Implications of a 5′-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5′-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56°C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37°C for 1 h and it was destroyed completely by heating at 100°C for 30 min. When the heated (56°C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5′-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35 000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggests that the previously reported undetectability of 5′-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5′-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5′-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Properties of Extracellular Enzymes from Aphanomyces astaci and Their Relevance in the Penetration Process of Crayfish Cuticle

In culture filtrates from the crayfish plague parasite, Aphanomyces astaci, protease and a low level of hyaluronidase activity were found. The hyaluronidase activity was highest at pH 6.5 or above and at about 23°C. The protease activity had a broad pH-optimum, between pH 7 and at least pH 10, and was partially denatured at 30°C. However, when incubated for 30 min with the substrate, casein, the activity increased logarithmically up to about 35–40°C and had an apparent optimum at 45–50°C. The proteases from the parasitic as well as from two less proteolytic, saprophytic Aphanomyces species were predominantly constitutive and were excreted mainly by the older mycelia. Proteases from the parasite and a saprophyte did not reach full activity until 10–30 min after substrate addition. No lipase activity was found in the case of the mycelium of the parasitic species. However, esterase was apparently present inside germinating zoospores. The native enzymes of A. astaci could degrade freeze-dried soft cuticle from crayfish. The relevance of the different enzymes of A. astaci for the penetration process within the cuticle of crayfish is discussed.     

Physicochemical Properties of Niigata Waxy Rices

The crude lipase powder has been purified 216-fold in specific activity by means of pH adjustment, DEAE-Cellu1ose, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 column chromatography and the recovery of the activity was 30%. The purified lipase was confirmed to be homogeneous with disc electrophoresis and ultracentrifugal analyses. The purified lipase was stable in the pH range from 7.0 to 10.0. Optimal pH for the lipolysis of polyvinyl alcohol-emulsified olive oil at 45°C was 8.0 and optimal temperature was 60°C. The purified lipase was stable up to 60°C and retained 55% of full activity after heating at 70°C for 20 min.     

Properties and Substrate Specificities of Four Esterases from Aspergillus niger NRRL 337

Properties and substrate specificities of four esterases (Esterase-I, -II, -III, -IV) from Aspergillus niger were studied. Esterase-I and Esterase-II were found to be markedly stable to heat. When Esterase-I was assayed at 35°C using methylacetylsalicylate as a substrate, even after heating at 100°C for 15 min 60% of its activity remained. However, Esterase-I scarcely hydrolyzed the substrate at 70°C or over, because of a reversible change in conformation by heating as found by CD measurement. The maximum activity of Esterase-I was found at 55°C at 20 min of reaction time. Esterase-II was stable up to 80°C and had an optimum temperature for reaction at 80°C, but was irreversively inactivated by heating for 15 min at 90°C.

The four esterases hydrolyzed aliphatic esters of short chain fatty acids and acetyl esters of phenols, but neither methyl esters of aromatic carboxylic acids nor acetyl esters of aromatic alcohols.     

Virus Meets Liposome

Abstract

Sendai virus was the first virus to encounter liposomes. Gangliosides when incorporated into liposomes act as Sendai virus receptors even at 0–4°C. When receptor-containing liposomes are incubated with virus at 37°C, they envelop the virus. At 37°C liposomes also fuse with Sendai virus membrane.

Virus binding initially involves weak adhesion, which may allow the virus to “browse” the cell, and which is followed by adhesion strengthening. MicrogrΔpHs of Sendai virus fusion with liposomes after one minute at 37°C indicate that fusion occurs at the very curved leading edge of the region of the liposome enveloping virus. A model of fusion is proposed that emphasizes the role of the curvature and membrane tension in this localized region of “host” membrane. The curvature assists close approach and destabilizes the outer monolayer. The proposed intermediates are consistent with the “stalk” hypothesis.     

