N-carboxymethylchitosan inhibition of aflatoxin production: Role of zinc

Aqueous Solutions of N-carboxymethylchitosan (NCMC) suppressed both growth and aflatoxin production byAspergillus flavus andA. parasiticus in submerged culture (Adye and Mateles A&M). Test media were amended with various concentrations of zinc (15, 30, 45, 60 uM), and NCMC solution (0.62 uM). After 8 days incubation NCMC-treated cultures showed marked reduction of aflatoxin production and fungal growth. Enhanced levels of zinc did not overcome the NCMC-mediated inhibition of fungal growth or aflatoxin production.     

Lymphocytes from children and infants mediate natural cell mediated cytotoxicity, (cytotoxicity)

Summary Lymphocytes from infants and young children were tested for natural cell mediated cytotoxicity (NCMC) against K562 and CCRF-CEM. NCMC by lymphocytes from pediatric donors of all ages was equivalent to that mediated by lymphocytes from adults. Since it has been suggested that the biological function of NCMC is to effect immunological surveillance against cancer, the appearance of NCMC effector cells early in development is consistent with early mobilization of the policing mechanism.This work was supported by Grant CA 25765 from the National Cancer InstitutePartially supported by a Grant from the Concern Foundation.     

Augmentation of human natural cell-mediated cytotoxicity by recombinant human interleukin 2

Human peripheral blood mononuclear cells (PBMC) demonstrated increased natural cell-mediated cytotoxicity (NCMC) activity after only 5 min of exposure to purified recombinant human IL 2 or interferon (IFN)-gamma. The mechanism of NCMC augmentation by treatment with IL 2 is not entirely dependent on IFN-gamma production because: a) IL 2 was found to augment NCMC activity at levels which did not induce detectable IFN-gamma; b) IL 2 required only 5 min of exposure to PBMC to augment NCMC activity, whereas 3 hr of contact were required to demonstrate detectable IFN-gamma levels; c) the levels of NCMC enhancement by treatment with IL 2 exceeded the amount of NCMC enhancement that could be due to IFN alone; d) anti-recombinant IFN-gamma, which totally eliminated the augmentation of NCMC enhancement by IFN-gamma, only partially reduced the augmentation of NCMC activity by IL 2; and e) combination treatment of PBMC with IL 2 and IFN-gamma resulted in a synergistic enhancement of NCMC. The results strongly support the conclusion that augmentation of NCMC by IL 2 and IFN-gamma involve overlapping mechanisms.     

Presence of two subunit types in ribulose-1,5-bisphosphate carboxylase from blue-green algae.

Ribulose-1,5-bisphosphate car?ylase (E.C. 4.1.1.39) from 2 blue-green algae, Plectonema boryanum and Anabaena variabilis, was isolated by sucrose density gradient centrifugation. Both enzymes had a sedimentation value of about 18s, similar to that of Chromatium enzyme. The presence of two subunits (A, B) in the algal enzyme was demonstrated by Nadodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the two subunits was determined: for Plectonema A, 5.4 × 10⁴ and B, 1.3 × 10⁴ and Anabaena A, 5.2 × 10⁴ and B, 1.3 × 10⁴, respectively. The car?ylase reaction catalysed by the algal enzyme was similar to the higher plant enzyme in exhibiting the Mg²⁺-effect, the optimal pH shifting from alkaline to neutral by elevating the concentration of Mg²⁺ in the assay mixture. The rabbit antisera developed against the spinach ribulose-1,5-bisphosphate car?ylase and its catalytic oligomer exhibited significant inhibitory effects on the car?ylation reaction catalysed by the algal enzyme.     

