Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational spectrum and recombinogenic effects induced by aminofluorene adducts in bacteriophage M13

Double-stranded replicative form (RFI) DNA of bacteriophage M13 strain M13mp10 which carries partial lacZ gene has been modified in vitro to various extents with N-hydroxy-2-amino-fluorene (N-OH-AF) and then transfected into E. coli cells. High-performance liquid chromatography (HPLC) analysis results demonstrate that the sole adduct (95%) formed in modified DNA is N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF). Approximately 20 adducts per RFI molecule constitute 1 lethal event when plaque-forming ability is assayed on E. coli cells which have received no prior SOS induction. The mutagenicity of dG-C8-AF adducts was assayed by measuring loss of beta-galactosidase activity as a function of adducts per molecule. A dose-dependent increase in Lac- mutants was observed, with a 4-fold increase in mutants per survivor at 30 adducts/molecule. The mutations produced, characterized by DNA sequencing, occur predominantly at either G or C positions different from those observed in the spontaneous mutant spectrum. Restriction-mapping results show that in our assay system, dG-C8-AF adducts induce a previously unreported recombinogenic activity.     

The catecholamine binding site of the beta-adrenergic receptor is formed by juxtaposed membrane-spanning domains

The catecholamine binding domain of the turkey erythrocyte beta-adrenergic receptor was mapped by determining the sites of covalent labeling of the purified receptor by two beta-adrenergic photoaffinity reagents, [125I]iodocyanopindolol-diazirine (ICYP-da) and [125I] iodoazidobenzylpindolol (IABP). Both labels were incorporated at two separate sites. By sequencing a labeled peptide, one site of labeling was found to lie at Trp330 in the extracellular half of the seventh membrane span. This position is homologous to the retinal attachment site in rhodopsin. The second labeled site was isolated on an 8000-Da peptide and immunoprecipitated using sequence-directed antibodies. This site lies in membrane spans 3-5. Labeling of the two sites was equal using ICYP-da and 3-10-fold greater in the span 7 site using IABP. These data indicate that the catecholamine binding site is formed from the juxtaposition of span 7 and spans 3-5 in a tertiary structure probably similar to that of rhodopsin.     

Dissociation between increased surface expression of gp165/95 and homotypic neutrophil aggregation

Whether homotypic neutrophil aggregation depends on the quantitative increase of gp165/95 molecules (Mac 1, CR3) recruited to the cell surface during activation was studied using mAb of the CD11b group that recognize distinct epitopes encoded by the alpha-subunit of this glycoprotein. After the addition of antibody MN41, neutrophils did not aggregate in response to a chemoattractant, FMLP. Blockade of preexisting surface gp165/95 by mAb MN41, followed by removal of the excess antibody from the mixture, was used to show that the molecules of gp165/95 newly expressed in response to stimulation by a chemoattractant were incapable of effectively mediating the induced cell-cell interactions of aggregation. Flow cytometry studies confirmed that binding of unlabeled antibody MN41 did not block further increases in surface expression of gp165/95 after stimulation with FMLP. These data suggest that molecules of gp165/95 exhibit two functionally distinct forms, one, present on the surface of freshly isolated neutrophils, that becomes competent to mediate the aggregation response upon activation by a stimulus and a second form that can be translocated to the cell surface by the stimulus but is greatly diminished if not lacking in the ability to participate in that aggregation event.     

Evidence for two forms of phospholipase A2 in human semen

The molecular weight of the active unit of phospholipase A₂ (PA₂) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA₂ activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA₂ activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA₂ was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA₂ activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA₂ when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA₂ was found and was sensitive to changes in temperature.     

Experimental formulations ofBacillus sphaericus for the control of anopheline and culicine larvae

Summary Four experimental formulations ofBacillus sphaericus Neide (2362 isolate) were evaluated for larvicidal activity against culicine and anopheline larvae in several natural and artificial habitats. A granular formulation (5% primary powder) was tested against natural populations of mosquitoes in two simulated habitats in Florida and in maturing and reflooded rice fields in Louisiana. Larvae ofCulex quinquefasciatus Say were reduced by 97 and 99% after application of the granules at the rate of 10 kg/ha to polluted tanks and 2.5 kg/ha to sod-lined potholes, respectively. Anopheline andPsorophora columbiae (Dyar and Knab) larvae were reduced by 68 and 92–100%, respectively, after application of 5 kg granules/ha to rice fields. A flowable concentrate (12.8% primary powder) applied to unpolluted and organically enriched habitats in Florida at 0.25 kg/ha reduced populations ofCulex spp. by 93–100% and 99%, respectively. Sustained-release briquets (5% primary powder) applied at the rate of one half briquet/1.8 m² sod-lined potholes reduced larval populations ofCx. quinquefasciatus by 88–95% for up to 2 weeks in open sunlight. Sustained-release pellets (30% primary powder) applied to small woodland pools in Memphis, TN at the rate of four pellets/pool virtually eliminated larval populations ofCx. restuans Theobald for over 8 days. Variable persistance of larvicidal activity was noted for the other treatments depending on the formulation, target species and habitat.     

A review of the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency

Hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) is a purine salvage enzyme that catalyses the conversion of hypoxanthine and guanine to their respective mononucleotides. Partial deficiency of this enzyme can result in the overproduction of uric acid leading to a severe form of gout, whilst a virtual absence of HPRT activity causes the Lesch-Nyhan syndrome which is characterised by hyperuricaemia, mental retardation, choreoathetosis and compulsive self-mutilation. The HPRT-encoding gene is located on the X chromosome in the region q₂₆–q₂₇ and consists of nine exons and eight introns totalling 57 kb. This gene is transcribed to produce an mRNA of 1.6 kb, which contains a protein encoding region of 654 nucleotides. With the advent of increasingly refined techniques of molecular biology, it has been possible to study the HPRT gene of individuals with a deficiency in HPRT activity to determine the genetic basis of the enzyme deficiency. Many different mutations throughout the coding region have been described, but in the absence of precise information on the three-dimensional structure of the HPRT protein, it remains difficult to determine any consistent correlation between the structure and function of the enzyme.     

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.