Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

A host-vector system for an Arthrobacter species

An efficient host-vector system has been developed for an industrial strain of Arthrobacter sp. (NRRL B3728)used for glucose isomerase production. Protoplasts of Arthrobacter were generated by treating the cells with 0.5 mg lysozyme ml(-1) for 60 min in a solution containing 0.5 M-sucrose. Around 30% of the protoplasts regenerated on agar containing 0.5 M-sodium succinate as osmotic stabilizer. Three hybrid vectors, PBL2100, pCG1100 and pCG2100, were constructed by combining the Escherichia coli plasmid pBR322, a kanamycin- resistance gene from pNCAT4 and a cryptic plasmid from either Brevibacterium lactofermentum NCIB 9567 or Corynebacterium glutamicum NCIB 10026. These vectors transformed the protoplasts and expressed the kanamycin-resistance gene for screening. They contain a number of unique restrictions sites for cloning of foreign DNA. The transformation frequency of this system was 10(5)-10(6) transformants per micrograms of input plasmid and ws constant up to 5 micrograms of DNA. the probability of a plasmid transforming a plasmid transforming a protoplast was in the range 10(-5)-10(-6). The copy number of pBL2100 was around 5 per cell and those of pCG1100 and pCG2100 were around 33 per cell. Deletion mutants were generated from pCG2100. One of them, pCG2120, was able to transform protoplasts of strain NRRL B3728. Plasmids pBL2100 and pCG2100 were structurally stable in cells of NRRL B3728 but could not be maintained in non-selective medium. They segregated at a rate of 12.2 and 2.2% per generation respectively.     

Tryptophan enhancement of somatic embryogenesis in rice

Cereal embryos can produce two types of callus. One type, termed “embryogenic,” consists of small meristematic-like cells and gives rise to many plants by somatic embryogenesis if placed on a suitable regeneration medium. The other is termed “nonembryogenic” and consists of long tubular cells which gives rise to few or no plants. High concentrations of tryptophan increased the formation of embryogenic callus in three rice cultivars (Oryza sativa L. Calrose 76, Pokkali, and IR 36) but not in four others (Mahsuri, Bg 400-1, H₄, and Giza 159). The best concentration of tryptophan for Pokkali and Calrose 76 was 100 micrograms per milliliter, and for IR 36, 50 micrograms per milliliter. Indoleacetic acid at 100 micrograms per milliliter promoted an effect similar to that of tryptophan on Calrose 76. The difference between japonica (Calrose 76, Giza 159) and indica (Pokkali, IR 36) varieties is not the causal factor for the difference in response to tryptophan. Kinetin does not appear to be a requirement for embryogenic callus formation in Calrose 76. Plant regeneration from Calrose 76 embryogenic callus occurred at low levels in media containing no hormones. 6-benzyladenine, or 2,3,5-triiodobenzoic acid but not indoleacetic acid at 0.1 to 0.5 micrograms per milliliter significantly increased regeneration.     

R-plasmid transfer in a wastewater treatment plant.

Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium 10 micrograms of ampicillin, 30 micrograms of chloramphenicol, 10 micrograms of streptomycin, 100 micrograms of sulfadiazine, or 30 micrograms of tetracycline. Mean transfer frequencies for laboratory matings were 2.1 X 10(-3). In situ matings for primary and secondary settling resulted in frequencies of 4.9 X 10(-5) and 7.5 X 10(-5), respectively. These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents.     

Mating Type Interaction in Closterium peracerosum-strigosum-littorale: Mating Induced Protoplast Release

Mating type interaction between heterothallic strains of Closteriumperacerosum-strigosum- littorale was studied during the conjugationprocess. When vegetative cells of opposite mating types weremixed under the nitrogen depleted mating conditions, the formationof conjugation-papilla and the release of the protoplast withinthe distended papilla from the gametangial cell were shown inboth the paired and unpaired cells. The protoplasts of pairedcells fused to form zygotes, while those of unpaired cells broke.When cells were mixed at a ratio of around 5 to 3 (mating typeplus to mating type minus), sexual activation occurred at thehighest frequency. Mixing at a ratio of 1 to 3, however, inducedthe highest frequency of disrupted cells. Mating type plus cellswere found to be broken specifically when direct contact withmating type minus cells was prevented. This disruption seemedto be mediated by a factor secreted from mating type minus cells. (Received March 20, 1981; Accepted August 18, 1981)     