Natural cell-mediated cytotoxicity in normal human peripheral blood lymphocytes and its in vitro boosting with BCG

Summary Natural cell-mediated cytotoxicity (NCMC) against K-562 human erythroleukemic cells was monitored in an overnight chromium release assay using normal human peripheral blood lymphocytes (PBL) as effector cells. Two hundred and ten normal individuals were tested from 3 to 24 times over a period of 3 years. The level of NCMC was shown to vary from 4% to 46% lysis at an effector-to-target cell ratio of 5/1; males had higher levels of activity than females (P<0.001). A group of individuals with low natural killer (NK) cell activity (below the 90% tolerance limit) was identified in replicate experiments and 60% of them were young women (ages 20–39). In vitro boosting of NK activity with Bacillus Calmette-Guérin (BCG) was also studied; overall, 56% of normal individuals responded positively to BCG. There was a significant (P<0.0001) correlation between the unstimulated level of NCMC and the in vitro boosting with BCG, as 63% of individuals with a normal level of NK activity could be boosted as against only 19% of persons with low NK activity. We have also established the in vivo relevance of this in vitro test by determining the degree of correlation between responses to in vitro boosting with BCG and a positive or negative reaction in a hypersensitivity skin test using 5 IU of PPD (purified protein derivative of BCG). Our results indicate that NCMC is an individual trait that varies little under physiological conditions, and that the response to BCG is a characteristic property of the effector lymphocyte, depending primarily on the unstimulated level of NCMC.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

The growth-limiting factor of a population of cyanobacteria in the hypertrophic Dutch Lake Brielle

Summary Some methods to identify nutrient deficiencies in natural populations of algae are described. A bloom of the cyanobacteriumMicrocystis aeruginosa in Lake Brielle, The Netherlands, is taken as example of a natural situation. Some results obtained from continuous cultures ofM. aeruginosa contribute to the understanding of the characteristics of the natural population. Attention is paid to nutrient levels of the lake, pigment contents and N:P ratio of the algal population, the primary productivity and the uptake kinetics of nitrate and ammonia. The natural algal population of the lake showed a preferential use of ammonia. It is assumed that the growth-limiting factor forM. aeruginosa under the prevailing temperature is light.     

Indomethacin does not influence natural cell-mediated cytotoxic response to endurance exercise.

Natural cell-mediated cytotoxicity (NCMC) has been shown to be attenuated during recovery from high-intensity or prolonged exercise. Two theories have been proposed to explain the transient suppression of NCMC: prostaglandin-induced inhibition of natural killer (NK) cell activity or a numerical redistribution of NK cells. This study was designed to examine the effects of oral indomethacin (a prostaglandin inhibitor) on NCMC before and after 1 h of high-intensity running (85% maximal oxygen uptake). A secondary purpose was to compare whole blood and isolated peripheral blood mononuclear cell assay procedures for assessing NCMC. Ten male distance runners completed two trials that were preceded by either 48 h of indomethacin (Indo; 150 mg/day) or no treatment (control). NK (CD3(-)/CD16(+)/CD56(+)) cell concentrations were significantly elevated postexercise but were not affected by Indo. NCMC was significantly suppressed at 1.5 h of recovery relative to preexercise only with the whole blood assay procedure. Indo was not found to influence NCMC, leukocyte, or lymphocyte subset concentrations. Mean cytotoxic response was significantly greater with the whole blood method.     

An improved cheap culture medium for the blue-green microalga Spirulina

Summary The possibility of using crude seasalt, and saltpeter as the basis of a culture medium for the blue-green alga Spirulina maxima, was investigated. Saltpeter appears to be an excellent nitrogen and micronutrient-source and is very cheap. The effect of the nutrient level on algal growth, and nitrogen conversion efficiency, was studied. The results suggest the possibility of using this medium in a large-scale algal culture.     

Enzymatic hydrolysis of penicillin V to 6-aminopenicillanic acid byFusarium oxysporum

Summary A strain ofFusarium oxysporum was identified as having an intracellular penicillin V acylase activity (penicillin V amidohydrolase EC 3.5.1.11). Activity was induced by phenoxyacetic acid and had a good tolerance for high substrate and product concentrations. Washed cells could be used repeatedly for the complete hydrolysis of 5% penicillin V solutions. The enzyme was partially purified and concentrated from disrupted cells by fractional precipitation with water miscible solvents.     

