Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(o)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(o)/G(1), blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(o)/G(1), and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEG-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta -dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(o)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms. (C) 2001 Elsevier Science Inc. All rights reserved.

Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases, Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer, The monomeric unit of the protein was chemically synthesized in three parts (positions 1-51, 54-99, and 102-127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

The effect of high-pass filtering on TEOAE in 2-month-old infants

The aim of the present study was to investigate the effect of high-pass filtering on TEOAE obtained from 2-month-old infants as a function of filter cut-off frequency, activity states and pass/fail status of infants. Two experiments were performed. In Experiment 1, 100 2-month-old infants (200 ears) in five activity states (asleep, awake but peaceful, sucking a pacifier, feeding, restless) were tested by use of TEOAE technology. Five different filter conditions were applied to the TEOAE responses post hoc. The filter conditions were set at 781 Hz (default setting), 1.0, 1.2, 1.4 and 1.6 kHz. Results from this experiment showed that TEOAE parameters, such as whole-wave reproducibility (WR) and signal-to-noise ratio (SNR) at 0.8 kHz and 1.6 kHz, changed as a function of the cut-off frequency. The findings suggest that the 1.6 kHz and 1.2 kHz filter conditions are optimal for WR and SNR pass/fail criteria, respectively. Although all infant recordings appeared to benefit from the filtering, infants in the noisy states seemed to benefit the most. In Experiment 2, the high-pass filtering technique was applied to 23 infants (35 ears) who apparently failed the TEOAE tests on initial screening but were subsequently awarded a pass status based on the results from a follow-up auditory brainstem response (ABR) assessment. The findings showed a significant decrease in noise contamination of the TEOAE with a corresponding significant increase in WR. With high-pass filtering at 1.6 kHz, 21/35 ears could be reclassified into the pass category.

Surface diffusion and adsorption of hydrocarbons in activated carbon

Adsorption and diffusion in a porous media were studied theoretically and experimentally with a differential transient permeation method. The porous medium is allowed to equilibrate at some specified loading, and then the time trajectory of the permeation process is followed after a small difference between the pressures at the end faces of the porous medium is introduced at time t = 0 +. Such a trajectory us. time would contain adsorption and diffusion characteristics of the system. By studying this for various surface loadings, pore and surface diffusions can be fully characterized. Mathematical modeling of transient permeation is detailed for pure gases or vapors diffusion and adsorption in porous media. Effects of nonlinearity of adsorption isotherm, pressure, temperature and heat effects were considered in the model. Experimental data of diffusion and adsorption of propane, n-butane and n-hexane in activated carbon at different temperatures and loadings show the potential of this method as a useful tool to study adsorption kinetics in porous media. Validity of the model is best tested against the transient data where the kinetics curves exhibit sigmoidal shape, which is a result of the diffusion and adsorption rate during the initial stage of permeation.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

A role for Rho in smooth muscle phenotypic regulation

The role of the small GTP-binding protein Rho in the process of smooth muscle cell (SMC) phenotypic modulation was investigated using cultured rabbit aortic SMCs. Both Rho transcription and Rho protein expression were high for the first 3 days of culture ("contractile" state cells), with expression decreasing after change to the "synthetic" state and peaking upon return to the contractile phenotype. Activation of Rho (indicated by translocation to the membrane) also peaked upon return to the contractile state and was low in synthetic state SMCs. Transient transfection of synthetic state rabbit SMCs with constitutively active Rho (vall4rho) caused a dramatic decrease in cell size and reorganization of cytoskeletal proteins to resemble those of the contractile phenotype; alpha-actin and myosin adopted a tightly packed, highly organized arrangement, whereas vimentin localized to the immediate perinuclear region and focal adhesions were enlarged. Conversely, specific inhibition of endogenous Rho, by expression of C3 transferase, resulted in the complete loss of actin and myosin filaments without affecting the distribution of vimentin. Focal adhesions were reduced in number. Thus, Rho plays a key role in regulating SMC phenotypic expression.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression

The c fins gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c -fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSS) were identified within mouse intron 2. Sequences of these DHSS were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fins intronic regulatory element (FIRE), which is even more highly conserved than the c-fins proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Spl, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high and low-level c -fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fins gene.

Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation

The microphthalmia transcription factor (MITF), a basic-helix-loop-helix zipper factor, regulates distinct target genes in several cell types. We hypothesized that interaction with the Ets family factor PU.1, whose expression is limited to hematopoietic cells, might be necessary for activation of target genes like tartrate-resistant acid phosphatase (TRAP) in osteoclasts. Several lines of evidence were consistent with this model. The combination of MITF and PU.1 synergistically activated the TRAP promoter in transient assays. This activation was dependent on intact binding sites for both factors in the TRAP promoter. MITF and PU.1 physically interacted when coexpressed in COS cells or in vitro when purified recombinant proteins were studied. The minimal regions of MITF and PU.1 required for the interaction were the basic-helix-loop-helix zipper domain and the Ets DNA binding domain, respectively. Significantly, mice heterozygous for both the mutant mi allele and a PU.1 null allele developed osteopetrosis early in life which resolved with age. The size and number of osteoclasts were not altered in the double heterozygous mutant mice, indicating that the defect lies in mature osteoclast function. Taken in total, the results afford an example of how lineage-specific gene regulation can be achieved by the combinatorial action of two broadly expressed transcription factors.

