver was incubated at 37°C for 24 hrs., the activity of the total acid phosphatase decreased to about 50 % of the initial activity. During a 16 days' incubation at 4°C the total acid phosphatase activity of bovine liver, however, remained unchanged.     

The effects of injections of D,L-5-hydroxytryptophan on the efflux of endogenous serotonin and 5-hydroxyindoleacetic acid from a synaptosomal fraction.

Abstract— The effects of i.p. injections of SO mg/kg d,l-5-hydroxytryptophan (5-HTP) and saline alone on the in uitro release of endogenous serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were studied using preparations of axon terminals (P₂ isolated from the telencephalon of rats. The level of 5-HT was 2-fold greater and the level of 5-HIAA was 5-fold greater in the P₂ fraction isolated from rats given the d,l-5-HTP injection than from rats given saline injections. At 37°C the in vitro efflux of 5-HT and 5-HIAA from the P₂ fractions of animals injected with 5-HTP 30min before killing was approx 3 times higher than the saline control group. The amount of 5-HT and 5-HIAA released at 37°C was 3–5 times higher than the amount released at 0°C for both the 5-HTP and saline injected rats. Increasing the concentration of potassium ions in the media to 55 mm significantly increased the release of 5-HT but not 5-HIAA in both groups of animals. The amount of 5-HT released by 55mm-K⁺ was about 2-fold higher from the P₂ fraction isolated from rats given 5-HTP injections with respect to those given saline injections. The potassium stimulated release of 5-HT was calcium dependent. The data thus indicate that injection of 50 mg/kg d,l-5-HTP in rats can cause an increase in the level of 5-HT and 5-HIAA in a crude synaptosomal fraction and that as a result of this increase, there is a temperature dependent increased release of 5-HT and 5-HIAA under normal resting membrane conditions. There is also an increased release of 5-HT as a result of membrane depolarizing conditions induced by elevated potassium levels which is calcium dependent.     

Prostaglandin endoperoxides promote calcium release from a platelet membrane fraction in vitro

A calcium sequestering platelet membrane fraction was prepared and the effect of arachidonic acid, PGG₂ and PGH₂ on calcium content evaluated. At 4°C, 6.7–16.7 μM arachidonic acid caused significant release of calcium from preloaded vesicles. Such release was completely inhibited by aspirin pretreating the platelets from which the membrane fraction was prepared. γ-linolenic acid, not a substrate for prostaglandin synthesis, did not cause calcium release. At 37°C, after a 5 minute calcium loading of the membrane vesicles, arachidonic acid, PGG₂, and PGH₂ caused release of calcium. Calcium release by the PGG2 and PGH₂ was only slightly inhibited by aspirin. Imidazole, which prevented conversion of the prostaglandin endoperoxides to thromboxanes, also only slightly inhibited calcium release. Other prostaglandins including PGD₂, PGE₁, PGE₂ and PGD₂ had no effect on the calcium content of the vesicles. These studies suggest that PGG₂ and PGH₂ may exert their effects on platelets by mobilizing calcium from an internal membrane store to make it available to promote platelet activation.     

Aspirin inhibits Ca2t+-stimulated fatty acid release from human washed platelets

Ca²⁺ at 2mM concentration stimulates the release of saturated and unsaturated fatty acids from intact washed platelets incubated at 37°C with stirring. Aspirin at a concentration of 0.4mM inhibits both cyclo-oxygenase activity and fatty acid efflux induced by Cat+. Thus, in intact washed platelets, aspirin reduces formation of cyclo-oxygenase products by direct inhibition of the enzyme and by reducing the availability of precursor arachidonate.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Variability and stability of selected components in rainbow trout Salmo gairdneri serum and the precision of automated analysis in measuring these components*

Eighteen components in rainbow trout serum were tested for variability among individuals and stability during storage. In addition, the precision of an automated serum analysis system was determined. Stability of serum components was observed over 42 days at temperatures of 25° C, 4° C and - 10° C. Components tested included: albumin, total protein, blood urea nitrogen, cholesterol, chloride, glucose, potassium, sodium, cholinesterase, alkaline phosphatase, lactic dehydrogenase, a-hydroxybutyrate dehydrogenase, glutamic pyruvic transaminase, phosphohexose isomerase, inorganic phosphorus, calcium, creatinine, and creatine phosphokinase. Fish serum was generally more stable than human serum when stored at 25° C and 4° C and similar in stability at - 10° C. Precision of analytical methodologies was excellent for all components measured except creatine phosphokinase.     

