Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.     

Rat thymus reconstitution after thymocyte destruction by glucocorticoid treatment--from the view of endogenous DNA synthesis and soybean lectin responsiveness in thymocytes

The dynamic process in rat thymocyte restoration after their destruction by glucocorticoid (GC) administration was examined. Thymus weight and thymocyte count became minimal 4-5 days after the administration. Then the thymus took a course of recovery. Endogenous DNA synthesis in thymocytes, reflecting their proliferation within thymus, decreased for 4 days but began to increase 6-8 days after GC treatment. Thymocyte responsiveness to soybean lectin (SBL), a possible stimulator for T-cell-precursors, showed elevation 4-5 days after the treatment. A marked decrease of lymphocytes in the cortex and unclearness of cortico-medullary junction were observed 2-3 days after GC treatment. Clusters of small lymphoid cells, which possibly contained SBL-responding cells, were found in the subcapsular area 4 days after the treatment and successively, large lymphocytes became visible in the same area. Thereafter, small lymphocytes in the cortical mid and deep zones increased, and cortico-medullary junction was restored. These histological features are discussed from the view of correspondence with the dynamic changes of endogenous DNA synthesis and SBL responsiveness in the thymocytes after GC administration.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Rhombomere formation and hind-brain crest cell migration from prorhombomeric origins in mouse embryos

Prior to rhombomere development, structures called prorhombomeres appear in the mammalian hindbrain. This study clarifies the developmental relationship between prorhombomeres and their descendent rhombomeres and hindbrain crest cells in mouse embryos by focal dye injections at various levels of prorhombomere A (proRhA), proRhB, and proRhC, as well as at their boundaries. ProRhA gives rise to two rhombomeres, rhombomeres 1 and 2 (r1 and r2), as well as to crest cells that migrate into the first pharyngeal arch, including the trigeminal ganglion. ProRhB develops into r3 and r4 and produces crest cells populating the second arch and acousticofacial ganglion. The anterior portion of proRhC gives rise to r5 and r6 and to crest cells migrating into the third pharyngeal arch and the IXth ganglion; its posterior portion develops into r7 and releases crest cells into the fourth pharyngeal arch region as well as the Xth ganglion. These results suggest that the boundaries between prorhombomeres serve as lineage restrictions for both hind-brain neuroepithelial cells and for segmental origins of crest cell populations in mouse embryos. The Hox code of the mouse head can be schematized in a much simpler way based on this prorhombomeric organization of the hind-brain, suggesting that prorhombomeres primarily underlie mammalian hind-brain segmentation.     

冬虫夏草模式标本的研究

对冬虫夏草Ｃｏｒｄｙｃｅｐｓｓｉｎｅｎｓｉｓ（Ｂｅｒｋ．）Ｓａｃｃ．的模式标本进行了形态学和有关文献的研究,井与近邻的同种标本进行了比较;该模式现存英国皇家植物园,邱国标本馆,系１８７９年由Ｍ．Ｊ．Ｂｅｒｋｅｌｅｙ研究的标本。     

An increase of acidic isoform of catalase in red blood cells from HIV(+) population

A systemic oxidative stress of HIV (+) individuals has been recognized from a low glutathione level and a high level of inflammatory cytokines such as TNF. Previously, we demonstrated that the catalase enzyme activity in HIV (+) population is significantly altered depending on the cell types; the level was significantly high in red blood cells while the enzymes in white blood cells were remarkably low (Res Commun Subs Abuse 16: 161–176, 1995). In this study, we further characterized the difference in RBC catalase molecules between HIV (+) and control population. We have found that RBC from HIV (+) population, whether they were asymptomatic or symptomatic, contained a significantly elevated catalase protein accompanied by the enzyme activities, and that the majority of the elevated protein were acidic pl of the molecules with an identical subunit mass of approximately 60 KDa. These results suggest that catalase is induced prior to and/or during erythroid differentiation lineage in HIV (+) population as a somatic defense to respond and compensate for a systemic oxidative stress and for an anemic condition. (Mol Cell Biochem 165: 77–81, 1996)     

Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

Antitumor effects of human recombinant interleukin-1α and etoposide against human tumor cells: mechanism for synergismin vitro and activityin vivo

Recombinant human interleukin 1α (rh IL-1α) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell linesin vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1α. The purposes of this study were to test our hypothess that these events were critical to the synergy between rhIL-1α and VP-16, to determine whether rhIL-1α and VP-16 synergize to increase superoxide (SO) anion radical productionin vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combinaton against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1ra) before exposure to rhIL-1α, VP-16 and rhIL-1α plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1α, VP-16, and rhIL-1α+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1α and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1α and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1α and VP-16. However, when A375/S0D15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfecton were exposed to rhIL-1α and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1α and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1α and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combnation of IL-1α and VP-16 might prove useful for the treatment of malignant diseasein vivo, if the increased toxicity can be reduced or managed. The US Government’s right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged.