The basic flow equations of electrophysiology in the presence of chemical reactions: I. Development of the equations

Starting from the basic flux equation, it is possible to obtain an integral form relating the current componentsI _i at an arbitrary pointr ₂ to the distribution of mobilities and concentrationsc _i, potential forces\(\bar \mu \), and chemical productivityp _i without any restrictive assumptions such as constant mobilities, constant field, steady state, or electrical neutrality. The equation is

$$\begin{gathered} I_i (r_2 ) = G_i (r_2 )\left[ {\Delta \bar \mu _i - \int_{r_1 }^{r_2 } {z_i } FA\left( {p_i - dc_i /dt} \right)\left( {\frac{1}{{G_i (r)}}} \right)dr} \right]; \hfill \\ G_i (r) = 1/\int_{r_1 }^r {\frac{{dr}}{{z_i^2 F^2 c_i u_i }}.} \hfill \\ \end{gathered} $$     

Food mixing by generalist grasshoppers: Plant secondary compounds structure the pattern of feeding

Plant secondary compounds can prevent feeding by phytophagous insects or, if ingested, can be poisonous to them. Less attention has been paid to the additional effects they have on feeding behavior when they are only weakly deterrent or not deterrent at all. Experiments were carried out on the generalist grass-hopperSchistocerca americana. Individuals were presented either with two cakes of high-quality artificial food with a single deterrent compound added or with two cakes, each with a different added deterrent compound. The deterrents consisted of single plant secondary compounds that were either marginally or strongly deterrent. There were profound differences in feeding behavior between those individuals given identical and those given different cake types, including longer feeding bouts on single cakes when a choice of different cake types was available. The behavioral effects demonstrate that the presence of secondary compounds in one food can influence the patterns of feeding on other available foods and suggest that such chemicals could impact foraging activities in a complex manner.     

Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Examination of lymphokines induced in mice following immunization with recombinant simian virus 40 large tumor antigen

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes.     

The paracellular pathway in toad urinary bladder: Permselectivity and kinetics of opening

Summary Determination of serosa-to-mucosa fluxes of Na, K, and Cl yields information about the properties of the shunt pathway in toad urinary bladder. We show that measurement of these fluxes at 30-sec intervals following an abrupt increase in mucosal osmolality yields evidence on the rate of opening of the path and of its permselectivity. The relationship between the fluxes of any pair of these ions indicates that the shunt is paracellular both before and after the increase in conductance effected by hyperosmolality and that the transepithelial PD affects the permselectivity properties (at 0 mV,P _K/P _Na/P _Cl= 10.710.57; at +25 mV,P _K/P _Na/P _Cl=10.710.99). The relationship between any of the fluxes and the total transepithelial conductance is linear and yields an estimate of cellular conductance (the intercept of this regression on the conductance axis) which is in accord with that measured electrically. These studies provide information on tight junction permeability to nonelectrolytes, as well. Finally, they provide new information about the role of the shunt path as a controlling influence on transepithelial sodium transport and raise the possibility that, in both leaky and tight epithelia, differences in transepithelial conductance from tissue to tissue, organ to organ, and species to species may be due, in the absence of edge damage, to changes in conductance of the paracellular pathway.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.