Epifluorescence and video analysis of vacuole motility and development in stomatal cells of allium.

The vacuole in stomatal cells of Allium undergoes major changes in shape during differentiation, switching from a globular form in new guard mother cells to a network of interconnected tubules and chambers, and then back to a globular form as guard cells mature. In addition, vacuolar network elements exhibit characteristic movements and rearrangements.

SLC6A4 variation and citalopram response.

The influence of genetic variations in SLC6A4 (serotonin transporter gene) on citalopram treatment of depression using the Sequenced Treatment to Relieve Depression (STAR*D) sample was assessed. Of primary interest were three previously studied polymorphisms: 1) the VNTR variation of the second intron, 2) the indel promoter polymorphism (5HTTLPR or SERT), and 3) a single nucleotide polymorphism (SNP) rs25531. Additionally, SLC6A4 was resequenced to identify new SNPs for exploratory analyses. DNA from 1914 subjects in the STAR*D study were genotyped for the intron 2 VNTR region, the indel promoter polymorphism, and rs25531. Associations of these variants with remission of depressive symptoms were evaluated following citalopram treatment. In white non-Hispanic subjects, variations in the intron 2 VNTR (point-wise P = 0.041) and the indel promoter polymorphism (point-wise P = 0.039) were associated with remission following treatment with citalopram. The haplotype composed of the three candidate loci was also associated with remission, with a global p-value of 0.040 and a maximum statistic simulation p-value of 0.0031 for the S-a-12 haplotype, under a dominant model. One SNP identified through re-sequencing the SLC6A4 gene, Intron7-83-TC, showed point-wise evidence of association, which did not remain significant after correction for the number of SNPs evaluated in this exploratory analysis. No associations between these SLC6A4 variations and remission were found in the white Hispanic or black subjects. These findings suggest that multiple variations in the SLC6A4 gene are associated with remission in white non-Hispanic depressed adults treated with citalopram. The mechanism of action of these variants remains to be determined.

Translation of pharmacogenetics into clinically relevant testing modalities.

Pharmacogenetics (PGx) relies on the genetic makeup of an individual to predict drug response and efficacy, as well as potential adverse drug events. Significant advances in PGx research have been made since inherited differences in response to such drugs as isoniazid and succinylcholine were explored in the 1950s, and the clinical utility and application of PGx are especially apparent in some subspecialty areas of chemotherapeutic, psychotropic drug, and anticoagulant therapies.

Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase.

Single crystals of the non-fluorescent flavoprotein (NFP) purified from Photobacterium leiognathi strain S1 have been grown from ammonium sulphate solutions using the hanging drop vapour diffusion technique. The crystals grow as thin (0.06 mm) plates and belong to the orthorhombic space group C222(1): a = 57.06(3) A, b = 92.41(6) A, c = 99.52(6) A. There is one NFP monomer per asymmetric unit and crystals diffract to 2.2 A spacings on film. A complete native data set to 2.5 A resolution has been collected on a San Diego Multiwire Detector system at the University of Alberta and a heavy-atom derivative search is presently in progress.

Secretion of functional Renilla reniformis luciferase by mammalian cells.

The soft coral Renilla reniformis luciferase enzyme is a monomeric soluble intracellular protein that is used increasingly as a marker of gene expression. Here the Renilla luciferase gene was engineered to encode a protein product secreted by mammalian cells. The 5' end of the Renilla luciferase gene was fused in frame with the 3' end of a short DNA sequence encoding the signal peptide from human interleukin-2 (IL-2) protein. This construct was cloned under transcriptional control of the cytomegalovirus (CMV) promoter in a mammalian expression vector. Simian COS-7 cells were transiently transfected with the construct, and light emission was measured from cell lysates and from cell culture media. The results of these experiments indicated that Renilla luciferase was secreted as a functional enzyme by mammalian cells. The advantages and disadvantages of secreted Renilla luciferase as a marker of gene expression in comparison to other secreted protein markers are discussed.

