Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates

Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.     

Genetic Analysis of Rapid Tolerance to Ethanol's Incoordinating Effects in Mice: Inbred Strains and Artificial Selection

Ethanol tolerance, a decrease in drug responsiveness with repeated administrations, is an important diagnostic criterion for alcoholism. Rapid tolerance develops within 8-24 hours of an initial ethanol exposure and shares many similarities with chronic tolerance. The genetic contribution to rapid tolerance to ethanol-induced ataxia was estimated using a panel of inbred strains of mice. Strains differed significantly in the degree of rapid tolerance development, which had a broad-sense heritability estimate of 0.11. Artificial selection was carried out to develop lines of mice that would show High (HRT) and Low (LRT) levels of Rapid Tolerance. Starting with HS/Npt mice, derived from a systematic cross of eight inbred strains, a significant response to selection was seen in replicate 1 by the third selection generation. No difference was found in replicate 2. Heritability estimates after the fourth generation were 0.25 for HRT-1 mice and 0.06 for LRT-1 mice. HRT-1 and LRT-1 mice also differed significantly in chronic tolerance development to four doses of ethanol. These studies provide evidence for a genetic contribution to rapid tolerance and support a genetic link between rapid and chronic tolerance to ethanol's ataxic effects.     

Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

Behavioral Profiles of Genetically Selected Aggressive and Nonaggressive Male Wild House Mice in Two Anxiety Tests

Artificially selected aggressive (SAL) and non-aggressive (LAL) male house mice were tested in a hexagonal tunnel maze and light-dark preference (LD) box to determine if the bidirectional selection for aggressive behavior leads to a coselection for different levels of trait anxiety. The tunnel maze consists of an open, brightly lit central arena surrounded by a complex system of interconnecting tunnels. As in the LD box, animals which spend less time and are less active in the brightly illuminated section of the maze are considered to have higher anxiety levels. In the tunnel maze, the LAL mice showed more exploration and spent more time in the central arena than the SAL animals, but only during the final 2 min of the 6-min test. This reduced preference for the central arena was not due to general inactivity or a failure of the SAL to find the central arena and indicates a higher level of state anxiety in the aggressive animals. In contrast, no anxiety-like differences were found in the LD box, either for the percentage of time spent in the light compartment or for the number of crossings. SAL males actually showed higher levels of moving and rearing, and lower levels of freezing, than did LAL males.     

The topography of expanded uncrossed retinal projections following neonatal enucleation of one eye: differing effects in dorsal lateral geniculate nucleus and superior colliculus

The topographic organization of the uncrossed retinal projections to the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) was studied in normal adult hooded rats and in rats subjected to unilateral ocular enucleation on the day of birth. Sections were stained for anterograde degeneration products following discrete retinal lesions at various locations. The projection from the temporal crescent to the dLGN in neonatally enucleated rats had an expanded but topographically normal organization, with the nasotemporal and dorsoventral retinal axes displaying polarities identical to those in normal adults. Neonatal enucleation permits the remaining uncrossed retinogeniculate projection to extend primarily along the "lines of projection" into neuropil normally recipient of binocularly conjugate crossed projections. In the SC, the dorsoventral axis of the temporal crescent showed a normal polarity, but the nasotemporal axis failed to display any topographic organization. Retinal loci in the temporal crescent projected throughout the rostrocaudal extent of the ipsilateral SC. Retinal lesions placed outside the temporal crescent failed to produce any substantial degeneration in ipsilateral dLGN or SC. These topographically distinct effects in dLGN and SC following unilateral eye removal on the day of birth are discussed in the context of differing constraints upon axonal ingrowth and connectivity during early development, which may normally bring about the characteristically distinct features of retinogeniculate and retinocollicular organization.     

小型中医经络检测仪的研制

在传统中医经络理论的基础上，结合先进的电子技术设备，设计了一种小型中医经络检测仪。此检测仪通过采集人体穴位经电流测试(给一定电流刺激)所反射出来的信息，经微控制器处理后，能够精确显示或打印出人体的健康状况。检测仪具有方便、快捷等优势，易推广普及。     

Tumor necrosis factor-{alpha} up-regulates Bcl-2 expression and decreases calcium-dependent apoptosis in human B cell lines

