C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

Summary The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 m diameter), a characteristic of so-called unspecialized peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to -ATPase and stearoyl-ACP 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.Abbreviations CAT chloramphenicol acetyltransferase - CHO cells Chinese hamster ovary cells - DAB 3,3-diaminobenzidine - GUS -glucuronidase - ICL isocitrate lyase - KSRM lysine-serine-arginine-methionine - MS malate synthase - PBS phosphate-buffered saline - PTS peroxisome targeting signal - SKL serine-lysine-leucine - tobacco BY-2 Bright Yellow-2 Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Age,Experience, and the Response of Streamside Salamander Hatchlings to Chemical Cues from Predatory Sunfish

Previous work has shown that streamside salamander larvae (Ambystoma barbouri; Ambystomatidae) exhibit an adaptive ‘sink to the bottom’ response to chemical cues from predatory green sunfish (Lepomis cyanellus; Centrarchidae), that is, larvae sink to the bottom more quickly (thus minimizing exposure time to sunfish predation) when they are dropped into water with sunfish chemicals (as compared to Ashless controls). Here, we examined this anti-predator behaviour in early hatchlings and the effects of age and experience on subsequent expression of this behaviour. Hatchlings responded significantly to fish chemical cues within the first 18 h after hatching. Age did not significantly influence this response, i.e. regardless of age (1, 7, or 14 days after hatching) larvae showed a significant response during their first exposure to fish chemical cues. Experience also did not significantly influence the larval response to fish chemicals i.e., repeated exposures over 2 weeks did not significantly influence the magnitude of the response. Finally, comparisons of 3 siblingships detected significant variation among siblingships that might reflect genetic variation in this behaviour.     

A linkage map of diploid Avena based on RFLP loci and a locus conferring resistance to nine isolates of Puccinia coronata var. ‘avenae’

An F₂ oat population was produced by crossing the diploid (n=7) species Avena strigosa (CI 3815) with A. wiestii (CI 1994), resistant and susceptible, respectively, to 40 isolates of Puccinia coronata, the causal agent of crown rust. Eighty-eight F₂ individuals were used to construct an RFLP linkage map representing the A genome of cultivated hexaploid oat. Two hundred and eight RFLP loci have been placed into 10 linkage groups. This map covers 2416 cM, with an average of 12 cM between RFLP loci. Eighty-eight F₃ lines, derived from F₂ individuals used to construct the map, were screened for resistance to 9 isolates of P. coronata. One locus, Pca, was found to confer a dominant resistance phenotype to isolates 203, 258, 263, 264B, 290, 298, 325A, and 345. Pca also conferred resistance to isolate 276; however, an unlinked second gene may also be involved.Journal Paper No. 15143 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3134 and 2447     

Inhibition of Immune Complex-Induced Inflammation by A small Molecular Weight Selectin Antagonist

The anti-inflammatory effect of a small molecular weight antagonist of P- and E-selectin-dependent cell adhesion was examined. The glycolipid sulphatide was shown to block the adherence of thrombin-activated rat platelets to HL-60 cells. This interaction is known to be dependent on P-selectin. The rat dermal reverse passive Arthus reaction was used to assess the effect of sulphatide on a neutrophil dependent inflammatory response. Sulphatide dosedependently blocked both the vascular permeability increase and cell infiltration after intraperitoneal administration. These results show that a small molecular weight compound which blocks P- and E-selectin dependent adhesion in vitro can effectively block the inflammation due to immune complex deposition. A compound with this type of profile may have therapeutic potential in the treatment of immune complex mediated diseases.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.     

A simple model of human foveal ganglion cell responses to hyperacuity stimuli

We developed a physiologically plausible model of the first steps of spatial visual information processing in the fovea of the human retina. With the predictions of this model we could support the hypothesis that, for moderate contrasts ( 40%), hyperacuity is mediated by the magnocellular (MC-) pathway. Despite the lower sampling density in the MC pathway, as compared to the parvocellular (PC-) pathway, the information that is transferred by the MC ganglion cells is sufficient to achieve thresholds comparable to those of human subjects in psychophysical tasks. This is a result of the much higher signal-to-noise ratio of the MC pathway cell signals. The PC pathway cells do not transfer enough information for hyperacuity thresholds.