Failure of (SJL/J x PL/J)F1 hybrid mice to develop immunologic tolerance to V beta 17a+ T cells results in F1 anti-SJL mixed lymphocyte reaction.

It has been reported previously that spleen cells from (SJL x PL) F1 hybrid mice are not tolerant to SJL parental cells as assessed by a one-way MLR. The possibility that the F1 anti-SJL reaction was due to the effect of lymphokines produced by the irradiated SJL T cells in response to I-Eu expressed on the F1 hybrid cells was eliminated since inclusion of anti-I-E mAb was without effect. Cell separations showed the responder cells to be plastic and nylon wool nonadherent Ia- T cells. Separation of the SJL spleen cells showed that the stimulator cells were nonadherent, passed through a nylon wool column, and were Ia-. the F1-anti-SJL MLR was blocked 70 to 90% by inclusion of mAb KJ23a in the culture medium that indicated that the stimulatory cell population was V beta 17a+ T cells. This was confirmed by the use of V beta 17a+ and V beta 17a-T cell clones as stimulators. To determine whether failure to develop tolerance to this T cell subset in F1 hybrid mice might be responsible for the F1-anti-parent MLR, (SJL x PL)F1 mice were treated at birth and 48 h thereafter with anti-I-E mAb for 7 wk. Spleen cells from antibody-treated F1 mice were nonreactive with irradiated SJL parental cells in contrast to spleen cells from control mice which indicated that V beta 17a+ T cells were eliminated by negative selection before the development of tolerance.     

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

Summary Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.     

Time-resolved infrared spectroscopy of electron transfer in bacterial photosynthetic reaction centers: dynamics of binding and interaction upon QA and QB reduction.

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [M?ntele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5 microseconds, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor QA and its environment upon formation of QA-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [M?ntele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; M?ntele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., M?ntele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., M?ntele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120 microseconds were observed and attributed to QA----QB electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes of QA and QB and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C = O and C = C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-microseconds signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered drug translocation mediated by the MDR protein: Direct,indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ltpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential () and/or intracellular pH (pH_i) and therebyindirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pH_i, or -dependent manner. A third model proposes that the protein alternates between drug pump and Cl^– channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pH_i, , and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.     

Cell-Cell Recognition during Fertilization in a Red Alga, Antithamnion sparsum (Ceramiaceae, Rhodophyta)

A gamete recognition mechanism in Antithamnion sparsum Tokidais proposed based on experiments using various lectins and carbohydrates.Spermatial binding to trichogynes is inhibited by pre-incubationof spermatia with concanavalin A (ConA) and/or L-fucose, whiletrichogyne receptors are blocked by the complementary carbohydrate-methyl D-mannose and/or the lectin Ulex europaeus agglutinin(UeA1). Binding inhibition (40–50%) was observed with10–50 mM carbohydrates and 25–50 µg ml^-1 lectins.The inhibitory effects of ConA and UeA1 is partially reversed(to 80–90% of controls) by addition of -methyl D-mannoseand L-fucose, respectively. Lectin binding to spermatial surfaceswas visualized by Fluorescein isothiocyanate (FITC) conjugatedConA, whereas carbohydrate receptors along the trichogyne andspermatium were localized with -mannosylated-FITC-albumin andL-fucosylated-FITC-albumin, respectively. These results suggestthat gamete recognition in Antithamnion sparsum is mediatedby a double-docking recognition system consisting of spermatiapossessing surface L-fucose receptors and -methyl D-mannosemoiety, and trichogynes possessing the complementary receptors. (Received December 5, 1995; Accepted April 22, 1996)     

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 11 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC₁ generation using both single-point ANOVA and interval mapping. A minimum number of 2–4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC₁ population.     

Identification of Selected Gamma-Ray Induced Deficiencies in Zebrafish Using Multiplex Polymerase Chain Reaction

The ease with which mutations can be generated in zebrafish makes this vertebrate an important resource for developmental genetics and genome studies. We have developed a PCR-based screening method that allows the efficient identification of gamma-ray induced deficiencies targeted to selected sequences. We describe three mutants characteristic of our findings and show that these mutations include deletions and translocations that can affect as much as 1% of the genome. These deficiencies provide a basis for analyzing the functions of cloned zebrafish genes using noncomplementation screens for point mutations induced by high-efficiency chemical mutagenesis.     

Granula motion and membrane spreading during activation of human platelets imaged by atomic force microscopy.

The redistribution of platelet constituents during activation is essential for their physiological function of maintaining hemostasis. We report here about real time investigations of the activation of native human platelets under physiological conditions from the initial formation of filopodia to the fully spread form by atomic force microscopy. We followed the trafficking of granules and their interaction with the plasma membrane within single cells. Our results show movement of certain granula towards the lamellipodia. Analysis of this rearrangement and the subsequent enlargement of the platelet surface reveals details of the membrane spreading process. Images of living cells are presented that show the distribution of cytoskeletal components and membrane-bound filaments at a resolution of better than 50 nm. The local minimum forces between the tip and the platelets were estimated to be smaller than 60 pN. A model for the elastic contributions of the glycocalix to the tip/membrane interaction was developed using the theory of grafted polymers.