Group I and Epstein–Barr virus-negative Burkitt's lymphomacell lines and the B104 lymphoma cell line which expresses aphenotype of immature B cells undergo apoptosis after cross-linkingof their surface Ig receptors or after exposure to a calciumionophore. We show here that tumor necrosis factor (TNF)- protectsthese B cell lines against Ca²⁺-dependent apoptosis. Protectionwas associated with up-regulatlon of bcl-2 mRNA and proteinexpression. The increase of Bcl-2 expression induced by TNF-was inhibited by chelerythrine, a specific inhibitor of proteinkinase C (PKC), suggesting that Bcl-2 expression was dependenton PKC activation. Furthermore, we show that phorbol estersand cyclosporin A (CsA), which prevent Ca²⁺-dependent apoptosis,up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expressionis controlled by calcineurin since we have shown that FK506but not rapamycin had the same effect on Bcl-2 expression, whereasokadaic acid, an inhibitor of phosphatases 1, 2A and 2C, wasineffective. These data provide direct evidence that TNF- preventsCa²⁺-dependent apoptosis by a Bcl-2-dependent mechanism mediatedby PKC.     

Constitutive and cytokine-regulated expression of presenilin-1 and presenilin-2 genes in human neural cell lines

To investigate the role of pleiotropic neuronal and glial cytokines in the regulation of presenilin (PS) gene expression in human neural cells, both presenilin-1 (PS1) and presenilin-2 (PS2) mRNA levels were analysed by Northern blotting in SK-N-SH neuroblastoma, IMR-32 neuroblastoma, NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N) and U-373MG astrocytoma cells following exposure to proinflammatory cytokines (TNF-alpha, IFN-gamma, or IL-1beta), anti-inflammatory cytokines (IL-10 or TGF-beta1), dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate (PMA). The constitutive expression of PS1 (3.0 kb) and PS2 (2.3 kb) mRNA was identified in all these cell lines, in which PS1 mRNA levels were unaltered following treatment with any cytokines and factors examined. By contrast, PS2 mRNA expression was upregulated substantially in SK-N-SH cells by exposure to TNF-alpha and in U-373MG cells by treatment with IFN-gamma, whereas it was downregulated in both NTera2-N and U-373 MG cells following exposure to IL-1beta or PMA. The levels of PS2 mRNA remained unchanged in IMR-32 cells after these treatments. These results indicate that PS1 and PS2 genes are expressed constitutively in a panel of human neural cell lines where PS2 mRNA expression is affected by a distinct set of cytokines via cell type-specific mechanisms that do not alter PS1 mRNA levels, suggesting the existence of separated regulatory systems controlling the expression of PS1 and PS2 genes in human neural cells.     

In vitro schedule-dependency of myelotoxicity and cytotoxicity of Ecteinascidin 743 (ET-743)

Background: Ecteinascidin (ET-743) is a marine derived compound with an interesting preclinical profile currently completing phase I clinical trials. The present study was undertaken to compare the toxicity of different schedules of ET-743 against human hemopoietic progenitors and tumour cell lines.Materials and methods: Human hemopoietic progenitors and solid tumour cell lines were incubated with ET-743 for one hour, 24 hours and one hour daily for five consecutive days to define by comparison an in vitro therapeutic index. Additional experiments were set up to assess whether incubation for 24 hours or five days could change either the sensitivity of cells or the activity of ET-743.Results: Prolonged or repeated exposures were more toxic than a single one hour exposure (P < 0.001), but due to the higher sensitivity to prolonged exposure of several tumor cell lines, prolonged treatment yielded a more favorable in vitro therapeutic index. After incubation for 24 hours, ET-743 showed a significantly (P < 0.01) lower inhibiting capacity. Incubation before treatment rendered progenitors more resistant, but incubation after treatment increased their sensitivity, so that overall the toxicity of ET-743 on hemopoietic cells appears to be close to AUC dependency.Conclusions: Despite the possible effect of some experimental artefacts, prolonged exposure could represent the best schedule of administration of ET-743.     

Virology studies and cell lines for prostate cancer

Immunological and biochemical probes for viral genomes and products, growth in cell culture, co-culture methods to activate latent genomes, use of activating agents, and electron microscopy have been used in efforts to demonstrate RNA viruses in prostate cancer. Despite findings of C-type particles and p30 antigens, the role of RNA viruses appears to be secondary, with activation of the virogene being a relatively uncommon occurrence. No compelling evidence for Herpes II or cytomegalovirus as etiologic agents has emerged, despite their common presence in the urogenital tract. Though the search for integration of fragments of viral genome into host DNA is still in progress, it appears unlikely that these viruses would account for a significant number of prostate carcinomas. Progress has been achieved in developing simple, reliable, primary culture methods for human prostatic tissue, using explants or dispersed cells. Three cell lines, all from metastatic foci, have been established, are characterized, and are available for distribution. One neonatal cell strain retains many properties of normal prostate.