Hair cell regeneration after acoustic trauma in adult Coturnix quail

Recovery of hair cells was studied at various times after acoustic trauma in adult quail. An initial loss of hair cells recovered to within 5 percent of the original number of cells. Tritium-labeled thymidine was injected after this acoustic trauma to determine if mitosis played a role in recovery of hair cells. Within 10 days of acoustic trauma, incorporation of [3H]thymidine was seen over the nuclei of hair cells and supporting cells in the region of initial hair cell loss. Thus, hair cell regeneration can occur after embryonic terminal mitosis.     

Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets

Damage to the vessel wall is a signal for endothelial migration and replication and for platelet release at the site of injury. Addition of transforming growth factor-beta (TGF-beta) purified from platelets to growing aortic endothelial cells inhibited [3H]thymidine incorporation in a concentration-dependent manner. A transient inhibition of DNA synthesis was also observed in response to wounding; cell migration and replication are inhibited during the first 24 hours after wounding. By 48 hours after wounding both TGF-beta-treated and -untreated cultures showed similar responses. Flow microfluorimetric analysis of cell cycle distribution indicated that after 24 hours of exposure to TGF-beta the cells were blocked from entering S phase, and the fraction of cells in G1 was increased. The inhibition of the initiation of regeneration by TGF-beta could allow time for recruitment of smooth muscle cells into the site of injury by other platelet components.     

Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells

The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro with [3H]thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis. Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus, apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA breakdown and preventing apoptosis in erythroid progenitor cells.     

Quinacrine: mechanisms of antimalarial action

Two new interesting modes of action of quinacrine have been discovered. The first concerns a dose-related inhibition of uptake of [8-(3)H] adenosine into host cells of parasitized blood. Second, the drug inhibits the incorporation of tritiated adenosine triphosphate primarily into RNA but also into DNA of the erythrocyte-free malarial parasite Plasmodium berghei.     

DNA synthesis and mitosis in well-differentiated mammalian cardiocytes

Incorporation of [(3)H]thymidine into nuclei of heart cells of 2-day-old rats indicates that neonatal cardiac cells containing well-aligned myofibrils synthesize DNA. In these highly differentiated cells, neither the presence of contractile proteins nor their organization into myofibrils inhibits either DNA synthesis or mitosis.     

Sequence-specific isotope effects on the cleavage of DNA by bleomycin

Bleomycin is a metal- and oxygen-dependent DNA cleaver. The chemistry of DNA damage has been proposed to involve rate-limiting abstraction of the 4'-hydrogen. A DNA fragment has been prepared that contains [4'-2H]thymidine residues of high isotopic content. Primary kinetic isotope effects have been directly observed at individual thymidine residues with DNA sequencing technology.     

Stimulation of cells by antibody

Tumor cell lines exposed to immunoglobulins specific for cell surface antigens developed increased cellular incorporation of [(125)I]iododeoxyuridine and [(3)H]thymidine (up to 200-fold increases over cells treated with normal rabbit immunoglobulins). Antibody-stimulated cells multiplied more rapidly and lived longer than control cells in tissue culture. These observations were made both with cells substituted with 2,4,6-trinitrophenol and purified antibody against 2,4,6-trinitrophenol, and with several cell lines and their respective whole-cell antibodies. Antibodies that were stimulatory at low concentrations were cytotoxic at high concentrations. These observations may have significance in regard to enhancing effects of antibodies on tumor cell growth in vivo.     

Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA

A rat dopamine (DA) transporter complementary DNA has been isolated with combined complementary DNA homology and expression approaches. The DA transporter is a 619-amino acid protein with 12 hydrophobic putative membrane-spanning domains and homology to the norepinephrine and gamma-aminobutyric acid transporters. The expressed complementary DNA confers transport of [3H]DA in Xenopus oocytes and in COS cells. Binding of the cocaine analog [3H]CFT ([3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane) to transfected COS cell membranes yields a pharmacological profile similar to that in striatal membranes.     

