Enhanced detection of glycoproteins in polyacrylamide gels

A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Mathematical models for mixing in deep-jet bioreactors: Calculation of parameters

The macroscopic mathematical model based on compartments with ideal mixing zones and tanks-in series was evaluated. Based on the experimental data obtained in a 300 dm³ pilot reactor and the dependence of mixing time on the volume of liquid phase, we have found mathematical relations between the ratio of vessel diameter to liquid level, adjustable parameters of model and the mixing time.List of Symbols V dm³ total volume of bioreactor - V _g dm³ total volume of liquid - V ₁ dm³ volume of ideally mixed zone in the vessel - V ₂ dm³ volume of macromixer in inner circulation flows - V ₃ dm³ volume of liquid phase in the pump - V ₄ dm³ volume of liquid phase in the pipe between the vessel and the pump - V ₅ dm³ volume of liquid phase in the pipe between the pump and air input system included falling jet - V _LT dm³ volume of liquid in the tank - V _LC dm³ volume of liquid in the circulation system - F _E dm³/s inner volumetric circulation flow rate across the macromixers - F _cir dm³/s external volumetric circulation flow rate, pumping capacity - t _A s time interval of the pulse application - t _AA s time point of the pulse application related to the free choosen starting point of the experiment - t _m s mixing time - t _c s circulation time - t _end s end time of simulation - C _*,* kg/m³ concentration of tracer in the indicated compartment - C ₀ kg/m³ concentration of the tracer before the injection - C _t kg/m³ concentration of the tracer at the indicated time - C kg/m³ theoretical concentration of the full mixed tracer - C _sim kg/m³ calculated concentration of tracer during numerical integration method - i index of an arbitrary tank - D _T m diameter of bioreactor - D 1/s dilution rate - H _L m level of liquid in the unaerated vessel - vector of inhomogenities     

HIV promoter activity in primary antigen-specific human T lymphocytes

Human retroviruses, such as the HIV, infects human T cells, and efficient HIV replication occurs primarily in activated T cells rather than resting cells. Increased HIV production is likely caused by the activation of the retroviral promoter, and the HIV promoter may be regulated by intracellular signals induced during immune stimulation. To examine the regulation of retroviral promoter activity in normal, Ag-specific primary T lymphoblasts, a heterogeneous population of primary human T cells was transfected with either the HIV promoter or a promoter from a different retrovirus, Rous sarcoma virus (RSV) by protoplast fusion technique. Transfected T cells responded normally to Ag or mitogen stimulation, and activation of these T cells increased both the HIV and RSV promoter activity. Promoter activity was assessed by using transient expression assays after the T cells were restimulated with Ag, mitogen, or IL-2. In situ hybridization of transfected human T cells showed that 68 to 95% of activated lymphocytes expressed CAT mRNA directed by HIV or RSV. Thus, protoplast transfection of primary T cells was efficient in that the majority of cells expressed CAT message. By deletion of different regions of the HIV promoter, the enhancer region was identified as necessary for effective HIV promoter activity. In addition these deletion studies identified a region that negatively affects HIV promoter activity in primary T cells. Cotransfection of the HIV promoter with the HIV transactivator protein, tat, increases HIV promoter activity in both resting and activated primary human T cells only when the tat target sequences were present.     

Mutagenicity of vinyl chloride in man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies

The mutagenic effects of vinyl chloride monomer in man were studied in the lymphocyte culture with 3 methods: the chromosome aberration assay, the micronucleus assay and the sister-chromatid exchange method. Compared with control, values obtained by these tests are increased in workers occupationally exposed to vinyl chloride. In relation to non-smokers, smokers exposed to vinyl chloride show significant increases in sister-chromatid exchange frequencies. The problem of correlating the results of the chromosome aberration assay with micronucleus and sister-chromatid exchange frequencies is discussed.     

Thehigh growth(hg) Locus Maps to a Deletion in Mouse Chromosome 10

Thehigh growth(hg) locus in mice produces a 30–50% increase in weight gain of homozygous individuals. Here we report that the microsatellite markerD10Mit69is deleted in high growth mice. The deletion ofD10Mit69was uncovered in a screen of the high growth mouse and its progenitor strains for available markers in thehgregion. We demonstrate thathgandD10Mit69cosegregate in a cross of congenic strains C57BL/6J-hghg× C57BL/6J. These results suggest that deletion of a region aroundD10Mit69is responsible for the high growth effect. MarkerD10Mit69will be utilized as an entry point to physical cloning of thehg-containing segment. A dense map of markers aroundhgconstructed here should allow identification of markers in homologous regions in domestic animals and humans, which may be utilized to assess the role of thehglocus in these other species.