Nerve conduction theory: Some mathematical consequences of Bernstein's model

The generally accepted permeability theory of nerve conduction is presented in mathematical form. The resulting velocity formula is found to agree well with data on squid giant axon, but predicts velocities considerably too high in the case ofNitella. The dependence of velocity on fiber diameter is discussed for both medullated and non-medullated nerve, it being shown theoretically that velocity is proportional to the square root of diameter for non-medullated and to the diameter for medullated nerve. The equations relating the shape of the action spike to the observed permeability changes are given but are not solved.     

Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks

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Gap junctional intercellular communication in cells isolated from urethane-induced tumors in A/J mice

Studies using normal or neoplastically transformed established mouse lung epithelial cell lines revealed a reduction in gap junctional, intercellular communication (GJIC) with transformation. To determine the stage in tumor development at which GJIC is interrupted, we used the well-established model of lung tumors induced in strain A/J mice by urethane. In this system, tumor development follows a well-characterized pattern; hyperplasias, adenomas, and carcinomas are manifested at approximately 8, 16, and 40 weeks after urethane treatment, respectively. GJIC levels were examined using a novel technique where cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse that opens transient pores on the plasma membrane is applied in the presence of the fluorescent dye, Lucifer yellow, causing dye penetration into cells growing on the conductive part of the slide. Migration of the dye through gap junctions to nonelectroporated cells growing on the nonconductive area is then microscopically observed under fluorescence illumination. Unexpectedly, primary cells cultured from urethane-induced tumors, even late stage carcinomas, possessed extensive GJIC immediately upon isolation. Upon passage for several months however, these cells lost GJIC. These results suggest that the molecular changes that lead to the formation of the tumor in vivo are not sufficient to interrupt gap junctions. Propagation of tumor cells in culture induces additional alterations that can lead to gap junction closure.     

Rapid protein production using CHO stable transfection pools

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post‐transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence‐activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three‐fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin‐opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Substrate specificity of human endonuclease III (hNTH1). Effect of human APE1 on hNTH1 activity

Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg:A) (Marenstein, D. R., Ocampo, M. T. A., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2001) J. Biol. Chem. 276, 21242-21249). When Tg is opposite guanine (Tg:G), the two activities are of the same specific activity as the AP lyase activity of hNTH1 against Tg:A (Ocampo, M. T. A., Chaung, W., Marenstein, D. R., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2002) Mol. Cell. Biol. 22, 6111-6121). We demonstrate here that hNTH1 was inhibited by the product of its DNA N-glycosylase activity directed against Tg:G, the AP:G site. In contrast, hNTH1 was not as inhibited by the AP:A site arising from release of Tg from Tg:A. Addition of human APE1 (AP endonuclease-1) increased dissociation of hNTH1 from the DNA N-glycosylase-generated AP:A site, resulting in abrogation of AP lyase activity and an increase in turnover of the DNA N-glycosylase activity of hNTH1. Addition of APE1 did not abrogate hNTH1 AP lyase activity against Tg:G. The stimulatory protein YB-1 (Marenstein et al.), added to APE1, resulted in an additive increase in both activities of hNTH1 regardless of base pairing. Tg:A is formed by oxidative attack on thymine opposite adenine. Tg:G is formed by oxidative attack on 5-methylcytosine opposite guanine (Zuo, S., Boorstein, R. J., and Teebor, G. W. (1995) Nucleic Acids Res. 23, 3239-3243). It is possible that the in vitro substrate selectivity of mammalian NTH1 and the concomitant selective stimulation of activity by APE1 are indicative of selective repair of oxidative damage in different regions of the genome.     

Treatment with non-hypercalcemic analogs of 1,25-dihydroxyvitamin D3 increases responsiveness to 17beta-estradiol, dihydrotestosterone or raloxifene in primary human osteoblasts

Pretreatment with 1 nM 1,25-dihydroxyvitamin D(3) (1,25), or non-hypercalcemic Vitamin D analogs, upregulated the response of creatine kinase (CK) to 17beta-estradiol (30 nM E(2)), raloxifene (3000 nM RAL) or dihydrotestosterone (300 nM DHT) in primary human bone cells. Previously, we reported that these osteoblast-like cells responded to gonadal steroids in a sex specific manner. Bone cells derived from pre-menopausal women showed greater stimulation of CK specific activity by E(2) than bone cells from post-menopausal women; in male-derived cells no age related difference was found. In this study, we treated cells derived from female or male bones, at different ages, with the side chain modified analogs of Vitamin D: CB 1093 (CB), EB 1089 (EB), MC 1288 (MC) and the demonstrably non-calcemic hybrid analog JK 1624 F2-2 (JKF), by daily addition of 1 nM, for 3 days. On day 4, cells were incubated with sex steroids for 4h and cell extracts were prepared. Pretreatment with JKF or CB significantly upregulated the response to 30 nM E(2) in all female-derived cells and to 300 nM DHT in mature male-derived cells. In cells from older males, only JKF caused augmentation of DHT action. Bone cells from pre- or post-menopausal females responded to 3000 nM RAL by increased CK activity to the same extent as to 30 nM E(2); however, RAL and E(2), when applied together, resulted in mutual annihilation of their agonist activities. Vitamin D analogs prevented the antagonistic effect of RAL in the presence of E(2), possibly due to increased numbers of ERs. Both estrogen receptors, alpha (ERalpha) and beta (ERbeta), were expressed in male- as well as in female-derived cells. However, only in female-derived cells were ERalpha and ERbeta upregulated by pretreatment with Vitamin D analogs. This study raises the possibility of testing combined Vitamin D analog and estrogen replacement treatment for post-menopausal women to prevent osteoporosis.