Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells

A group of mAb recognizing the 200- and/or 220-kDa determinants (CD45R) of the leukocyte common Ag such as 2H4, WR16, MD4.3, and SN130 cross-block each other showing that they recognize a closely related epitope. The antibody UCHL1 reacts with a 180-kDa determinant of the leukocyte common Ag and exhibits a reciprocal T subset distribution pattern to the CD45R group. Peripheral blood T cells were 40% positive for UCHL1 and 58 to 65% positive for the CD45R antibodies; less than 1% of cells stained for both. On activation of CD45R+,UCHL1- T cells by PHA, up to 40% of cells became positive for both CD45R and UCHL1 by day 3. By day 7, CD45R+,UCHL1- cells fell from 90 to less than 21% whereas UCHL1+,CD45R- cells rose from 2 to 93%. Conversely, PHA-stimulated UCHL1+,CD45R- cells remained UCHL1+,CD45R- during the 7 days in culture showing that phenotypic change was unidirectional from CD45R+ to UCHL1+. In primary allogeneic mixed lymphocyte reactions, activated CD45R+ T cells also showed a change to UCHL1+. When these cells were rechallenged by the original alloantigen, the UCHL1+ cells showed 7- to 20-fold greater proliferation than the CD45R+ cells on day 3 after rechallenge. The recovery of virtually all alloantigen induced secondary proliferative response in the UCHL1+,CD45R- T cell population suggests that UCHL1 identifies a primed population of T cells which may include memory cells.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Replication of R-factor R1 in Scherichia coli K-12 at different growth rates.

The R-factor R1drd-19 mediates resistance to beta-lactam antibiotics via a beta-lactamase. A strain of Escherichia coli K-12 carrying R1drd-19 was grown at different growth rates by using different carbon sources. The specific rate of production of the R1 beta-lactamase increased linearly with the growth rate and with the gene dosage. The content of R1 deoxyribonucleic acid was estimated by alkaline sucrose gradient centrifugation and by analysis of the specific rate of beta-lactamase synthesis in nutritional shift-up experiments and was found to decrease fivefold when the growth rate was increased from 0.4 to 1.8 doublings per h. The number of R1 molecules per cell decreased from six to two in the same growth range. The presence of the plasmid affected the mean cell size significantly; at a growth rate of 0.4 doublings per h the R-+ cells were on the average 50% bigger than the R-minus cells, whereas the effect was less than 10% at a growth rate of 1.8 doublings per h. Several reports in the leterature state that the initiation mass of chromosome replication is constant. In this paper it is shown that the initiation mass of R1 replication is proportional to the growth rate. Thus, the replication of the plasmid R1 and of the chromosome are independently regulated processes. It is argued that plasmid replication is under negative control.     

Role of a thymic stromal cell clone in inducing the stage-specific differentiation of various subpopulations of double negative thymocytes

The monolayer of a thymic stromal cell clone termed MRL104.8a induced the differentiation of adult double negative (DN) thymocytes (CD3-4-8-) through a CD3-4-8+ intermediate into CD3- (or dull) 4+8+ stages. DN thymocytes were separated into three subpopulations depending on their cell-surface expression of Pgp-1 and IL-2R, namely, Pgp-1+IL-2R-, Pgp-1-IL-2R+, and Pgp-1-IL-2R-. The present study investigated the requirements of the MRL104.8a monolayer for inducing the differentiation of these DN thymocyte subpopulations. The following were revealed: i) the MRL104.8a monolayer failed to induce the differentiation of a Pgp-1+IL-2R- subpopulation; ii) whereas a Pgp-1-IL-2R+ subpopulation did not express either CD4 or CD8 Ag when cultured in medium, culturing this subpopulation on the thymic stromal cell monolayers resulted in the expression of CD8 but not CD4 Ag; and iii) a Pgp-1-IL-2R- DN subpopulation obtained through less extensive treatments with anti-CD4 and anti-CD8 antibodies in the presence of C before sorting procedures spontaneously differentiated into double positive cells in medium. In contrast, most of DN cells with the same phenotype obtained through extensive anti-CD4 and -CD8 treatments before sorting failed to express CD4 and/or CD8 Ag in medium but could differentiate through a CD3-4-8+ into more mature stages only when they were cultured on the thymic stromal monolayer. These results indicate differential requirements of thymic stromal cells for the differentiation of various DN subpopulations with qualitatively distinct phenotypes and different magnitudes (very low vs almost zero levels) of CD4/CD8 expression.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