Green-fluorescent protein mutants with altered fluorescence excitation spectra.

Using random mutagenesis and visual selection of fluorescent clones, we have isolated a T203I and a E222G mutant of the Aequorea green-fluorescent protein. Each mutant has one of the two fluorescence excitation bands of the wild type deleted and retains the other without a wavelength shift. This finding is consistent with each excitation band corresponding to a distinct spectroscopic state of the chromophore. Both mutations are single amino acid exchanges which in the linear sequence are located remotely from the chromophore but in the folded protein may be situated in its vicinity. We conclude that the mutations influence the fluorescence properties by changing the interactions between the chromophore and its protein environment.

A sandwich type acridinium-linked immunosorbent assay (ALISA) detects soluble ErbB1 (sErbB1) in normal human sera.

The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression. In addition to the ErbB1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB1, i.e., sErbB1. Overexpression of ErbB1 in a variety of tumors has led us to hypothesize that sErbB levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers. To address this hypothesis we have developed an acridinium-linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media. This assay can also detect full-length ErbB1 in cell and tissue extracts. Our ALISA is characterized by high sensitivity (intra-assay LLD < 1 fmol/ml), a broad linear range (approximately 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV. Our ALISA does not detect full-length ErbB2, ErbB3, or ErbB4; or p105 soluble ErbB2. We report that serum sErbB1 levels of healthy women (median = 3716 fmol/ml), ranging in age from 43 to 76 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years. Additional analyses do not indicate that serum sErbB1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA. Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a approximately 110-kDa protein. We conclude that our ALISA is measuring the relative levels of this p110 sErbB1 analog in normal human sera. Our ALISA, therefore, should be useful for measuring the levels of ErbB1 and sErbB1 molecules in tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.

Validation of a single nucleotide polymorphism genotyping assay for the human serum paraoxonase gene using electronically active customized microarrays.

OBJECTIVES: Develop a microarray based genotyping assay to detect SNPs in human paraoxonase (PON I) and compare its accuracy with DNA sequencing and RFLP based assays. DESIGN AND METHODS: Amplicons spanning the polymorphic regions of interest were genotyped by sequencing, RFLP, and microarray technology. Validation parameters included precision, linearity of signal response, and carryover between adjacent sites on the microarray. RESULTS: A 100% correlation in results obtained using DNA sequencing, RFLP and microarray technology was observed. CONCLUSIONS: The microarray technology provides an accurate and reliable assay platform with applicability in high throughput genotyping studies.

Dynamic fluorescence study of the interaction of lumazine protein with bacterial luciferases.

The equilibrium association of lumazine protein from Photobacterium phosphoreum with luciferases from either P. phosphoreum or an aldehyde-requiring dark mutant of Vibrio harveyi is measured from changes of the rotational correlation time which is derived from the decay of the lumazine ligand's fluorescence anisotropy. The rotational correlation time of lumazine protein is 23 ns (2 degrees C, 0.25 M Pi) and is increased on addition of luciferase due to the formation of a higher molecular weight complex. The V. harveyi luciferase exhibits full competence for the association and a 1:1 stoichiometry with a Kd in the range 40-90 microM. At lower ionic strength (0.05 M Pi), the Kd increases but is reduced again by the addition of dodecanol or dimyristoyllecithin. In contrast, tetradecanal, a substrate for the bioluminescence reaction, exerts no influence on the association. The equilibration rate is found to be too slow and for both luciferases the Kd values are too high for the interaction of the native proteins to account quantitatively for the spectral shifting of the bioluminescence by lumazine protein.

Encapsulated radiophosphorescent standards for day-to-day photometer calibration.