Adaptive response of human lymphocytes to low concentrations of radioactive thymidine

When human lymphocytes were cultured with [3H]thymidine, which acts as a source of low-level chronic radiation, and then exposed to 150 rad of x-rays at 5, 7, 9, or 11 hours before fixation, the yield of chromatid aberrations was less than the sum of the yields of aberrations induced by [3H]thymidine and x-rays separately. Often fewer aberrations were found after exposure to radiation from both sources than were found after exposure to x-rays alone. At the same fixation times, nonradioactive thymidine did not affect the yield of x-ray-induced aberrations. The same phenomenon occurred at earlier fixation times, after exposure to 30 or 40 rad of x-rays and [3H]thymidine. This response is analogous to the adaptive response to alkylating agents whereby prior treatment with small doses for a long period reduces the damage occurring from large doses of similar agents given for a short time.     

Human-mouse somatic cell hybrids with single human chromosome (group E): link with thymidine kinase activity

Mouse somatic cells lacking thymidine kinase were mixed in culture with human diploid cells lacking hypoxanthine guanine phosphoribosyl transferase, and hybrid cells were isolated and maintained in a selective medium containing hypoxanthine, aminopterin, and thymidine. The hybrid cells at the time of isolation had karyotypes consisting predominantly of mouse chromosomes but with one human chromosome, a submetacentric member of group E, apparently giving thymidine kinase to the hybrid cell. However, after long-term propagation in the selective medium this chromosome has been lost, although cells continue to show thymidine kinase activity as demonstrated by the incorporation of (3)H-thy-midine into DNA in the hybrid cell. The hybrid cells have only mouse electro-phoretic variants for glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase, suggesting that the human genetic loci for these enzymes are not represented in the hybrid genome and may be unlinked to that for thymidine kinase.     

Requirement of nuclear prolactin for interleukin-2--stimulated proliferation of T lymphocytes

Prolactin (PRL) is necessary for the proliferation of cloned T lymphocytes in response to interleukin-2 (IL-2). Translocation of PRL into the nucleus occurs during IL-2--stimulated mitogenesis. Therefore, the function of intranuclear PRL in T cell proliferation was tested. Eukaryotic expression vectors were prepared to express wild-type PRL [PRL(WT)], PRL that lacks the signal sequence for translocation into the endoplasmic reticulum [PRL(ER-)], and chimeric PRL in which the signal peptide was replaced with the sequence that directs the nuclear translocation of the SV40 large T antigen [PRL(NT+)]. Expression of these constructs in a T cell line (Nb2) responsive to PRL and IL-2 resulted in localization of PRL in the extracellular milieu, cytoplasm, or nucleus, respectively. Stimulation with IL-2 alone resulted in a five- to tenfold increase in the incorporation of [3H]thymidine by cells expressing PRL(NT+) or PRL(WT) as compared to PRL(ER-) or the parental Nb2 cells. Only the PRL(NT+) clone proliferated continuously with IL-2 stimulation in the presence of antiserum to PRL. These results demonstrate that nuclear PRL is necessary for IL-2--stimulated proliferation and suggest that a peptide hormone can function in the nucleus without binding to its cell surface receptor.     

Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA

Both interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) induce proliferation of cultured fibroblasts and smooth muscle cells. These polypeptide mediators are released by activated macrophages and other cell types in response to injury and are thought to have a role in tissue remodeling and a number of pathologic processes. Analysis of the kinetics of [3H]thymidine incorporation by cultured fibroblasts demonstrated that the response to IL-1 is delayed approximately 8 hours relative to their response to PDGF. IL-1 transiently stimulated expression of the PDGF A-chain gene, with maximum induction after approximately 2 hours. Subsequent synthesis and release of PDGF activity into the medium was detected as early as 4 hours after IL-1 stimulation, and downregulation of the binding site for the PDGF-AA isoform of PDGF followed PDGF-AA secretion. Antibodies to PDGF completely block the mitogenic response to IL-1. Therefore, the mitogenic activity of IL-1 for fibroblasts and smooth muscle cells appears to be indirect and mediated by induction of the PDGF A-chain gene.     

A portal blood factor as the humoral agent in liver regeneration

It was demonstrated, by use of extracorporeal cross-circulation, that a humoral factor responsible for liver regeneration does not arise from the liver remnant. While intact livers of normal rats incorporated [methyl-(3)H]- thymidine in proportion to the amount of liver removed in the partner, the greatest response occurred after a total hepatectomy. Evidence from portacaval-shunted,partially hepatectomized animals connected to normal members indicates that the factor is in portal blood and that the onset of regeneration is the result of a quantitative imbalance between the available portal blood factor and the number of liver cells present.     