Immunoregulatory T lymphocytes in man. Soluble antigen-specific suppressor-inducer T lymphocytes are derived from the CD4+CD45R-p80+ subpopulation

Regulation of the immune response in man is largely dependent on interactions between cells of the cluster designation 4+ (CD4+) helper/inducer sublineage and the CD8+ suppressor/cytotoxic sublineage. When cultured with autologous antigen-primed CD4+ lymphocytes, CD8+ cells differentiate into suppressor T cells (Ts) that specifically inhibit the response of fresh autologous CD4+ cells to the priming antigen only. The current study was undertaken to analyze the roles in this suppressor circuit of subpopulations of the CD4+ sublineage distinguished from one another on the basis of their binding (or lack of binding) to monoclonal antibodies against molecules p80 (Leu8) and CD45R (p220/Leu18/2H4). When examined for the proliferative responses to alloantigenic stimuli, each of the four: CD4+p80+, CD4+p80-, CD4+CD45R+, and CD4+CD45R- populations proliferated vigorously, synthesized interleukin 2 (IL-2) and interferon and released soluble IL-2 receptors. However, the responses to soluble antigens such as Candida and diphtheria toxoid were exhibited by CD4+CD45R-, CD4+p80+, and CD4+p80- cells, but not by CD4+CD45R+ cells. When examined for their ability to induced CD8+ Ts in the Candida-driven suppressor-induction culture system, only CD4+p80+ and CD4+CD45R- cells induced strong suppression. Further, when CD4+CD45R- cells were separated into CD4+CD45R-p80+ and CD4+CD45R-p80- subpopulations, despite the ability of both subpopulations to respond to Candida, only CD4+CD45R-p80+ cells induced autologous CD8+ Ts. Activated CD8+ Ts suppressed not only proliferation but also the release of soluble IL-2 receptors by autologous antigen-activated CD4+ cells. Thus, the antigen-specific suppressor-inducer T cells appear to be derived from the CD4+CD45R-p80+ (Leu3+, Leu8+, 2H4-) subpopulation of the CD4+ sublineage.     

Restriction and modification activities from Streptococcus lactis ME2 are encoded by a self-transmissible plasmid, pTN20, that forms cointegrates during mobilization of lactose-fermenting ability.

A self-transmissible (Tra+) plasmid encoding determinants for restriction and modification activities (R+/M+) from Streptococcus lactis ME2 was isolated and characterized. The 28-kilobase (kb) plasmid (pTN20) was detected in lactose-fermenting (Lac+) transconjugants generated from matings between S. lactis N1, and ME2 variant, and a plasmid-free recipient, S. lactis LM2301. The plaquing efficiencies of prolate- and small isometric-headed phages were reduced on transconjugants containing either pTN20 (R+/M+ Tra+) or 100-kb plasmids encoding Lac+, R+/M+, and Tra+. Lac+ transconjugants which harbored pTR1040 (Lac+) and pTN20 (R+/M+) were phenotypically R-/M- and transferred Lac+ at low frequency in subsequent matings to give rise to 100-kb R+/M+ plasmids. R+/M+ activities and high-frequency conjugal transfer ability were detected in Lac+ transconjugants that contained pTR1041 (Lac+) and pTN20 (R+/M+). No 100-kb R+/M+ plasmids were recovered after these matings, suggesting that pTR1041 was mobilized by pTN20 through a process that resembled plasmid donation. pTR1041 was identical to pTR1040 but contained an additional 3.3-kb DNA fragment. These data suggested that phenotypic expression of R+/M+ and Tra+ is affected by coresident Lac+ plasmids. Restriction enzyme analysis and hybridization reactions demonstrated that the 100-kb R+/M+ plasmid was formed by a cointegration event between pTR1040 (Lac+) and pTN20 (R+/M+ Tra+) during conjugal transfer via a conductive-type process. This is the first report that defines self-transmissible restriction and modification plasmids in the lactic streptococci.     

