Loss of transferrin receptors following induced differentiation of HL-60 promyelocytic leukemia cells

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.     

Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor

Reduction of iron in diferric transferrin is inhibited by monoclonal antibodies to the transferrin receptor which bind at sites other than the high affinity transferrin binding site. These antibodies include B3/25, GB16 and GB22. Two antibodies which bind at the high affinity site for transferrin, 42/6 and GB18, do not inhibit iron reduction by transplasma membrane electron transport. The results are consistent with the proposal that differric transferrin reduction or stimulation of transmembrane NADH oxidase activity involves a site different from the high affinity diferric transferrin binding site. A synergistic action of antibodies with epitopes at the tight binding site involved in iron uptake and the antibodies which inhibit electron transport, B3/25 and GB16, can explain the increased inhibition of growth observed when both 42/6 and B3/25 are added to proliferating cells.     

Unscheduled DNA synthesis in primary cultures of human placentae.

Ultraviolet light-induced unscheduled DNA synthesis in primary cultures of human placentae examined as a function of radiation-dose and repair-incubation period was found to be dependent upon cell type and independent of gestational age. Primary cultures obtained by continuous harvesting of enzymatically released cells from fragments of 11-week and term specimens contained cytotrophoblasts and fibroblasts. Fibroblasts exhibited 3-fold more repair than did cytotrophoblasts from the same organ at both 11 weeks and term.     

Trophoblast transferrin and transferrin receptors in the host--parasite relationship of human pregnancy.

Transferrin and specific transferrin receptors are demonstrated on the microvillous surface of syncytiotrophoblast in human immature and term placentae by immuno histological techniques with the use of light and electron microscopy. That the distribution of transferrin is limited to the materno-foetal interface supports the hypothesis that binding of maternal transferrin to trophoblast receptors is involved in the process of iron transport to the foetus. Parallel studies with baboon placentae demonstrate the presence of trophoblast receptors which bind both baboon and human transferrin, thereby putting forward an experimental model which might be used to test the biological significance of placental transferrin receptors in primates. In addition, investigation of a large number of human cell lines shows that many transformed cells, but no normal cells (such as blood lymphocytes) or cells from primary culture (such as neonatal foreskin fibroblasts), possess the ability to bind transferrin to their membranes. These findings suggest that transferrin receptors may play important biological roles in addition to that of iron transport from mother to foetus. One such role could be the limitation of iron in intervillous spaces, thus depriving iron-requiring microorganisms of iron, hence serving as a non-specific factor of resistance for placentae. Another role for foetal transferrin receptors on trophoblasts could be to bind maternal transferrin at the materno-foetal interface, thus frustrating maternal immunosurveillance. This is similar to a mechahism used by schistosomes in the host-parasite relation where host proteins are bound by the parasite to escape immunological recognition. The presence of transferrin receptors on transformed cells suggests that this mechanism might also be employed by tumour cells. Finally, in view of previous studies which show that transferrin is required by stimulated lymphocytes to pass from the G1 to the S phase of cellular replication, it is proposed that trophoblast transferrin receptors could limit the amount of transferrin in intervillous spaces and thus impede the proliferation and possible cytotoxicity of maternal activated lymphocytes at the materno-foetal interface.     

Phylogenetic and DNA methylation analysis reveal novel regions of variable methylation in the mouse IAP class of transposons

Background

Select retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these “metastable epialleles,” nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, A^vy and Cabp^IAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%).

Results

Utilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes A^vy and Cabp^IAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p?=?0.040 and p?=?0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p?>?0.99).

Conclusions

Combined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.     

Purification and characterization of a doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) reductase from rat liver plasma membranes

Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.     

In vivo analysis of the 3' untranslated region of GB virus B after in vitro mutagenesis of an infectious cDNA clone: persistent infection in a transfected tamarin

GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3' untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3' terminal sequence. Our investigators previously demonstrated that the 3' terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.     

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.     

Development of a genetic linkage map and identification of homologous linkage groups in sweetpotato using multiple-dose AFLP markers

Sweetpotato genomic research is minimal compared to most other major crops despite its worldwide importance as a food crop. The development of a genetic linkage map in sweetpotato will provide valuable information about the genomic organization of this important species that can be used by breeders to accelerate the introgression of desired traits into breeding lines. We developed a mapping population consisting of 240 individuals of a cross between ‘Tanzania’, a cream-fleshed African landrace, and ‘Beauregard’, an orange-fleshed US sweetpotato cultivar. The genetic linkage map of this population was constructed using Amplified Fragment Length Polymorphism (AFLP) markers. A total of 1944 (‘Tanzania’) and 1751 (‘Beauregard’) AFLP markers, of which 1511 and 1303 were single-dose markers respectively, were scored. Framework maps consisting of 86 and 90 linkage groups for ‘Tanzania’ and ‘Beauregard’ respectively, were developed using a combination of JoinMap 3.0 and MAPMAKER/EXP 3.0. A total of 947 single-dose markers were placed in the final framework linkage map for ‘Tanzania’. The linkage map size was estimated as 5792 cM, with an average distance between markers of 4.5 cM. A total of 726 single-dose markers were placed in the final framework map for ‘Beauregard’. The linkage map length was estimated as 5276 cM, with an average distance between markers of 4.8 cM. Duplex and triple-dose markers were used to identify the corresponding homologous groups in the maps. Our research supports the hypothesis that sweetpotato is an autopolyploid. Distorted segregation in some markers of different dosages in this study suggests that some preferential pairing occurs in sweetpotato. However, strict allopolyploid inheritance in sweetpotato can be ruled out due to the observed segregation ratios of the markers, and the proportion of simplex to multiple-dose markers. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This paper is a portion of a dissertation submitted by Jim C. Cervantes-Flores.