Waste water treatment using saline cultures of microalgae

Summary Survival of microorganisms (Escherichia coli has been used as an example) is affected by a combination of salinity and high pH induced by the active photosynthesis of marine microalgae (Aphanotece or Dunaliella sp.). This effect can be applied to create a more efficient wastewater treatment process using algal stabilization ponds.     

Brine organisms and the question of habitat-specific adaptation

Among the well-known ultrasaline terrestrial habitats, the Dead Sea in the Jordan Rift Valley and Don Juan Pond in the Upper Wright Valley represent two of the most extreme. The former is a saturated sodium chloride-magnesium sulfate brine in a hot desert, the latter a saturated calcium chloride brine in an Antarctic desert. Both Dead Sea and Don Juan water bodies themselves are limited in microflora, but the saline Don Juan algal mat and muds contain abundant nutrients and a rich and varied microbiota, includingOscillatoria,Gleocapsa,Chlorella, diatoms,Penicillium and bacteria.In such environments, the existence of an array of specific adaptations is a common, and highly reasonable, presumption, at least with respect to habitat-obligate forms. Nevertheless, many years of ongoing study in our laboratory have demonstrated that lichens (e.g.Cladonia), algae (e.g.Nostoc) and fungi (e.g.Penicillium,Aspergillus) from the humid tropics can sustain metabolism down to –40°C and growth down to –10°C in simulated Dead Sea or Don Juan (or similar) media without benefit of selection or gradual acclimation. Non-selection is suggested in fungi by higher growth rates from vegetative inocula than spores. The importance of nutrient parameters was also evident in responses to potassium and reduced nitrogen compounds.In view of the saline performance of tropicalNostoc, and its presence in the Antarctic dry valley soils, its complete absence in our Don Juan mat samples was and remains a puzzle.We suggest that adaptive capability is already resident in many terrestrial life forms not currently in extreme habitats, a possible reflection of evolutionary selection for wide spectrum environmental adaptability.     

In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins.     

Storage in water ofRhizobium phaseoli cells harvested from continuous culture and its effect on biological nitrogen fixation

Summary Taken cells from cultures ofRhizobium phaseoli grown at different dilution rates were tested for plant growth promotion in a bioassay withPhaseolus vulgaris, before and after storage in water for one month. Cells grown at higher dilution rates showed a greater loss of effectiveness     

The monoclonal antibody CJA3 down-regulates the susceptibility of human tumor cell lines to natural cell-mediated cytotoxicity

While developing monoclonal antibodies (MoAb) to colorectal carcinoma (CRC) cells, we noted that one MoAb, termed CJA3, down-regulated natural cell-mediated cytotoxicity (NCMC) against CRC cell lines SW480 and SW620. The MoAb CJA3 was developed by immunizing a BALB/c mouse with fresh human colorectal adenocarcinoma cells. The antigen recognized by the MoAb CJA3 was expressed on several solid tumor cell lines and on one of the six lymphoreticular cell lines tested, but was not detected on normal peripheral blood lymphocytes (PBL). SDS-PAGE analysis of the antigen immunoprecipitated by the MoAb CJA3 from the CRC cell lines SW480 and SW620 and from the melanoma cell line MALME-3M revealed a component with a m.w. of 150,000. Preincubation of CRC cell lines SW480 and SW620 with the MoAb CJA3 for 16 hr reduced their susceptibility to NCMC by about 50%. Kinetic experiments showed that prolongation of the incubation of target cells with the MoAb CJA3 resulted in a time-dependent increase in the amount of MoAb bound. Maximum binding of the MoAb CJA3 was reached after 4 hr of incubation. The increase in antigen expression chronologically paralleled the decrease in NCMC target cell sensitivity, suggesting that the membrane alterations induced by the MoAb CJA3 were important for NCMC against these two cell lines.     

