Antigen-induced murine B cell lymphomas. I. Induction and characterization of CH1 and CH2.

Two unusual murine lymphomas, designated CH1 and CH2, were produced in the newly developed double congenic strain of mice, B10 H-2a H-4b p/Wts. Both tumors lack the T cell-specific antigen (thy-1), but express cell surface immunoglobulin and the H-2K, H-2D, and Ia specificities determined by the H-2a haplotype. Further studies have demonstrated that these tumors represent "early" B cells in that they express surface IgM (mu heavy and lambda light chains), but do not bear surface delta, gamma, or alpha heavy chains. CH1 and CH2 lack surface C3 receptors and results from assays for Fc receptors have proven variable. A competition radioimmunoassay directed against the gp71 group-specific antigen of Friend leukemia virus has shown that there is a murine leukemia virus associated with these tumors, however, we have been unable to establish a causal relationship between the virus and this malignancy. A comparison of the surface characteristics of these tumors with other mammalian B cell lymphomas is presented.     

Biogenic amines in the two-spotted spider mite

Whole body extracts of the two-spotted spider mite (Tetranychus urticae Koch) were analyzed using a 16-channel electrochemical array high performance liquid chromatography (HPLC)-based detection system that allows the simultaneous isolation and identification of a variety of biogenic amines. The spider mite extracts were found to contain the biogenic amines octopamine, dopamine, and 5-hydroxytryptamine (5-HT), as well as several precursors and metabolites including tyrosine, tyramine, tryptophan, and N-acetyl octopamine. Differences in the levels of biogenic amines were observed between eggs and the adult stages and between males and females. This is the first direct determination of biogenic amines in the Tetranychidae and the first demonstration of 5-HT in any mite species. © 1994 Wiley-Liss, Inc.     

Calcium clearance mechanisms of mouse sperm

The spermatozoon is specialized for a single vital role in fertilization. Past studies show that Ca2+ signals produced by the opening of plasma membrane entry channels initiate several events required for the sperm to reach and enter the egg but reveal little about how resting [Ca2+]i is maintained or restored after elevation. We examined these homeostatic mechanisms by monitoring the kinetics of recovery from depolarizing stimuli under conditions intended to inhibit candidate mechanisms for sequestration or extrusion of Ca2+ from the cytosol. We found that the Ca2+-ATPase pump of the plasma membrane performs the major task of Ca2+ clearance. It is essential in the final stages of recovery to achieve a low resting [Ca2+]i. With immunomethods we found a approximately 130-kD plasma membrane Ca2+-ATPase protein on Western blots of whole sperm extracts and showed immunolocalization to the proximal principal piece of the flagellum. The plasma membrane Na+-Ca2+ exchanger also exports Ca2+ when [Ca2+]i is elevated. Simultaneous inhibition of both mechanisms of extrusion revealed an additional contribution to clearance from a CCCP-sensitive component, presumably sequestration by the mitochondria. Involvement of SERCA pumps was not clearly detected. Many aspects of the kinetics of Ca2+ clearance observed in the presence and absence of inhibitors were reproduced in a mathematical model based on known and assumed kinetic parameters. The model predicts that when cytosolic [Ca2+] is at 1 microM, the rates of removal by the Ca2+-ATPase, Na+-Ca2+-exchanger, mitochondrial uniporter, and SERCA pump are approximately 1.0, 0.35, 0.33, and 0 micromole l(-1) s(-1), rates substantially slower than those reported for other cells studied by similar methods. According to the model, the Na+-Ca2+ exchanger is poised so that it may run in reverse at resting [Ca2+]i levels. We conclude that the essential functions of sperm do not require the ability to recover rapidly from globally elevated cytosolic [Ca2+].     

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.     