Hemagglutination activities of purified pertussis toxin and filamentous hemagglutinin against erythrocytes from various animals

The hemagglutinating (HA) activities of purified pertussis toxin (PT) and filamentous hemagglutinin (FHA) were evaluated against unfixed and glutaraldehyde-fixed erythrocytes from ox, goose, horse, monkey, sheep, chicken, and rabbit. Both PT and FHA showed HA activities against fixed and unfixed erythrocytes from all the animals studied. The HA titers of FHA were higher than those of PT. The HA activities of FHA and PT were not destroyed completely even after heating these preparations at 56 C for 30 min. A simple test for the assay of PT in culture supernatants of Bordetella pertussis on the basis of HA activity has been described.     

The protection,stabilization and reactivation of estradiol receptors in human myometrial cytosol

Protection of estradiol receptor binding sites in human mymetrial cytosol is achieved though the agency of dithiothreitol (Cleland's reagent) (lmM) at 4°C and ? 20°C storage temperatures. Prolonged storage of cytosol at 4° C without the protective reagent results in substantial loss of binding activity. This is partially restored after Cleland's reagent (lmM) addition. However, cytosol inactivated by heating at 60°C for 30 min cannot be reconstituted in this way.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus).

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Production and Purification of Thermostable β-Glucosidase from Mucor miehei YH-10

A thermophilic fungus, Mucor miehei YH-10, isolated from manure was selected to produce thermostable β-glucosidase among 207 isolates. When Mucor miehei YH-10 was grown on wheat bran medium, the maximal accumulation of thermostable β-glucosidase was obtained after 4 days at 50°C, The β-glucosidase had an optimal temperature of 60°C and retained 73% of original activity after heating at 95°C for 5 min. The β-glucosidase was fractionated by Sephadex G-100 chromatography into two components during the purification steps. These components were further purified by consecutive column chromatographies until they were homogeneous on disc electrophoresis. One retained 56% of original activity after heating at 95°C for 5 min, whereas the other was completely inactivated after heating at 80°C for 5 min.     

Effect of heat on zoospore motility and multiplication of Olpidium radicale and O. brassicae

Whereas zoospores of six cultures of O. brassicae were immobile after treating in glycine solution at 35–40°C for 10 min, those of a cucurbit culture of O. radicale were immobile only after heating at 45°C. Further, O. radicale multiplied in cucumber at 20°, 30° and 35°C, whereas O. brassicae multiplied at 20°, but not 30° or 35°C. Such differences in reaction to temperature might sometimes be useful in separating mixtures of the two species in common hosts such as cucumber.     

High temperature during storage favours infection potential of resting spores of Polymyxa graminis of Indian origin

The infection potential of sporosori of Polymyxa graminis involved in the transmission of the Indian peanut clump virus (IPCV) was assessed by culturing bait plants exposed to various concentrations of sporosorus suspensions and then determination of the numbers of plants that became infected. Storage of air-dried inoculum at temperatures above 30°C resulted in an increase in the infection potential compared to that of sporosori stored at 15°C or 20°C. In contrast, when the sporosori were stored at -20°C or freeze-dried, their infection potential was low. These results confirm the adaptation of P. graminis isolates associated with IPCV transmission to the tropical environment. The implication of storage temperature for the epidemiology of Indian peanut clump virus and for the assessment of the infection potential of the vector in the soil is discussed.     

Biological activity of Friend spleen focus-forming virus after cold storage

Two stocks of Friend spleen focus-forming virus (SFFV) were prepared, one in saline and the other in Eagle's medium with 2% fetal calf serum, and the effects of different freezing, storage and thawing temperatures were determined for the recovery of infectious virus from each diluent. Once frozen, virus maintained its titer at ?70 and at ?170 °C for up to 13 weeks, while it lost titer at ?13 °C more rapidly if it had been prepared in saline than in medium. However, during the freezing process lower ambient temperatures (?70 and ?170 °C) gave lower virus yields than a higher temperature (?13 °C) did. Similarly, rapid thawing (in a 37 °C water bath) was less efficient than slow thawing (in 4 or 20 °C air) for the recovery of infectious SFFV, This study illustrates the importance, for efficient recovery of leukemogenic activity from stored murine leukemia virus stocks, of the temperature used for freezing or thawing, as well as for storage.     

