Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.     

Determination of the source of immunoreactive relaxin in the cat

We have recently reported the secretory profile of relaxin throughout gestation in the cat. Because the appearance of relaxin begins at about Day 20 (Day O = ovulation) and because implantation begins shortly before this at Days 13-14, we hypothesized that relaxin was of feto-placental origin. To test this hypothesis, we used 4 experimental groups: 1) Control (laparotomy-only at Day 23 or 42, n = 4); 2) Early Ovariectomy (Ovx, bilateral ovariectomy between Days 23 and 26, n = 4); 3) Late Ovx (bilateral ovariectomy between Days 40 and 44, n = 4); 4) Tissue Removal (removal of feto-placental units, uterus, and one ovary on Days 16, 21, 28 and 35, n = 1 per day). Pregnancies were maintained in both Ovx groups by progesterone administration. Relaxin secretory patterns in Ovx groups were similar to the Control data. Relaxin was detectable in plasma beginning at about Day 20, with maximum concentrations reached by Day 30. Relaxin concentrations were highest (immunoactivity per mg tissue) in homogenates of placental tissues as compared to luteal, fetal, or uterine tissues. Altogether, these data indicate that the feto-placental unit is the source of relaxin in the cat.     

Properties of the binding of follicle-stimulating hormone to the fetal rat testis

Basic properties of the binding of [131I]-labeled rat FSH ([131I]rFSH) to the testicular homogenates of fetal rats were analyzed by micro-radioreceptor assay. Specific binding of FSH was detectable in the testicular preparations from 15.5-day fetuses, but it was very low. After 17.5 days of gestation, specific FSH binding was apparent in the testis and was effectively displaced by rat FSH but not by rat LH. The Scatchard plot analyses of the binding of FSH to the testicular preparations of fetuses showed straight lines similar to those of postnatal rats, suggesting the presence of a single class of binding sites. The mean dissociation constant (Kd) for FSH receptors in 17.5-day fetuses was 0.413 +/- 0.043 nM, which was significantly greater than that in postnatal rats at 50 days of age. However, the Kd in 19.5-day fetuses was not significantly different from those in 17.5-day fetuses and postnatal rats due to its considerable variance. The capacity of FSH binding sites was 0.51 +/- 0.01 fmol/testis in 17.5-day fetuses, which was significantly less than those of 19.5-day fetuses and postnatal rats.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

A Ca2+- and Voltage-Dependent Cl- -Sensitive Anion Channel in the Chara Plasmalemma: A Patch-Clamp Study

The inside-out patch-clamp technique was applied to the plasmolyzedplasmalemma of inter-nodes of Chara corallina without enzymatictreatment. We found two different types of channel activitythat were CP^–-sensitive. Both types of channel were Ca²⁺-dependent.However, the one that exhibited greater dependence on Ca²⁺ ionswas the focus of our studies, and we named it the Ca²⁺-dependentCP^–-sensitive anion channel. When the concentration ofCa²⁺ ions on the cyto-plasmic side was 1.0 µM, the Ca²⁺-dependentCP^–-sensitive channel opened most frequently between approximately–80 and –100 mV. At 10 µM Ca²⁺, it openedless frequently, and at 0.1 µM Ca²⁺ it scarcely openedat all. These observations indicate that the anion channel ofinterest is voltage-dependent over a restricted range of concentrationsof Ca²⁺ ions. The dependence on Ca²⁺ and voltage of the channelcan explain the behavior of the excitable Ca²⁺-activated Cl^–channel in the Chara plasmalemma. The channel activity was blockedby several antagonists of calmodulin. ⁴ Present Address: Department of Biology, College of GeneralEducation, Osaka University, Toyonaka, 560 Osaka, Japan (Received October 8, 1990; Accepted April 4, 1991)     

Detection of 1-nitropyrene in yakitori (grilled chicken)