Ovine interleukin 12 has biological activity on ovine and human activated peripheral blood mononuclear cells

Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits that form the biologically active p70 molecule, and is a potent inducer of the pro-inflammatory cytokine IFN-gamma. In this study the coding sequence for ovine interleukin 12 p35 and p40 subunits was derived by RT-PCR cloning. Ovine p35 and p40 cDNA sequences show a high level of similarity at the nucleic acid and protein levels when compared to corresponding bovine and human sequences. In particular, cysteine residues and N-linked glycosylation sites are conserved between species. Secretion of the IL-12 heterodimer from CHO cells co-transfected with ovine p35 and p40 cDNA was shown by immunoprecipitation of a 60 and 66 kDa protein from transfectant supernatant. In addition, the supernatant from co-transfected cells augmented the proliferation of Con A-activated ovine peripheral blood mononuclear cells (PBMNC). Cross-species activity was shown by the enhancement of proliferation of human phytohaemagglutinin (PHA)-activated PBMNC. Supernatants from co-transfectants of hu p35/ov p40 and ov p35/hu p40, to generate chimeric heterodimers, also demonstrated stimulatory activity. Human and chimeric IL-12-induced proliferation of activated PBMNC was inhibited using an anti-human IL-12 polyclonal antibody, however this antibody showed minimal inhibition of ovine IL-12. This study suggests that ovine IL-12 has biological properties similar to its human counterpart.     

Network theory and metapopulation persistence: incorporating node self‐connections

Network analysis is gaining increasing importance in conservation planning. However, which network metrics are the best predictors of metapopulation persistence is still unresolved. Here, we identify a critical limitation of graph theory‐derived network metrics that have been proposed for this purpose: their omission of node self‐connections. We resolve this by presenting modifications of existing network metrics, and developing entirely new metrics, that account for node self‐connections. Then, we illustrate the performance of these new and modified metrics with an age‐structured metapopulation model for a real‐world marine reserve network case study, and we evaluate the robustness of our findings by systematically varying particular features of that network. Our new and modified metrics predict metapopulation persistence much better than existing metrics do, even when self‐connections are weak. Existing metrics become good predictors of persistence only when self‐connections are entirely absent, an unrealistic scenario in the overwhelming majority of metapopulation applications. Our study provides a set of novel tools that can substantially enhance the extent to which network metrics can be employed to understand, and manage for, metapopulation persistence.     

In vitro maturation of bitch oocytes from advanced preantral follicles in synthetic oviduct fluid medium: serum is not essential

The ability of oocytes from preantral follicles to mature in vitro was assessed using a synthetic oviduct fluid (SOF) medium. Advanced preantral follicles (approximately 210 microm diameter) were isolated from the ovaries of domestic bitches and assigned to one of four treatment groups: (1) SOF (n = 230); (2) SOF + 3 mg/ml bovine serum albumin (+BSA, n = 220); (3) SOF + 20% fetal bovine serum (+FBS, n = 227); or (4) SOF + 3 mg/ml BSA + 20% FBS (+BSA+FBS, n = 232), then cultured for up to 72 h. A group of control follicles was not cultured (n = 103). The percentages of oocytes reaching metaphase I to metaphase II stages (MI to MII) did not differ between treatments at each culture period. Within treatments, the percentages of oocytes at MI to MII stages did not differ with duration of culture. However, when compared to the control group (0.97%) the percentages of oocytes at MI to MII increased (P < 0.05) in the SOF group after 48 h (10.0%) and 72 h (12.2%) of culture. In the +BSA (10.1%) and +FBS (9.7%) groups, the percentages of oocytes at MI to MII increased (P < 0.05) above control values only after 72 h of culture. The percentage of oocytes at MI to MII did not significantly increase in the +BSA+FBS group (3.9,6.6 and 7.6% at 24,48 and 72 h of culture, respectively) compared to the control group. These results indicate that under the described conditions supplementation of culture medium with BSA or FBS is not essential, and the simple medium SOF can support nuclear maturation of a small proportion of bitch oocytes in vitro.     

Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I⁻, Fe(CN)⁴⁻₆, W(CN)⁴⁻₈) are poor reducing agents for Z⁺· whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors.

A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z⁺·. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner surface.