Effect of dimethylsulphoxide on the functional characteristics of isolated perfused rat liver

Exposure of rat liver, perfused with 7% BSA in Krebs-Ringer bicarbonate buffer, to 1.4 m Me₂SO at 35 °C had no effect on the release of potassium from the livers, but the rate of urea synthesis fell from 0.6 to 0.1 μmol/min. Bile production also decreased and the total amount collected during perfusion was only half that produced by controls. After perfusion for 4 hr at 35 °C control livers and those exposed to Me₂SO started to release GOT into the perfusate but livers exposed to the cryoprotective compound released the enzyme at a faster rate.Exposure of livers to Me₂SO at 5 °C resulted in potassium being released at a slower rate (0.98 μmol/min) than from cooled controls (1.19 μmol/min) and urea synthesis was decreased from 0.8 to 0.2 μmol/min. Bile production also declined but, because bile flow normally ceases during hypothermia, the effect on this aspect of liver function was probably less than was found at 35 °C. Release of GOT from livers exposed to Me₂SO at 5 °C was quite different from that observed at 35 °C; the enzyme appeared in the perfusate after about 8 hr and it was present in much lower concentration than was found with appropriately cooled controls which started to release the enzyme after 6 hr.Thus, exposure of rat liver to Me₂SO at 5 °C appears to be slightly less damaging than exposure at 35 °C and it may even have a beneficial effect on some aspects of liver function in vitro.     

Release of ascorbate from a synaptosomal fraction of rat brain

We have studied factors controlling the release of endogenous ascorbate from synaptosomes prepared from various regions of the rat brain. Ascorbate was spontaneously released from synaptosomes, and this efflux could be enhanced by incubation at 37°C. A further additional ascorbate release could be induced by potassium depolarization or, in striatal, hippocampal and cortical synaptosomes, by incubation with the amino acid glutamate. Spontaneous, depolarization and glutamate-evoked ascorbate release were shown to occur by separate mechanisms. Glutamate-evoked ascorbate release occurred by a heteroexchange mechanism. In cerebellar synaptosomes there was no evidence for such heteroexchange; however, in synaptosomes of this brain region kainic acid induced ascorbate release, probably by acting on excitatory amino acid receptors. The results are discussed in relation to the changes in extracellular brain ascorbate occurring in vivo.     

The Impact of Hydrothermal and Dilute Acid Pretreatments and Inorganic Metals on Thermal Decomposition of Agricultural Residues and Agricultural Residue Ash Properties

The impacts of hydrothermal and dilute acid pretreatments and alkali and alkaline earth metals (AAEMs) on the thermal degradation of biomass were studied. Besides, the influence of these pretreatments on the biomass ash properties was investigated. The influence of pretreatments on the biomass thermal degradation was manifested in the removal of potassium out of the biomass. The presence of potassium in the biomass catalyzed cellulose thermal degradation and increased the char percentage at temperatures higher than 380 °C. Pretreatments were effective at removing the potassium from biomass and dramatically reduced the char percentage at temperatures higher than 380 °C. It was found that the best burning temperature for biomass ash production was 500 °C because at this temperature the thermal degradation of biomass was completed under pure combustion. It was shown that when burning biomass in oxygen-limited environments, removing AAEMs, particularly potassium, will improve the quality of ash as a potential candidate for supplementary cementitious materials for concrete application.     

Effect of preservation at low temperature (4 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

When polymorphonuclear neutrophils were stored at 4 °C for up to 2 weeks, the maintenance of the integrity of PMNs was examined by determining changes in enzyme activity, enzyme release, stimulated superoxide anion generation, and sensitivity to hypotonicity. Until at least 3-day storage, no changes were observed in enzyme activity, enzyme release, and stimulated superoxide anion generagion. After 1-week storage, the ability of PMNs to generate superoxide anions decreased considerably and the extracellular release of lactate dehydrogenase (LDH) was observed. After 2 weeks of storage, this LDH release and inhibition of O₂^?-generating ability of PMNs increased further, although enzyme activities were only slightly affected except for acid p-nitrophenyl phosphatase. The resistance of PMNs to hypotonic solutions decreased with increasing preservation time at 4 °C.     

An enzymatic method for the production of 6-oxohexanoic acid from 6-aminohexanoic acid by an enzyme oxidizing ω-amino compounds from Phialemonium sp. AIU 274

An enzymatic method for 6-oxohexanoic acid production was developed using 6-aminohexanoic acid and an ω-amino group-oxidizing enzyme (ω-AOX) from Phialemonium sp. AIU 274. 6-Oxohexanoic acid was produced from 6-aminohexanoic acid with 100% yield by incubation with 0.3 U of the ω-AOX and 20 U of catalase at 30 °C for 30 h in 0.1 M potassium phosphate buffer (pH 7.0).     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report.