Solid, unquenched, radiophosphorescent standards for use in the day-to-day calibration of bottom viewing photometers (luminometers) were prepared by encapsulating commercially-available phosphor powders that are excited to phosphoresce by the beta- decay of 63Ni (t0.5 = 96 yr) or 14C (t0.5 = 5730 yr). The radionuclides are physically adsorbed on the phosphors by precipitation either as a "basic nickel carbonate" or as barium carbonate. The radioactive phosphors are then deposited by centrifugation as a thin layer at the bottom of the vials or tubes that are normally used in the photometer. The phosphor layer is infiltrated with a plastic resin and embedded. A light absorbing layer is subsequently cast over the phosphor layer to prevent stray light excitation of phosphorescence. The encapsulated photometer standards have remained mechanically and photometrically stable since their fabrication, which in some cases is 3 years ago. An equivalent level of visible luminescence emitted from the standards of up to 2.3 x 10(10) photons.s-1 was achieved by using an appropriate amount of radioactivity and the proper phosphor. The phosphor used in the standards could be chosen such that the radiophosphorescence emission spectrum corresponded approximately to the chemiluminescence or bioluminescence spectrum under investigation.

Simultaneous genotyping of multiple polymorphisms in human serotonin transporter gene and detection of novel allelic variants.

The serotonin transporter, called SLC6A4, SERT or 5-HTT, modulates neurotransmission by removal of serotonin from the synapse of serotonergic neurons, facilitating serotonin reuptake into the presynaptic terminus. Selective serotonin reuptake inhibitors block the action of the serotonin transporter and are used to treat depression and other neuropsychiatric disorders. Three polymorphisms in the 5-HTT gene have been implicated in treatment response and neuropsychiatric disorders. A 44-bp promoter ins/del polymorphism (5-HTTLPR) produces primarily long and/or short alleles due to either 14 (short) or 16 (long) repeats of variably conserved 20-23 bp units. Also implicated, a 17-18 bp variable number tandem repeat found in intron2 (StIn2) is expressed as triallelic content with 9, 10, or 12 repeats (StIn2.9, StIn2.10 or StIn2.12). Finally, a single nucleotide polymorphism rs25531 located within the promoter polymorphic-linked region alters the function of the long promoter allele. We developed a PCR-based fragment analysis assay, which is analyzed on an ABI sequencer, whereby we are able to detect all three genotypes simultaneously. Using this technique, we identified novel sequences, which demonstrate promoter repeat regions containing (1) a 17 repeat with rs25531 A/G polymorphism, (2) two with 18-repeat units, (3) one with 20-repeat units and (4) a 24-repeat sequence. The novel repeats were confirmed by direct sequencing of gel-purified amplicons.

Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum.

The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic phosphorus, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is "buried". Modification of this buried Cys and at least one Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.

Physical characterization of lumazine proteins from Photobacterium.

The physicochemical properties of Photobacterium lumazine proteins have been investigated. The molecular weights obtained by several physical techniques are in good agreement, and the averages are 2% and 8% higher than the minimum molecular weights from amino acid and ligand content. The average molecular weights, sedimentation coefficients, and molecular radii are respectively the following: Photobacterium leiognathi lumazine protein, 21 200 +/- 300, 2.18 S, and 22.9 A; Photobacterium phosphoreum lumazine protein, 21 300 +/- 500, 2.16 S, and 23.0 A. The hydrations of the lumazine proteins, estimated in several ways, indicate less hydration for P. leiognathi than for P. phosphoreum. The frictional ratios corrected for hydration give axial ratios less than 1.3 for both lumazine proteins. These values agree with those obtained by a combination of rotational and translational frictional parameters and elimination of the common hydrated volume terms. There is insufficient area on the exterior surface to accommodate hydration when the lumzine proteins are considered as smooth-surfaced ellipsoids. The required surface area can be accommodated however by surface roughness with a minimum of 30% internal water.

Free radical participation in bacterial bioluminescence.

The metastable intermediate II produced on reaction of bacterial luciferase with reduced flavin mononucleotide and O2, reacts with any of several stable free radicals to produce bioluminescence. The bioluminescence spectrum is very similar to that from the well-studied intermediate II and aldehyde reaction, and the number of photons per luciferase molecule reacted is at least 40% of the aldehyde reaction.