Multiplexed fluorescence detection in microfabricated devices with both time-resolved and spectral-discrimination capabilities using near-infrared fluorescence

We examined the feasibility of using a two-color time-resolved detection scheme with microdevices for DNA sequencing applications. A home-built dual-color optical-fiber-based time-resolved near-infrared (IR) fluorescence microscope successfully coupled lifetime discrimination with color discrimination, increasing fluorescence multiplexing capabilities. The instrument was constructed by using two pulsed-diode lasers (680/780-nm excitation) and two avalanche photodiodes as the basic building blocks. The data were processed using electronics configured in a time-correlated single-photon counting format. The use of near-IR fluorescence detection greatly simplified the hardware and allowed low detection limits (< 0.1nM). We examined the separation of a single-base tract on a microchip and compared the performance with that of conventional capillary gel electrophoresis. The microchip was fabricated in glass and contained an effective separation length of 7.0 cm. It was found that, without incorporating a solid-phase reversible immobilization cleanup procedure, the calculated lifetime of the dye label on the microchip was longer and the standard deviation was larger than those of the same sample analyzed using capillary electrophoresis. Using cleanup steps, the accuracy and precision of the measurements improved. Lifetimes of four near-IR dyes (AlexaFluor680, IRD700, IRD800, and IRD40) used in this study were determined to be 986 ps (RSD=2.1%), 1551 ps (RSD=1.8%), 520 ps (RSD=3.3%), and 788 ps (RSD=4.9%), respectively, in a microchannel filled with poly(dimethylacrylamide) (POP-6) gel. The lifetimes calculated using maximum likelihood estimators provided favorable precision on the microchip, where small numbers of photocounts were collected. An M13mp18 template was sequenced on the microchip using a two-color two-lifetime format with POP-6 as the sieving polymer. Read lengths of 294 bp with calling accuracies of 90.8 and 83.7% were achieved in each color channel. The relatively low calling accuracy and the short read length resulted primarily from the short separation channel, which yielded low electrophoretic resolution.     

A mutagenesis-enhancing activity of 12-O-tetradecanoylphorbol 13-acetate detected in Chinese hamster ovary cells

Tumour-promoting agents may bring about the completion of multi-step carcinogenesis by acting as enhancers of mutagenesis, recombinogens or clastogens. We report here that the classical mouse skin tumour promoter TPA, although non-mutagenic per se, can enhance the induction of OuaR CHO-K1 cell mutants by MNNG approximately 2-fold. This observation was made at a concentration approaching the compounds aqueous solubility limit which was non-cytotoxic. Mutagenesis enhancement was dependent on TPA being present throughout mutation expression and mutant selection. It was not accompanied by any modification of cell sensitivity to mutagen killing. In the same treatment protocol TPA did not enhance either EMS- or UV-induced mutagenesis. TPA exposure over 2 rounds of cell replication failed to produce an increase in the frequency of SCE in control or mutagen-treated CHO-K1 cultures. Likewise TPA exposure over 1 round of cell replication failed to produce an increase in the frequency of chromosomal aberrations. Apparently TPA is not a recombinogen or clastogen but in the right exposure regime is capable of acting to enhance mutagenesis by certain genotoxic agents, an action which may contribute to tumour promotion.     

Intersubunit location of the active site of ribulose-bisphosphate carboxylase/oxygenase as determined by in vivo hybridization of site-directed mutants

Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50.5-kDa subunits with two substrate binding sites per molecule of dimer. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interactions, we have used a novel in vivo approach for producing heterodimers from catalytically inactive, site-directed mutants of the carboxylase. When the alleles encoding these mutant proteins are placed separately into compatible plasmids and coexpressed in the same Escherichia coli host, activity is observed at about 20% of the wild-type level. Analysis of the carboxylase purified from these cells reveals the presence of heterodimers of the two mutant proteins. This interallelic complementation demonstrates that domains from each of the subunits interact to form a shared active site.     

A specific histochemical method for phospholipids and sulpholipids

Synopsis A new histochemical method is described for the polychromatic staining and identification of phospholipids and sulpholipids in tissue sections. It is an adaptation of a chromatographic method described previously (Kennedy & Collier, 1962). Sections are stained in a mixture of Diamond Sky Blue B, Diamond Cyanine R and formalin, followed by differentiation in 2-morpholinoethanol and bromine water, counterstaining in Cresyl Fast Violet and decolorization in lactic acid.The interpretation of the final colours in the test tissues was checked by extraction and chromatography of their lipid content.The staining of diverse animal and plant tissues is described and illustrated in colour.     