Uptake and Degradation of Cyclic AMP by Chloronema Cells

Suspension cultures of intact chloronema cells of the moss Funaria hygrometrica take up [³H]cAMP and degrade it rapidly. The increase in total radioactivity accumulated by the cells was linear up to 30 minutes. Initially, the major degradation products were 5′-AMP and adenosine, but later predominantly ADP and ATP. In spite of rapid degradation, the amount of extracellularly applied cAMP retained by the cells is about 4-fold higher than the maximum endogenous level of cAMP reported previously (Handa, Johri 1977 Plant Physiol 59: 490-496). The uptake showed a distinct dependence on the density of the culture. Cells at a lower cell density (1-2 milligrams per milliliter) accumulated 4 to 6 times more radioactivity than the cells at high density (>10 milligrams per milliliter). The cyclic nucleotide phosphodiesterase (cNPDE) activity of whole cells (18 milliunits per milligram protein) was comparable to that of protoplasts (23 milliunits per milligram protein), but about 4-fold lower than that of lysed protoplasts (80 milliunits per milligram protein), indicating an intracellular degradation of cAMP by chloronema cells.     

No neoplastic alteration of metabolically competent rat liver cells in vitro by chemical carcinogens: 3-methylcholanthrene, dimethylnitrosamine and Natulan

Epithelial rat liver cell line RL-19 was checked for aryl hydrocarbon hydroxylase and dimethylnitrosamine demethylase activity. Aryl hydrocarbon hydroxylase activity was found at the rate of about 14.5 pmoles 3-hydroxy-benzopyrene per min per mg protein. This activity was not inducible by 3-methylcholanthrene or by phenobarbital and was independent of the subculture level. From the 45th up to the 59th subculture the mean demethylase activity was about 1.08 nmoles HCHO per min per mg protein, but was decreased to 0.64 nmoles HCHO per min per mg protein at the 131st subculture. RL-19 cells were treated with 3-methylcholanthrene (0.5-1.0 microgram/ml), dimethylnitrosamine (100-400 micrograms/ml), or Natulan (50 micrograms/ml), respectively, for 7 to 10 days. During a 6 months subsequent cultivation no neoplastic changes were observed as revealed by morphological investigation, soft agar assay, and transplantation. It is suggested that metabolic competence for carcinogen activation is only one prerequisite for neoplastic alteration in vitro, and that RL-19 cells are refractory to the action of carcinogens in spite of their metabolic capacity.     

Parameters influencing the liposome-mediated insertion of fluorescein diacetate into plant protoplasts

Maximum uptake of liposome-encapsulated fluorescein diacetate by Daucus carota protoplasts was observed when 6 × 10⁶ protoplasts per milliliter were incubated with 2.4 × 10⁷ liposomes per milliliter for 1 hour. In the case of Nicotiana glutinosa protoplasts, optimum ratio of protoplasts to liposomes was 1:10, where 2.3 × 10⁵ protoplasts per milliliter were provided. Neutral and positive liposomes were found to be efficient vehicles to transfer their contents into plant protoplasts. When protoplasts treated with liposomes were cultured in a synthetic medium for 1 week, 20% resumed cell divisions.     

Eradication of biofilm cells of Staphylococcus aureus with tobramycin and cephalexin.