Effect of low-dose ultrasonic treatment on phystological variables inAnabaena flos-aquae andSelenastrum capricornutum

Summary Cell protein content in two species of cultured algaeAnabaena flos-aquae andSelenastrum capricornutum, was markedly enhanced by low-dose, short-duration ultrasonic treatment. Chlorophylla levels and¹⁴C-bicarbonate uptake rates were not affected by ultrasonic treatment in either species. Ultrasonically-activatedAnabaena cultures placed in media deficient in nitrogen and phosphorus produced more biomass per unit time, exhibited less cell-surface alkaline phosphatase activity per cell, and had a higher heterocyst frequency than non-sonicated, nutrient-deficient cultures. In contrast, sonicated, nutrient-deficientSelenastrum cultures grew more slowly and had higher alkaline phosphatase activity than non-sonicated variants. Collectively, the data suggest that key metabolic variables may be altered by ultrasonic treatment in algal cultures and that the magnitude and direction of change may be species-specific.     

Cellulose production by planktonic algae in lacustrine environments

Cellulose production by planktonic algae in a eutrophic pond and in an oligotrophic lake was estimated by comparing the amount of cellulose contained in intact algal cells with the amount of cellulose present in the water column. Cellulose contents of laboratory grown algal species representing the dominant cellulose producers were ranged from 2 to 39% of the total dry weight of cell mass, depending upon the species and stage of growth. The relative amounts of cellulose present in the water column ranged from 4 to 50% of the total dry weight of particulate matter.It was estimated that more than 30% of cellulose in the water column was actually contributed by the viable algal cells present during the algal bloom. Despite its algal origin, more cellulose was found in the water column than could be accounted for by the number of algal cells observed. The difference was due to the accumulation of cellulose from previous algal crops. This observation indicated that production of algal cellulose exceeded decomposition and little or no decomposition of cellulosic material took place in the water column.     

Human natural cell-mediated cytotoxicity

Summary The contribution of natural cell-mediated cytotoxicity (NCMC) to tumor-directed cytotoxicity is unknown. This study was undertaken to correlate changes in NCMC to a single target cell (K-562) with responses to therapy in patients with small cell carcinoma of the lung, resectable stage I non-small cell carcinoma of the lung, and stage I and II melanomas. Data from these studies suggested that these patients have depressed levels of NCMC to K-562 compared with a normal age-matched control population. However, NCMC levels appear to fluctuate with tumor burden, being highest in patients with large tumor masses and lowest in patients with no clinical evidence of tumor following a successful response to therapy.     

Experimente zum Bewegungsverhalten von einzelligen Fliesswasseralgen

1. A schematical model is given of the drifting and recolonisation of algal cells in an artificial channel with algal growth on the bottom when water free of algae was flowing through, as well as of the colonisation of a clean channel by algal cells, when water of a brook was flowing through. Both phenomena are compared with drift and colonisation in a natural channel by means of quantitative sampling.
2. Before settling down again after drifting away a single algal cell may cover a distance of less than 38 m only.
3. Multiple sampling over a period of 24 hours demonstrated diurnal periodicity in the drifting and colonisation of various algal species from running water.

     

Picophytoplankton dynamics in the equatorial Pacific: diel cycling from flow-cytometer observations

The French JGOFS cruise FLUPAC was conducted in October–November 1994 in the western and central equatorial Pacific Ocean. During a 7-day time series at 150 °W, in addition to conventional sampling (four times per day from discrete depths between 0 and 150 m), a high frequency (hourly) experiment was performed by continuously pumping water at 5 m below the surface over 24 h. Flow cytometric measurements allowed us to recognise and to follow separately the two major components of equatorial picophytoplankton, the Prochlorococcus and the picoeukaryotes. The hourly surface experiment confirmed the synchrony of Prochlorococcus cell division and showed that picoeukaryotes exhibited a similar behaviour. The main consequence is that the maximum potential growth rate of picophytoplankton is one doubling per day for both cell groups. The vertical profiles indicated that the diel cycling extends throughout the surface layer for both algal groups. The cells were observed to divide daily from late afternoon to the middle of the night, and then to disappear, probably as a result of grazing. In the surface layer, variations of abundance allowed us to estimate a growth rate of about 0.6 day¹. Mean cell light scattering parFS as measured by the cytometer indicated a decrease in cell size concurrent with cell division and an increase during the photosynthetic growth phase.