Examination of the intersubunit interaction between glutamate-48 and lysine-168 of ribulose-bisphosphate carboxylase/oxygenase by site-directed mutagenesis

The active site of ribulose-bisphosphate carboxylase/oxygenase is constituted from domains of adjacent subunits and includes an intersubunit electrostatic interaction between Lys 168 and Glu48, which has been recently identified by x-ray crystallography (Andersson, I., Knight, S., Schneider, G., Lindqvist, Y., Lundqvist, T., Br?ndén, C.-I., and Lorimer, G.H. (1989) Nature 337, 229-234; Lundqvist, T., and Schneider, G. (1989) J. Biol. Chem. 264, 7078-7083). To examine the structural and functional requirements for this interaction, we have used site-directed mutagenesis to replace Lys168 of the homodimeric enzyme from Rhodospirillum rubrum with arginine, glutamine, or glutamic acid. All three substitutions result in mutant enzymes with less than or equal to 0.1% of wild-type activity. The nonconservative substitution of Lys168 with a glutamyl residue precludes the formation of a stable dimer, explaining the consequential abolition of enzymic activity. Both the Arg168 and Gln168 mutant proteins are isolated as stable dimers, even though the latter obviously lacks an electrostatic interaction present in the wild-type enzyme. Despite the absence of overall carboxylase activity, these two mutant proteins serve as catalysts for the enolization of ribulose bisphosphate, as measured by exchange of the C3 proton with solvent. These observations, as well as ligand-binding properties of the mutant proteins, are consistent with Lys168 facilitating a catalytic step subsequent to enolization.     

Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates

Two Zn-Pc-peptide conjugates bearing either a short linker or a long PEG-linker between the macrocycle and a bifunctional peptide containing the nucleoplasmin and HIV-1 Tat 48-60 sequences have been synthesized in order to increase the Pc cell-targeting ability and to evaluate the effect of the linker. The presence of the peptide chain increased the water solubility of the Pc macrocycle and, consequently, its fluorescence in aqueous solutions. The highest fluorescence quantum yields were observed at low pH (5.0) for both conjugates and were always higher for the conjugate bearing the short linker. Both conjugates were found to have low dark cytotoxicity toward human HEp2 cells (IC50 > 77 microM) but were highly phototoxic (IC50 < 2 microM at 1 J cm-2). The conjugate bearing the long PEG-linker accumulated the most within cells (26 times more than the unconjugated Zn-Pc), followed by the short linker conjugate (17 times more than the unconjugated Zn-Pc). Both conjugates were found to localized preferentially within the cell lysosomes.     

Phenotypic variation in exopolysaccharide production in the marine,aerobic nitrogen-fixing unicellular cyanobacterium Cyanothece sp.

The aerobic nitrogen-fixing cyanobacterium, Cyanothece sp. BH68K produces non-mucoid variants defective in exopolysaccharide (EPS) production at a high frequency. The EPS-producing wild-type colonies (EPS⁺) have a characteristic smooth and shiny appearance which allows them to be easily distinguished from the EPS^- variants. When grown on agar plates lacking a source of combined nitrogen, the EPS^- variants exhibited a yellow phenotype typical of nitrogen starvation. These EPS^- variants showed varying degrees of reversion back to the EPS⁺ phenotype. After reversion, they exhibited normal diazotrophic growth on agar plates. Alcian blue and ruthenium red staining indicated that the EPS is an acidic polysaccharide, which is present as a loose network around the cell, and which can be completely removed by low speed centrifugation. The accumulation of EPS takes place mainly during the stationary phase. All EPS^- variants failed to produce any EPS. Analysis of growth of wild-type and EPS^- variants revealed that EPS production is beneficial for diazotrophic growth on solid medium, but not in liquid medium. In addition, EPS phenotypic alteration may have some advantage in the dispersal of cells from one place to another in the natural environment.K.J. Reddy. J. Tang and R.L. Bradley are, and B.W. Soper was, with the Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902; B.W. Soper is now with the Jackson Laboratory, Box 302, 600 Main St., Bar Harbor, ME 04609.     

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4⁺ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.     

Ion solvation and water structure in potassium halide aqueous solutions

The structure of water and the nature of ionic hydration is explored in aqueous solutions of potassium fluoride, chloride, bromide and iodide over a range of concentrations up to 4.8 ion pairs per 100 water molecules, using the combined techniques of neutron diffraction with hydrogen isotope substitution. The diffraction data are interpreted using the method of empirical potential structure refinement, which attempts to build a three-dimensional model of the scattering system consistent with the diffraction data. The water structure is strongly perturbed in the first hydration shells of both anion and cation, but is found to be only mildly perturbed outside of this region, with the largest effects occurring with the smallest anion and highest concentrations. For the potassium ion there are strong orientational correlations in the first hydration shell, with the water molecules lying with their dipole moments pointing almost directly away from the cation on average, but with an angular spread of approximately +/-60 degrees which is mildly dependent on the anion type present. For all the anions the water molecules in the first shell are strongly oriented with one O-H vector pointing directly towards the anion on average, with an angular spread of approximately +/-10 degrees for F(-), increasing to approximately +/-22 degrees for I(-). For both anions and cations the second hydration shell is much more disordered than the first, but there is a weak pattern of orientational correlation which becomes more pronounced with the larger anions. There is some evidence that the fluoride ion structures water significantly in its first hydration shell, but not beyond. The findings throw further light on recent findings that the orientational relaxation time for water outside the first shell of dissolved ions is the same as in the bulk liquid.