Rhodnius prolixus LIPOPHORIN: LIPID COMPOSITION AND EFFECT OF HIGH TEMPERATURE ON PHYSIOLOGICAL ROLE

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin‐layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55°C. When it was pretreated at 75°C, maximal inhibition of phospholipid transfer was observed after 3‐min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75°C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55°C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75°C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65°C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85°C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.     

Biological activity and conformational stability of the domains of plasma fibronectin

As measured by two assays of biological activity, fibronectin was readily denatured by heat. Both by the rat liver slice assay and by gelatin-latex agglutination, 90% of the activity disappeared in about 10 min at 60 °C. In contrast, immunological activity, as measured by microcomplement fixation, showed little change at 10 min and was at least 60% as great as unheated fibronectin after 20–50 min at 60 °C. Binding of heparin was unaffected by heating up to 52 min, but at very long times (48 hr at 60 °C), it also was lost. Differential scanning calorimetry of native fibronectin showed three endothermal denaturing transitions, at 68, 82, and 119 °C. Enthalpies of denaturation for the three transitions are approximately 2.6, 0.4, and 0.7 cal/g of flbronectin. These results are consistent with a three-domain structure for fibronectin. The domain which unfolds at 68 °C is associated with gelatin binding and cell. binding. The 82 °C domain appears to be associated with much of the immunological activity, and the 119 °C domain with heparin binding, as well as with some immunological activity. Residual immunological activity after loss of heparin binding may reside in nonordered portions of the molecule.     

Blocking of the induction and expression of immunologically functional T lymphocytes by rat antiactivated T-cell serum

A hybridoma, F133, that produces macrophage activation factor (MAF) after mitogen stimulation was developed by fusing the AKR-derived BW5147 thymoma with alloantigen-stimulated C3H/HeJ splenocytes. F133 supernatants were shown to contain MAF, migration inhibition factor, and a factor capable of suppressing the plaque-forming response to sheep erythrocytes but not lymphotoxin, interleukin II, or interferon. Both concanavalin A (Con A) and phytohemagglutinin (PHA) induced MAF production by F133. Time course and dose-response experiments showed that maximal concentrations of MAF were present 48 hr after stimulation with either 1.5 μg/ml Con A or 6 μg/ml PHA. F133 and normal splenocyte MAF preparations shared physicochemical properties in that heating at 100 °C for 30 min abolished MAF activity while 56 °C for 30 min or 100 °C for 2 min had little effect. In addition, both MAF preparations were dependent on the presence of lipopolysaccharide for macrophage activation and each was inactivated by pH 4.0 or pH 10 treatment while pH 6.0 and pH 8.0 had little effect. Also, pretreatment of both MAF preparations with either trypsin or chymotrypsin inactivated MAF activity.     

Selection of a Photosynthetic Bacterium Suitable for Hydrogen Production in Outdoor Cultures among Strains Isolated in the Seoul,Taegu, Sendai and Bangkok Areas

Very efficient hydrogen producing photosynthetic bacteria, strains SL1, SL3, SL16 and TG28 newly isolated in Korea, and strain KM113 newly isolated in the Sendai area, were found to be Rhodopseudomonas spp. To examine the stability of cell suspensions of the cultures for hydrogen production, which is closely associated with light absorption, we conducted larger scale cultures under periodic illumination (12-hr intervals) without stirring at 30°C using strains SL1 and Rhodopseudomonas sphaeroides B5, the latter was isolated in the Bangkok area. Both strains gave homogeneous cell suspensions throughout the incubation period and larger amounts of hydrogen were produced in a shorter period of time by both cultures than obtained with Rhodopseudomonas sp. TN3, an isolate from the Sendai area which was reported previously. With the cells of the new isolates and strains TN3 and B5 grown on glutamate-malate medium at 30°C, we measured hydrogen production at 20, 30 and 40°C in the same medium. Among them, strains SL1, SL16 and KM113 showed the highest hydrogen production activity at 30°C. The maximum hydrogen production rates with these strains were over 130 µ1/hr/mg dry cells, but at 40°C, the highest activity (138 µl/hr/mg dry cells) was obtained with strain B5. Since strain B5 also showed good activities at 20 and 30°C, we suggest that this strain might be suitable for hydrogen production in outdoor cultures.     