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.     

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.     

Characteristics of the ionic composition and ultrastructure of Bordetella pertussis in controlled regulation of the mineral element content in a growth medium

The content of trace elements necessary for the normal growth of bacteria was found to have no effect on the intracellular concentration of Ca2+ and Al3+. The content of Cu2+, Fe3+, Mn2+, Mg2+ was considerably reduced. The addition of Mg2+ at different concentrations to this culture medium stimulated the capacity of cells for accumulating not only Mg2+, but also some other ions. Their maximum intracellular concentration was observed when the concentration of Mg2+ in the culture medium was 41 mM. The growth of microbial cells in the standard culture medium containing Mg2+ at a concentration of 4 mM was accompanied by the increased consumption of elements actively participating in redox reactions (Cu2+, Fe3+, Mn2+). Shifts in the ionic composition of microbial cells were manifested by the morphological features of B. pertussis, linked with the increased synthesis of crystalloid structures. The influence of Mn2+, Al3+, Zn2+ at different concentrations on the ionic composition and morphology of B. pertussis was studied.     

An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

We examined the roles of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP3R isoforms (IP3R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba2+ than does capacitative calcium entry. This cation entry is absent in IP3R-KO cells. Expression of the type-3 IP3R (IP3R-3) in the IP3R-KO cells rescued not only agonist-dependent release of intracellular Ca2+, but also Ba2+ influx following receptor stimulation. Similar results were obtained with an IP3R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP3R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca2+ release or receptor-dependent Ba2+ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP3R.     

The evaluation of mixtures of yeast and potato extracts in growth media for biomass production of lactic cultures

The effectiveness of yeast extracts (YE) and potato extracts (PE) to promote growth of seven lactic cultures was evaluated by automated spectrophotometry (AS). Two aspects of the growth curve were analysed: (1) maximum biomass obtained (using ODmax) and (2) highest specific growth rate mu(max)) Eleven lots from the same PE-manufacturing process were examined for lot-to-lot variability. The ODmax values of three of the seven strains were significantly affected by lot source, but mu(max) was not significantly affected. The growth of bacteria was systematically lower in base medium containing 100% PE than in base medium containing 100% YE for both ODmax or mu(max) data, which could be related to the lower content in nitrogen-based compounds in PE. In AS assays, highest OD values for Lactobacillus casei EQ28, Lactobacillus rhamnosus R-011, Lactobacillus plantarum EQ12, and Streptococcus thermophilus R-083 were obtained with a mixture of PE and YE. Fermentations (2 L) were also carried out to determine the accuracy of AS to predict biomass levels obtained under fermentation trials. In these fermentations, replacement of 50% YE with PE was shown to enable good growth of S. thermophilus. With L. rhamnosus R-011, a high correlation (R2 = 0.95) was found between ODmax data obtained in the AS assays and that of the 2-L bioreactor when the same growth medium was used for both series of fermentations. However, AS was not as efficient when industrial media were used for the bioreactor assays. The relationship was still good for ODmax between AS data and that of the bioreactor data with L. rhamnosus R-011 in industrial LBS medium (R2 = 0.87), but was very poor with the S. thermophilus R-083 on Rosell #43 industrial medium (R2 = 0.33). Since PE cost 40% less than YE, there are strong economic advantages in considering such a partial replacement of YE by PE.     

Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153)

The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HB101 have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-1 than at 0.15 h-1. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-1, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene. Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that in complex media the plasmid is maintained longer by E. coli HB101 at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.     

