Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.     

The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.     

Ontogenetic Behavior and Migration of Chinese Sturgeon, Acipenser sinensis

The Chinese sturgeon, Acipenser sinensis, is an anadromous protected species that presently only spawns in the Yangtze River. Using laboratory experiments, we examined the behavioral preference of young Chinese sturgeon to physical habitat (water depth, illumination intensity, substrate color, and cover) and monitored their downstream migration. Hatchling free embryos were photopositive, preferred open habitat, and immediately upon hatching, swam far above the bottom using swim-up and drift. Downstream migration peaked on days 0–1, decreased about 50% or more during days 2–7, and ceased by day 8. Days 0–1 migrants were active both day and night, but days 2–7 migrants were most active during the day. After ceasing migration, days 8–11 embryos were photonegative, preferred dark substrate and sought cover. Free embryos developed into larvae and began feeding on day 12, when another shift in behavior occurred–larvae returned to photopositive behavior and preferred white substrate. The selective factor favoring migration of free embryos upon hatching and swimming far above the bottom may be avoidance of benthic predatory fishes. Free embryos, which must rely on yolk energy for activity and growth, only used 19 cumulative temperature degree-days for peak migration compared to 234 degree-days for growth to first feeding larvae, a 1:12 ratio of cumulative temperature units. This ratio suggests that sturgeon species with large migratory embryos, like Chinese sturgeon, which require a high level of energy to swim during migration, may migrate only a short time to conserve most yolk energy for growth.     

Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.     

Ginkgo biloba retains functions of both type I and type II flowering plant phytochrome

While the photoreceptor systems of flowering plants have been well studied, the origins of these gene families and their functions are only partially understood. To begin to resolve the evolutionary origins of angiosperm photoreceptor function, we have studied the photomorphogenic responses of the early diverging gymnosperm Ginkgo biloba. Here, we describe the effects of continuous white light, red light, far-red light, and blue light on stem length, chlorophyll accumulation, Lhcb mRNA accumulation, and plastid development. Differences in the efficacy of these light regimes on de-etiolation in Ginkgo suggest separate but complementary roles for red and blue light-sensing systems. Additionally, the unique manner in which developmental regulation occurs in Ginkgo reveals a far-red high irradiance response different from both angiosperm and other gymnosperm species. We conclude from these data that Ginkgo contains a functional complement to both flowering plant type I and type II phytochromes, as well as independent blue light-sensing system(s). The implications of these findings are discussed with respect to the evolution of higher plant photoreceptors.     

CDP840. A prototype of a novel class of orally active anti-inflammatory phosphodiesterase 4 inhibitors

The discovery, synthesis and biological activity of a series of triarylethane phosphodiesterase 4 inhibitors is described. Structure-activity relationship studies are presented for CDP840 (29), a potent, chiral, selective inhibitor of PDE 4 (IC(50) 4nM). CDP840 is non-emetic in the ferret at 30mgkg(-1) (po), active in models of inflammation and reverses ozone-induced bronchial hyperreactivity in the guinea pig.     

Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy

This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.     

Companion animal adoption study

To better understand the outcomes of companion animal adoptions, Bardsley & Neidhart Inc. conducted a series of 3 surveys over a 1-year period with dog and cat owners who had adopted their pet through either a (a) Luv-A-Pet location, (b) Adopt-a-thon, or (c) traditional shelter. This article suggests opportunities to improve owners' perceptions of their pets and the adoption process through (a) providing more information before adoption about pet health and behaviors, (b) providing counseling to potential adopters to place pets appropriately, and (c) educating adopters to promote companion animal health and retention. Results demonstrate that the pet's relationship to the family unit, such as where the pet sleeps and how much time is spent with the pet, is related to the amount of veterinary care the companion animal receives, and to long-term retention. Satisfaction and retention are attributed to the pet's personality, compatibility, and behavior, rather than demographic differences among adopters or between adoption settings. The age of the companion animal at adoption, the intended recipient, and presence of children in the home also play a role. Health problems were an issue initially for half of all adopted pets, but most were resolved within 12 months. Roughly one fourth of adopters who no longer have their companion animal said their pet died. Characteristics of pets that died support the contention that spaying and neutering profoundly affects a companion animal's life span. Although retention is similar for dogs and cats, mortality is higher among cats in the first year after adoption.     

Transfectant mosaic spheroids: a new model for evaluation of tumour cell killing in targeted radiotherapy and experimental gene therapy

Background

We describe an in vitro tumour model for targeted radiotherapy and gene therapy that incorporates cell population heterogeneity.

Materials and methods

Transfectant mosaic spheroids (TMS) and transfected mosaic monolayers (TMM) are composed of two cell populations derived from a single cell line. The cells of one population were transfected with the noradrenaline transporter gene (NAT), allowing active uptake of a radiolabelled targeting agent meta‐[¹³¹I]iodobenzylguanidine ([¹³¹I]MIBG); the other population of cells was derived from the same parent line and transfected with a marker gene – green fluorescent protein (GFP). After treatment with [¹³¹I]MIBG, cell kill was determined in TMM by clonogenic assay and in TMS by clonogenic assay and spheroid growth delay.

Results

We have used the TMS model to assess the ‘radiological bystander effect’ (radiation cross‐fire) conferred by the β‐emitting radiopharmaceutical [¹³¹I] MIBG whose cellular uptake is facilitated by the transfected gene encoding NAT. We show that cell killing by [¹³¹I]MIBG in both TMS and TMM cultures increased in direct proportion to the fraction of NAT‐transfected cells and that the degree of cell killing against fraction transfected was greater in TMS, suggestive of a greater bystander effect in the three‐dimensional culture system.

Conclusions

TMS provide a useful model for assessment of the effectiveness of targeted radiotherapy in combination with gene therapy when less than 100% of the target cell population is expressing the NAT transgene. Further, this novel model offers the unique opportunity to investigate radiation‐induced bystander effects and their contribution to cell cytotoxicity in radiotherapy and other gene therapy applications. Copyright © 2002 John Wiley & Sons, Ltd.