An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis

The cytosol fraction of rat testicular homogenates contained a specific androgen binding protein (t-ABP), indistinguishable from, the androgen binding protean in. the epididymis(e-ABP) in terms of its chromatographic behaviour by gel filtration, sedimentation rate, mobility on polyacrylamide electrophoresis and steroid specificity(5α-diaydrotestosterone(DHT) > testosterone > estradiol-17β > parogesterone and estradiol-17α).The stability of t-ABP as well was similar to that of e-ABP. The aBP-DHT complexes were stable between pH 6.5–10, exhibiting the highest degree of binding between pH 8–9. The binding activity persisted after heating at 50°C for 30 min., whereas heating at 60°C for 30 min. completely destroyed the binding. DHT did not significantly increase the stability towards pH and temperature denaturation. Binding activity was not reduced by 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS), whereas similar treatment with 1 nM N-ethyl-maleimide(NEM) or 1 mM Ellman's reagent reduced it by some 10–50 per cent. Exposure to 10 mM β-mercaptoethanol(βME) reduced the binding by 60–70 per cent. These studies strongly indicate that t-ABP and e-ABP are identical proteins.Hemicastration for 4 weeks eliminated the ABP from the epididymal cytosol fraction on the castrated side, indicating that this protein is produced by the testis.     

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.     

Interaction between photon flux density and elevated temperatures on photoinhibition in Alocasia macrorrhiza

The effects of light and elevated temperatures on the efficiency of energy conversion in PSII [?_PSII = (Fm′−Fs)/Fm′], pigment composition and heat tolerance of shade-acclimated Alocasia macrorrhiza were investigated. Leaf discs were exposed for 3 h to high light (HL; 1600 μmol photons · m⁻² · s⁻¹) or low light (LL; 20 μmol photons · m⁻² · s⁻¹) and a series of constant temperatures ranging from 30 to 49 °C. All HL treatments led to rapid and severe decreases in ?_PSII. During the 2-h recovery period (LL, 25 °C) following the HL treatments, fast and slow recovery phases could be distinguished. Leaf discs that had experienced HL and 30 °C recovered completely while no recovery of ?_PSII was seen after a 3-h exposure to HL and 45 °C. A 3-h exposure to 45 °C at LL led to a less severe decrease in ?_PSII and complete recovery was accomplished after less than 1 h. Under LL conditions a temperature of 49 °C was necessary to cause an irreversible decrease in ?_PSII, followed by necrosis the next day. Streptomycin had no effect on the degree of reduction and recovery in ?_PSII discs exposed to HL and 35–45 °C, but partially inhibited recovery in discs exposed to HL and 30 °C. Streptomycin led to a more severe decrease in ?_PSII at LL and 49 °C and completely inhibited recovery. Streptomycin had no effect on the conversion of the xanthophyll-cycle pigments during the treatment or the recovery. The epoxidation state was roughly the same in all leaf discs after a 3-h HL treatment (0.270–0.346) irrespective of the exposure temperature. The back-conversion of zeaxanthin into violaxanthin after a 2-h recovery period was only seen in leaf discs that had been exposed to HL and 30 °C. The thermotolerance of shade A. macrorrhiza leaves of 49.0 ± 0.7 °C (determined by fluorescence) coincided with the temperature at which damage occurred in leaf discs exposed to LL. However, under HL the critical temperature under which necrosis occurred was much lower (42 °C). The thermotolerance of A. macrorrhiza shade leaves could be increased by a short exposure (<20 min) to slightly elevated temperatures. Received: 11 June 1997 / Accepted: 9 September 1997