The functionally distinct subpopulations of human CD4+ helper/inducer T lymphocytes defined by anti-CD45R antibodies derive sequentially from a differentiation pathway that is regulated by activation-dependent post-thymic differentiation

The CD4+ helper/inducer T cell population is comprised of functionally distinct subsets identifiable by the HB11 (anti-CD45R) mAb. We have previously shown that the cells that provide help for antibody production express the CD4+CD45R- phenotype. In contrast, CD4+ CD45R+ cells have minimal, if any, helper cell functions; rather, these cells function as inducers of Ts cell activity. The lineal relationship of these phenotypically and functionally distinct CD4+ subsets is unknown. In the present studies, we have examined the hypothesis that the CD4+ subpopulations identifiable with anti-CD45R antibodies represent "virgin" or "memory" T cells sequentially derived from a common differentiation pathway but differing in their relative maturation. When freshly purified cells were tested, CD4+ CD45R+ cells had no Th cell function. However, after in vitro activation with PHA and propagation in IL-2, CD4+CD45R+ cells acquired the ability to provide help for antibody production. Moreover, this functional acquisition by these cells was accompanied by their conversion to the CD4+CD45R- phenotype. Analyses of the activation, growth kinetics, and functional dose-response characteristics of CD4+CD45R+ and CD4+CD45R- cells demonstrated that our findings did not result from the selective growth of CD4+ CD45R- cells contaminating the CD4+CD45R+ preparations. Thus, these data demonstrate that the "helper" and "suppressor-inducer" subsets of CD4+ cells identified by anti-CD45R antibodies are not comprised of fully mature, phenotypically and functionally stable T cells. Rather, these CD4+ subsets appear to represent cells at different maturational stages of an activation-dependent, post-thymic differentiation pathway.     

Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes

A proportion of Pgp-1+ cells in the thymus have been shown to have progenitor activity. In adult AKR/Cum mice the total Pgp-1+ population in the thymus differs from that of the bulk of thymocytes and is antigenically heterogeneous when examined by flow cytometry. Pgp-1+ thymocytes are enriched for several minor cell populations compared to total thymocytes: B2A2-, interleukin-2-receptor+ (IL-2R+), and Lyt-2-, L3T4-. However, these subsets are still a minor proportion of the Pgp-1+ cells, the majority being Lyt-2+ and/or L3T4+ and B2A2+. Pgp-1+ thymocytes also differ from the bulk of thymocytes in having lower amounts of Thy-1 and in showing a higher proportion of single positive (Lyt-2+, L3T4- or Lyt-2-, L3T4+) cells. Populations of adult thymocytes that are enriched in progenitor cells can be isolated by cytotoxic depletion using either anti-Thy-1 antibody (Thy-1 depletion) or anti-Lyt-2 and anti-L3T4 antibody (Lyt-2, L3T4 depletion). Pgp-1+ cells in progenitor cell-enriched populations are also phenotypically heterogeneous. Pgp-1+ cells in both populations may be IL-2R+ or IL-2R- and B2A2+ or B2A2-. The population of Pgp-1+ cells in progenitor cell-enriched populations in the adult differs from that of the fetus at 14 days of gestation in that in the 14-day fetus, most Pgp-1+ cells are IL-2R+. By Day 15 of gestation, distinct populations of Pgp-1+, IL-2R-; Pgp-1+, IL-2R+; and Pgp-1-, IL-2R+ cells are observed. In the 15-day fetus, as in the adult, many Pgp-1+ thymocytes express low to moderate levels of Thy-1. The total percentage of Pgp-1+ cells in the thymus varies among different mouse strains, ranging from 4 to 35% in the thymus of young adult mice. Pgp 1.1 strains contain more detectably Pgp-1+ thymocytes than Pgp 1.2 strains; however, there is variability in the proportion of Pgp-1+ cells, even among Pgp 1.2 strains. In contrast to AKR/Cum mice, the Pgp-1+ thymocyte population in BALB/c mice, which contain a high proportion of Pgp-1+ thymocytes, closely resembles the total thymocyte population.     

Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes

Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+-signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+-signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signalling.     

Heavy metals resistant plasmid-mediated utilization of solar by Pseudomonas aeruginosa AA301

Solar-degrading bacteria, Pseudomonas aeruginosa strains, were isolated from Egyptian soil by Mineral Salt Medium (MSM) supplemented with Solar (motor fuel) from different oil-contaminated sites in Sohag province. The strain AA301 of Pseudomonas aeruginosa showed appreciable growth in MSM medium containing high concentrations of Solar ranging from 0.5 to 3% (v/v), with optimum concentration at 1.5%. Solar was used as a sole carbon source and a source of energy by the bacterium. The ability to degrade Solar was found to be associated with a single 60-kb plasmid designated pSOL15. The plasmid-cured variant, which was obtained by culturing in LB broth with kanamycin, lost the plasmid indicative the ability to degrade Solar must depend on this plasmid. The wild type isolate, Pseudomonas aeruginosa AA301 and transformant strain, have maximum growth (OD600 = approximately 2) on Solar, however the plasmid-cured variant did not have any significant growth on Solar. Moreover, resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ was also 60-kb plasmid-mediated. Therefore, the strain AA301 could be good candidate for remediation of some heavy metals and oil hydrocarbons in heavily polluted sites.     