外源DNA导入酿酒酵母(Saccharomy cescerevisiae)2.558电转化条件的研究

[目的]探讨外源DNA导入酿酒酵母(Saccharomy cescerevisiae)2.558的电转化条件,初步确立提高其转化效率的方法。[方法]通过调整电转化的参数,将已构建好的转化载体转化至酿酒酵母2.558的感受态细胞中,对影响电转化的主要因素进行研究。[结果]采用对数生长中期菌体制备的感受态细胞,在质粒DNA加入量为1μg、电场强度为1.8 kV/cm、电阻为225Ω、用复苏缓冲液Ⅱ进行复苏培养的条件下,电转化效率达到最大值。[结论]该研究确定了酿酒酵母(Saccharomy cescerevisiae)2.558的最优电转化条件,为其遗传改造奠定了良好的技术基础。     

Mitogenesis in response to PDGF and bombesin abolished by microinjection of antibody to PIP2

The turnover of phosphatidylinositol 4,5-bisphosphate (PIP2) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including mitogens, but decisive evidence for the idea has not been obtained. In the present study, a monoclonal antibody to PIP2 was microinjected into the cytoplasm of NIH 3T3 cells before or after exposure to mitogens. The antibody completely abolished nuclear labeling with [3H]thymidine induced by platelet-derived growth factor and bombesin, but not by fibroblast growth factor, epidermal growth factor, insulin, or serum. The findings strongly suggest that PIP2 breakdown is crucial in the elicitation and sustaining of cell proliferation induced by some types of mitogens such as platelet-derived growth factor and bombesin.     

Transplanted precursors of nerve cells: their fate in the cerebellums of young rats

The multiplying cells of the external granular layer in 7-day-old rats were labeled with [(3)H]thymidine. Slabs of the cerebellum were transplanted into the same region of uninjected hosts of the same age. The transplanted, undifferentiated cells of the donors migrated actively in the cerebellar cortex of the hosts and apparently differentiated there into basket and granule cells.     

Blockade of deoxyribonucleic acid synthesis by deuterium oxide

Interference with deoxyribonucleic acid replication need not be a primary mechanism in the blockade of cell division by deuterium oxide, but current hypotheses on the molecular basis of the blockade do suggest that such interference might take place under appropriate conditions. Autoradiographic experiments support the suggestion, for whereas normal sea urchin eggs incorporate H(3)-thymidine into deoxyribonucleic acid from almost the beginning of development, cells immersed in deuterium-enriched media do not. Blockade of mitosis and inhibition of thymidine incorporation are simultaneously relieved when the eggs are returned to normal water.     

Interferon inhibits the establishment of competence in Go/S-phase transition

Addition of mouse interferon-alpha/beta (IFN) to confluent, quiescent BALB/c 3T3 (clone A31) mouse fibroblasts resulted in a block or delay in serum-induced activation of the cell cycle. It was necessary to add IFN within 6 hours after serum stimulation to inhibit nuclear labeling with [3H]thymidine. This is consistent with the time required for platelet-derived growth factor (PDGF) to induce cells to become competent to respond to additional growth factors present in platelet-poor plasma. Simultaneous addition of IFN with PDGF inhibited the PDGF-induced synthesis of a 29-kilodalton and a 35-kilodalton protein that normally occurs within 1 hour after PDGF addition. IFN also suppressed the general increase in protein synthesis that occurs by the fifth hour after PDGF addition. These results show that IFN antagonizes the action of PDGF, thereby interfering with the activation of Go cells for G1 traverse and S-phase entry.     

Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

Lability of host-cell DNA in growing cell cultures due to Mycoplasma

In HeLa-cell cultures chronically infected with Mycoplasma, (PPLO) host-cell DNA is unstable as detected by incorporation of H(3)-or C1 i-thymidine into DNA and subsequent release into the medium as acidsoluble radioactivity. This characteristic can be transmitted to PPLO-free cultures of strain L cells by inoculation with preparations of PPLO from the HeLa cells, although chronically infected cultures of L cells continue to multiply. In addition, virus preparations also may carry PPLO contamination through numerous passages.