Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of endotoxin to human monocytes and mouse macrophages: serum requirement

Specific binding of Bordetella pertussis and Neisseria meningitidis endotoxins to human monocytes and murine macrophages was demonstrated. Binding of B. pertussis endotoxin could be inhibited by endotoxins of Salmonella minnesota, Escherichia coli, and Klebsiella pneumoniae, the extent of inhibition being dependent on the origin of the lipopolysaccharides and on the origin of the mononuclear phagocytic cells. The binding of B. pertussis and N. meningitidis endotoxins which was mediated by the polysaccharide region of the endotoxins was serum dependent. The results indicated that the binding of endotoxin was promoted neither by natural antibodies directed against the endotoxin nor by proteins known to combine with endotoxins: immunoglobulins, albumin, or fibronectin; we have provided some evidence that complement components may play a role in the specific binding of endotoxins to the monocyte/macrophage membrane.     

Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences.

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.     

Regression analysis of the spontaneous spike activity of neurons in snail ganglia

Regression analysis of the spontaneous spike activity of neurons in Helix pomatia was carried out with the aim to establish the statistical parameters of this activity under constant experimental conditions and during longer time intervals. The activity of 38 randomly chosen neurons in visceral and parietal ganglia, penetrated by microelectrodes and activated either endogenously by pacemaker potentials or by synaptic inputs, was recorded during time intervals lasting from 20 min to 3 h. The main results of the statistical analyses are presented in the table where the parameters of both cell types are listed. The validity of the regression analysis applied here is discussed from the point of the possibility it offers for carrying on the data processing quickly and without applying complex calculating means. The results are also considered regarding the current interest of our research group.     

Intergeneric transformation betweenRhizobium andAzotobacter

Attempts were made to transform two strains ofAzotobacter, viz.A. chroococcum andA. vinelandii with the DNA ofRhizobium leguminosarum, R. japonicum, R. meliloti andR. trifoli. Resistance to streptomycin or crystal violet was used as marker for screening purposes. The frequency of transformation varied between 0.05 to 0.95 percent. The transformants also acquired a few other characters of the donor strains.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Increased sensitivity of the rapid hydrophobic grid membrane filter enzyme-labeled antibody procedure for Escherichia coli O157 detection in foods and bovine feces

Several strains of Escherichia coli O157:H7 artificially inoculated into vegetables and dairy products were recovered on hydrophobic grid membrane filters and enumerated by an enzyme-labeled antibody assay. The mean of the recoveries from 12 fresh vegetables was 108.8%, whereas that from 10 dairy products was 93.2%. Modified tryptic soy broth at 43 degrees C with shaking at 100 rpm provided optimum recovery of the organism from meat, with a sensitivity of less than or equal to 1 CFU/g, which is 10 times more sensitive than direct plating. The method performed equally well with vegetable and dairy products. Tryptic soy broth, however, under the same conditions gave the best results for fecal samples. Of 22 asymptomatic dairy cattle, reported as having positive Brucella titers when assayed with polyclonal antibodies, eight were found to contain E. coli O157 in their feces as demonstrated by the enzyme-labeled antibody assay by using monoclonal antibodies. This finding may explain some of the false-positive Brucella tests.