Kinetics of thymus repopulation by intrathymic progenitors after intravenous injection: evidence for successive repopulation by an IL-2R+, Pgp-1- and by an IL-2R-, Pgp-1+ progenitor

A kinetic study of thymus repopulation after intravenous injection of L3T4-, Lyt-2- thymocytes further depleted of IL-2R+ or Pgp-1+ cells indicates that donor cells within the thymus at Day 8 after injection descend primarily from an IL-2R+, Pgp-1- progenitor, while at Day 17 after injection most progeny of donor phenotype descend from an IL-2R-, Pgp-1+ progenitor. Repopulation studies in organ culture demonstrate that IL-2R+, Pgp-1+ cells also have progenitor activity. All three progenitors give rise to differentiated cell types normally present in the thymus. These results are consistent with the interpretation that the L3T4-, Lyt-2- population is composed of a heterogeneous collection of progenitors which repopulate the thymus with differing kinetics and imply that it will be difficult to establish lineage relationships within this population in the absence of a clonal assay for thymocyte progenitors.     

Autocrine production of insulin-like growth factor II using an inducible expression system results in reduced estrogen sensitivity of MCF-7 human breast cancer cells.

An inducible expression vector, utilizing the metal response elements from the human metallothionein IIA promoter and encoding human prepro-insulin-like growth factor II (IGF-II), was transfected into MCF-7 McG cells, an MCF-7 subline which exhibits an estrogen dependent phenotype in vitro and does not express detectable levels of IGF-II mRNA. Two stably transfected clones, designated MI5 and MI7, which expressed IGF-II mRNA in a Zn2+ regulated manner, were isolated. Clone MI5 did not secrete detectable levels of IGF-II activity, as determined by radioimmunoassay of conditioned medium, but clone MI7 secreted high levels of IGF-II activity in a Zn2+ inducible fashion. Clone MI5 and control clones transfected with the selection plasmid pSV2 neo and the control plasmid pSP65 continued to display an estrogen dependent phenotype in vitro. However, under both anchorage dependent and anchorage independent growth conditions, clone MI7 cells exhibited an estrogen responsive, rather than dependent, phenotype. Moreover, when grown in the presence of inducing concentrations of Zn2+, MI7 cells were either virtually (for anchorage dependent growth) or completely (anchorage independent growth) unresponsive to estradiol. Both the basal growth rate in the absence of metal ions and the Zn2+ induced increases in cell proliferation could be inhibited by the monoclonal antibody alpha-IR3, which blocks the binding site of the IGF-I receptor. The antiestrogens tamoxifen and 4-hydroxytamoxifen were found to enhance the growth stimulation resulting from Zn2+ induced IGF-II production in MI7 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of physical factors on the growth of insect cells in vitro. II. Sodium and potassium as osmotic pressure regulators of moth cell growth.

The tolerance of a cell line (IMC-HZ-1) from a moth, Heliothis zea, for the monovalent cations Na+ and K+ were defined. Cells shifted to media containing more than 70 mM of K+ showed decreased growth rates. No evidence was obtained for Na+ toxicity. The osmotic pressure tolerances were influenced by the K+ concentration of the medium. The richer the medium was in K+, the narrower was the spectrum of osmotic pressure tolerance. Once the limit of K+ tolerance was exceeded, the rate of decline of growth was linear with respect to further increases in K+. This rate of decline was independent of osmotic pressure. The initial responses of cells during one subculture (2 to 4 population doublings) in media differing from the standard medium (used to maintain the cell line) were not reliable indicators of the growth potential of the cells. Continued subculture in such media resulted in an upward trend in population growth rates in most cases.