The kinetics of growth and formation of biofilm by Staphylococcus aureus were investigated under iron-limited conditions in the chemostat. The population of planktonic cells reached 5.5 x 10(9) cells/mL 24 h after inoculation (D = 0.05 h-1) and remained constant throughout. The number of biofilm cells of S. aureus colonizing the silicone tubing increased exponentially from 6 x 10(4) to 2.7 x 10(7) cells/cm2 (6 days later) and continued to increase at a reduced rate to 2.7 x 10(8) cells/cm2 on day 13. Planktonic cells of S. aureus were susceptible to tobramycin and cephalexin. The planktonic cells could be successfully eradicated with a combination of 5 micrograms tobramycin plus 100 micrograms cephalexin per millilitre. Exposure of young biofilm cells of S. aureus to 5 micrograms tobramycin plus 100 micrograms cephalexin per millilitre resulted in a rapid loss of cell viability. The percentage of survival dropped to less than 0.0001% after exposure to these concentrations of antibiotics for 3 h. Old biofilm cells of S. aureus were found to be extremely resistant to these antibiotics. The cell viability was reduced to 0.09% after exposure to 10 micrograms tobramycin plus 100 micrograms cephalexin per millilitre. The results suggest that it is possible to eradicate S. aureus infection at the early stage with tobramycin plus cephalexin. Any delay in implementing antibiotic therapy is likely to result in the failure of the treatment. It is important to note that the concentrations of antibiotics required for the eradication of young biofilm cells must be determined for the treatment of device-associated infections.     

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl₂, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg²⁺ at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K_act(CO₂) of rubisco activation in the reconstituted system was 4 micromolar CO₂, compared to a K_act(CO₂) of 25 to 30 micromolar CO₂ for the previously reported spontaneous CO₂/Mg²⁺ activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO₂ and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.     

Teratogenic bioactivation of cyclophosphamide in vitro.

Day 10 rat embryos grown as cultured explants $\text{in vitro}$ developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.     

Increased sensitivity of heat-stressed Saccharomyces cerevisiae cells to food-grade antioxidants

Unheated and heat-stressed Saccharomyces cerevisiae cells were examined for their relative sensitivities to butylated hydroxyanisole (BHA), tertiary butylhydroquinone (TBHQ), and propyl gallate. Heated cells had significant (P less than or equal to 0.05) increases in sensitivity to 50 micrograms of BHA, 100 micrograms of TBHQ, and 1,000 micrograms of propyl gallate per ml as compared with unheated cells when surface plated on antioxidant-supplemented recovery agar. The rate of increase in size of colonies developed by heated cells was slower than that of unheated cells, and the presence of antioxidants in recovery agar enhanced this effect. Heat-stressed cells also had increased sensitivity to ethanol. Incubation temperatures of 15, 21, 30, and 37 degrees C for enumerating unheated cells had no significant effect on the numbers of colonies formed on unsupplemented recovery agar; however, incorporation of 100 micrograms of BHA, 200 micrograms of TBHQ, or 1,000 micrograms of propyl gallate per ml into agar resulted in significant decreases in the number of colonies formed by heated cells at various incubation temperatures. The detrimental effects of TBHQ and propyl gallate on repair of heat-injured cells are apparently expressed at a temperature higher than that observed for BHA. It is suggested that the adverse effects of antioxidants on repair of heat-injured S. cerevisiae cells may be associated with oxygen availability.     

Increased sensitivity of heat-stressed Saccharomyces cerevisiae cells to food-grade antioxidants.

Unheated and heat-stressed Saccharomyces cerevisiae cells were examined for their relative sensitivities to butylated hydroxyanisole (BHA), tertiary butylhydroquinone (TBHQ), and propyl gallate. Heated cells had significant (P less than or equal to 0.05) increases in sensitivity to 50 micrograms of BHA, 100 micrograms of TBHQ, and 1,000 micrograms of propyl gallate per ml as compared with unheated cells when surface plated on antioxidant-supplemented recovery agar. The rate of increase in size of colonies developed by heated cells was slower than that of unheated cells, and the presence of antioxidants in recovery agar enhanced this effect. Heat-stressed cells also had increased sensitivity to ethanol. Incubation temperatures of 15, 21, 30, and 37 degrees C for enumerating unheated cells had no significant effect on the numbers of colonies formed on unsupplemented recovery agar; however, incorporation of 100 micrograms of BHA, 200 micrograms of TBHQ, or 1,000 micrograms of propyl gallate per ml into agar resulted in significant decreases in the number of colonies formed by heated cells at various incubation temperatures. The detrimental effects of TBHQ and propyl gallate on repair of heat-injured cells are apparently expressed at a temperature higher than that observed for BHA. It is suggested that the adverse effects of antioxidants on repair of heat-injured S. cerevisiae cells may be associated with oxygen availability.     