Pieces of raw chicken with or without a marinating sauce were grilled over a city gas flame, extracted with benzene-ethanol (4:1) by ultrasonication and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor 1254-treated Sprague-Dawley rat liver (S9 mix). The basic fraction of yakitori without the sauce was more mutagenic than the other fractions for S. typhimurium strain TA98 in the presence of S9 mix. This is probably due to the presence of amino acid or protein pyrolysates. However, when the chicken was grilled with the sauce, the basic fraction showed lower mutagenicity for strain TA98 in the presence of S9 mix than did the same fraction without the sauce. The neutral fraction of yakitori with sauce showed high mutagenicity for strain TA98 in the absence of S9 mix, but low mutagenicity for strains TA98NR and TA98/1,8-DNP6, suggesting that this fraction might contain nitropyrenes (NPs). The neutral fraction of yakitori was analyzed by high-performance liquid chromatography (HPLC). The neutral fraction of the chicken grilled with the sauce for 3, 5 and 7 min contained 3.8, 19 and 43 ng, respectively, of 1-NP per gram of yakitori accounting for 3.0, 2.7 and 1.3%, respectively, of the total mutagenicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tripolyphosphate is an alternative phosphodonor of the selective protein phosphorylation of liver microsomal membrane

Two proteins (Mr = 145,000 and Mr = 130,000) of rat liver microsomal membrane are selectively phosphorylated in a characteristic biphasic time course by incubating the membrane with [gamma-32P]ATP in the absence of exogenously added Mg2+ (Lam, K. S., and Kasper, C. B. (1980) J. Biol. Chem. 255, 259-266). This endogenous phosphorylation system was solubilized with Triton X-100 and fractionated by chromatography with DEAE-cellulose and Sepharose 4B. The resulting preparation lacked both ATPase and inorganic pyrophosphatase activity, but retained its original character: the first phase occurred in the presence of ATP but the second phase was initiated after its depletion, implying the presence of a phosphodonor other than ATP. The putative phosphoryl donors were demonstrated to be ATP in the first phase and in the second phase tripolyphosphate, which is present in [gamma-32P]ATP preparations as a radioactive impurity. The latter conclusion was corroborated by results showing that tripolyphosphate purified from a commercial [gamma-32P]ATP and chemically synthesized [32P] tripolyphosphate were both capable of phosphorylating the two proteins and that the unlabeled tripolyphosphate competed effectively against the phosphodonor. A rapid dephosphorylation was observed in both phases upon removal of substrates during the reaction, indicating that there is a continuous turnover of the phosphoryl groups being transferred to the proteins. The second phase of phosphorylation maintained by the tripolyphosphate was shown to be reversibly inhibited by micromolar levels of ATP, ADP, and nonhydrolyzable analogues of these compounds. The implications of this unique phosphorylation system are discussed.     

Seasonal changes of glycogen/trehalose contents,supercooling points and survival rate in mature larvae of the overwintering soybean pod borer Leguminivora glycinivorella

The mature larvae of the soybean pod borer Leguminivora glycinivorella, spend over 9 months (October-next August) in the inactive state until pupation down to 3 cm below the surface in soil. Trehalose content of inactive larvae increases in early winter, attaining a maximum (ca 30 mg/g), and decreases in spring, with a concomitant decrease and increase of glycogen. The median supercooling points seasonally change from ?19.8°C (October) to ?25.0°C (February), and to ?17.0°C (June). The lower supercooling points in winter are in part due to the absence of unusually high values (> ?18°C). The increase in trehalose does not seem to be effective in depressing the supercooling points. The larvae are freeze-intolerant, but ambient temperatures in outdoor conditions are always above the supercooling points. The survival rates are very high throughout the inactive period.     

Ultrastructural detection of single-stranded DNA in rat liver nucleus by the electronmicroscopic immuno-peroxidase method

Single-stranded DNA (ssDNA) in nucleus of rat liver cell was detected electron-microscopically by an indirect immuno-peroxidase technique using anti-thymine antibody. Anti-thymine was made by immunizing a rabbit with thymine-bovine serum albumin (BSA) conjugate. Anti-thymine was prepared from anti-thymine-BSA serum by adsorption with insolubilized BSA. Immunochemical analysis of anti-thymine was performed by gel diffusion and confirmed the reactivity of antithymine with ssDNA. The enzymatic reaction products showing single-stranded DMA were detected in the chromatin areas of nuclei of both normal and regenerating rat liver cells. The specificity of the reaction product was checked by control experiments.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: II. Inductive property of membrane and effects of pH o and impermeable monovalent cations on breakdown phenomenon

Summary Changes in the chord conductanceG and the membrane electromotive forceE _m in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–V _m relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pH _o in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pH _o 7.In some cellsE _m shifted to a positive level as large as +50+70 mV during the breakdown phenomenon. Such a large positive shift ofE _m is caused mainly by the increase in conductance of Cl^– and partly Ca²⁺ and K⁺.