A comparative study has been made of platelets stored by freeze preservation following treatment with dimethyl sulfoxide (DMSO) or hydroxyethyl starch (HES) with fresh platelets and platelets stored at 4 °C for 48 hr. The indices studied were platelet recovery, pH, light microscope morphology, platelet Factor 3 (PF₃) availability and the hypotonic stress response. The DMSO preserved platelets gave a better response to hypotonic stress and incurred lesser degrees of membrane damage as demonstrated by PF₃ availability. There was however a significantly higher recovery of platelets treated with HES; with DMSO the osmotic damage inflicted during removal caused considerable lysis. Platelets frozen by DMSO or HES gave consistently better in vitro results than platelets stored at 4 °C for 48 hr. A preliminary clinical trial of HES preserved platelets has confirmed haemostatic effectiveness in vivo. HES being relatively nontoxic, platelets can be infused immediately after thawing and with minimal post thaw manipulation, thus maintaining a relatively closed system. It is concluded that cryopreservation with HES is a practical and effective means for long term platelet storage.     

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. III. Reappraisal of established methods

Established methods for analysis of components of lipopolysaccharides were assessed. Optimal release of glucosamine from lipopolysaccharide occurs after hydrolysis in 6 M hydrochloric acid at 100°C for 4 h and fatty acids are best released by treatment with boron trifluoride/methanol at 100°C for 6 h. The semicarbazide assay for 3-deoxy-d-manno-octulosonic acid was modified to give results comparable to those from the periodate/thiobarbituric acid method. It was concluded that each molecule of lipopolysaccharide from Salmonella minnesota R595 contains two octulosonic acid residues and only four fatty acids, on average. There are two amide-linked hydroxyacids, together with, on average, 0.5 residues of ester hydroxyacid and a total of 1.5 residues of ester-linked normal fatty acids. This conclusion differs from the accepted view of Salmonella lipid A, but is supported by NMR results.     

Cellular ion content changes during and after hyperthermia.

The potassium (K) level in mouse mastocytoma P815 cells undergoes a 40% reduction within 30 minutes of incubation at 43°C. It decreases further when the cells return to 37°C after a 60 minute 43°C incubation. A smaller change (20%) occurs after a 60 minute incubation at 41°C. Furthermore, nearly all of the lost K recovers in two hours after a subsequent incubation at 37°C. On the other hand, the sodium level in the cells increases by an amount much smaller than the potassium changes. However, the net loss of cations from the cells undergoing hyperthermia does not induce a simultaneous reduction of intracellular water volume.     

Interactions of lectins with CHO cell surface membranes. I. Competition studies indicate concanavalin A and WGA bind to discrete populations of sites

Human platelet-derived growth factor (PDGF) stimulates release of arachidonic acid from cellular phospholipids, synthesis and release of prostaglandins from the cell, and initiation of DNA synthesis in cultures of 3T3 Swiss mouse fibroblasts at similar concentrations with four independent preparations representing a million-fold range of purification. Stimulation of archidonic acid and prostaglandin release is an early event (beginning within minutes) in the response to PDGF treatment. Incubating cells with PDGF at 4°C followed by washing leads to activation of archidonic acid release on warming the cells to 37°C, consistent with binding of the factor to the cell surface. PDGF-stimulated arachidonic acid release, prostaglandin release, and initiation of DNA synthesis are all inhibited by phenylglyoxal at similar concentrations. These results suggest that activation of arachidonic acid release from phospholipids plays an essential role in the mechanism by which PDGF stimulates the initiation of DNA synthesis in 3T3 cells. The stimulation of initiation of DNA synthesis by PDGF does not appear to be mediated by the synthesis of prostaglandins or other known arachidonic acid metabolites because neither indomethacin (a fatty acid cyclooxygenase inhibitor) nor phenidone (a lipoxygenase inhibitor) inhibit initiation of DNA synthesis at concentrations which inhibit arachidonic acid metabolism. Although the activation of arachidonic acid release by PDGF is a calcium-dependent process, a simple calcium flux appears unimportant to the mechanism of activation. Evidence was also obtained against an involvement of sodium fluxes or proteolytic activity in the mechanism of stimulating arachidonic acid release by PDGF or serum.     

The effects of adenosine triphosphate and inorganic phosphates on the viability of stores rat skin.

Additions of ATP and inorganic phosphates to storage buffers, increase the viability of rat skin when stored at ?196 °C and at ?3 °C. The amino acid incorporation into the skin proteins, the α-[1-¹⁴C]-aminoisobutyric acid uptake by the skin and to a lesser extend the [6-³H]thymidine incorporation into DNA are protected by the phosphate compounds added to the storage medium. This stimulatory effect on the metabolic activity appears connected with the preservation and the protection of the oxidative phosphorylation, probably by providing the necessary phosphate radicals for the resynthesis of ATP. By simultaneously preventing potassium depletion during cooling and storage, the potassium phosphate compounds seem particularly suited to contribute to the preservation of the viability.