Identification of a new mating group and reproductive isolation in the <Emphasis Type="Italic">Closterium peracerosum–strigosum–littorale</Emphasis> complex

Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to compare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracerosum–strigosum–littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt^?) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt^? cells of mating group G was induced in the presence of mt⁺ cells of mating group I-E. In fact, the sex pheromone, protoplast-release-inducing protein of mating group I-E induced the release of protoplasts from mt^? cells of mating group G. When mt⁺ and mt^? cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E.     

Hexavalent chromium-resistant bacteria isolated from river sediments.

Hexavalent chromium [Cr(VI)] is a known carcinogen and mutagen; however, the actual mechanisms of Cr toxicity are unknown. Two approaches were used to isolate Cr(VI)-resistant bacteria from metal-contaminated river sediments. Diluted sediments were plated directly onto a peptone-yeast extract (PYE) medium containing 0 to 100 micrograms of Cr(VI) ml-1. Approximately 8.4 x 10(5) CFU g-1 were recovered on 0 microgram of Cr(VI) ml-1, whereas 4.0 x 10(2) CFU g-1 were recovered on PYE plus 100 micrograms of Cr(VI) ml-1. Alternatively, continuous culture enrichment techniques were employed using PYE and 100 micrograms Cr(VI) ml-1 input at dilution rates of 0.02 and 0.10 h-1. After six residence periods, 10(9) CFU were recovered on PYE agar containing 0 microgram of Cr(VI) ml-1 and 10(7) CFU on PYE agar plus 100 micrograms of Cr(VI) ml-1. Of 89 isolates obtained by direct plating onto PYE, 47% were resistant to 100 micrograms of Cr(VI) ml-1, and 29% were resistant to 250 micrograms of Cr(VI) ml-1. When the same isolates were plated onto PYE containing Cr(III), 88% were resistant to 100 micrograms ml-1 but only 2% were resistant to 250 micrograms ml-1. Cr, Co, Sb, and Zn were found in significantly higher concentrations at an industry-related contaminated site than at a site 11 km downstream. Total Cr in the sediments at the contaminated site averaged 586 micrograms (dry weight) g-1, and the downstream site averaged 71 micrograms (dry weight) g-1. The Cr recovered from acid-digested Ottawa River sediment samples was predominantly hexavalent. Five acid digestion procedures followed by atomic absorption spectroscopy were compared and found to be 30 to 70% efficient for recovery of Cr relative to neutron activation analysis. A population of aerobic, heterotrophic bacteria was recovered from sediments containing elevated levels of Cr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole     

Evaluating the relationship of metabolic activation system concentrations and chemical dose concentrations for the Salmonella spiral and plate assays

A factorial experimental design was used within this study to evaluate the influence of multiple metabolic activation system concentrations on the dose-response exhibited by promutagens (indirect-acting mutagens) in the Salmonella spiral and plate assays. The mutagenic activity of the three compounds used spanned three orders of magnitude. The mutagenic activity of the compounds ranged from 10 to 100 revertants/micrograms for acetylaminofluorene (2AAF) to more than 1000 revertants/micrograms for 2-aminoanthracene (2AA). Benzo [a] pyrene (BaP) activity was within an intermediate range (100-1000 revertants/micrograms). During a single experiment, a mutagen was tested in TA100 at 13 doses plus a negative control dose. Each dose was tested at 10 S9 concentrations. The S9 concentrations ranged from 0.1 mg protein/plate to 4 mg protein/plate in the standard plate assay and from 0.25 to 4.90 mg-equivalents in the spiral assay. The spiral Salmonella assay, an automated version of the standard assay, generates dose-response data from a concentration gradient on a single agar plate, thereby providing a straightforward approach to this type of study. This study demonstrates not only that even small differences in S9 concentrations can affect the measurement of mutagenic potency but that S9/compound interactions cannot be generalized through the use of interaction studies. This study also shows that spiral assay data and plate assay data for promutagens cannot be compared directly unless the S9 concentrations for all chemical doses are also comparable.     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.