The marginal dentition of the Australian lungfish, Neoceratodus forsteri (Osteichthyes : Dipnoi)

The dentary, a component of the transient marginal dentition found in the mandible of juveniles of the living Australian lungfish Neoceratodus forsteri, is a tooth plate exactly comparable to the tooth plates with radiating ridges that make up the marginal dentitions of Devonian dipnoans like Andreyevichthys, Orlovichthys and Ichnomylax. In N. forsteri, the dentary consists of two ridges, set almost in line with each other, and growing by the addition of cusps, of increasing sizes, to the extremity of each ridge. It is therefore equivalent to two ridges of a more normal tooth plate with radiating ridges. Despite its appearance, as a long row of sharp cusps ankylosed to a slender bone, and its position, embedded in soft tissue above the anterolabial margin of Meckel's cartilage, it is a tooth plate and is not comparable to the marginal dentitions of other vertebrates. Structure and development of the transient marginal dentition of this lungfish is another indication that dipnoans may not be the sister group of tetrapods.

Breeding dynamics of koalas in open woodlands

The spatial and breeding dynamics of koalas in sub-tropical woodlands at Blair Athol in central Queensland were intensively monitored between 1993 and 1998. Genetic relationships among koalas at this locality were studied to determine the breeding dynamics of males, including whether 'resident' or 'transient' males dominate as sires. Males and females were radio-collared and tracked periodically throughout each year of the study. Genotypes from hypervariable microsatellite loci identified uniquely all individuals and were used to analyse parentage as well as to determine population genetic parameters when compared with other regional localities. Koalas at Blair Athol comprise a population in genetic equilibrium. Gene diversity estimates show the population to be similar to other populations found in similar habitat in the region, and estimates of genetic differentiation among four regional populations showed that gene flow conforms to a model of isolation by distance. Analysis of parentage found that both resident and transient males sired about equal numbers of offspring. Familial DNA analysis revealed multiple paternities of successive young in this population. The conclusion from this study is that 'resident' status among males does not confer any advantage for parentage.

Economic and environmental impacts of pollution control in a system of environment and economic interdependence

The importance of the rate of change of the pollution stock in determining the damage to the environment has been an issue of increasing concern in the literature. This paper uses a three-sector (economy, population and environment), non-linear, discrete time, calibrated model to examine pollution control. The model explicitly links economic growth to the health of the environment. The stock of natural resources is affected by the rate of pollution flows, through their impact on the regenerative capacity of the natural resource stock. This can shed useful insights into pollution control strategies, particularly in developing countries where environmental resources are crucial for production in many sectors of the economy. Simulation exercises suggested that, under plausible assumptions, it is possible to reverse undesirable transient dynamics through pollution control expenditure, but this is dependent upon the strategies used for control. The best strategy is to spend money fostering the development of production technologies that reduce pollution rather than spending money dealing with the effects of the pollution flow into the environment. (C) 2001 Elsevier Science Ltd. All rights reserved.

Nocturnal activity in the green sea turtle alters daily profiles of melatonin and corticosterone

In nature, green turtles (Chelonia mydas) can exhibit nocturnal activity in addition to their typically diurnal activity cycle. We examined whether nocturnal activity in captive and free-living green turtles altered daily plasma profiles of melatonin (MEL) and corticosterone (CORT). In captivity, diurnally active green turtles expressed distinct diel cycles in MEL and CORT; a nocturnal rise was observed in MEL and a diurnal rise was observed in CORT. However, when induced to perform both low- and high-intensity nocturnal activity, captive green turtles exhibited a significant decrease in MEL, compared to inactive controls. In contrast, plasma CORT increased significantly with nocturnal activity, and further, the relative increase in CORT was correlated with the intensity of the nocturnal behavior. In free-living green turtles that performed nocturnal activity including: nesting, mate searching, and feeding/swimming behaviors, plasma profiles in MEL and CORT exhibited relatively little, or no, daily fluctuation. Our findings demonstrate that nocturnal activity in green turtles is often associated with MEL and CORT profiles that resemble those measured during the day. We speculate that these conspicuous changes in MEL and CORT during nocturnal activity could either support or promote behaviors that enable acquisition of transient resources important to the survival and reproductive success of green turtles. (C) 2002 Elsevier Science (USA).

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell. (C) 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 476-480, 2002; DOI 10.1002/bit.10126.

A normative study of distortion product otoacoustic emissions in six-year-old school children

Evoked otoacoustic emissions have demonstrated potential for application in the community-based hearing screening of paediatric populations. Distortion-product otoacoustic emissions (DPOAEs), as opposed to transient evoked otoacoustic emissions (TEOAEs), have not been extensively researched in this regard. The current study aimed to describe the range of DPOAE values obtained in a large cohort (1576 ears) of 6-year-old children in school settings and to examine possible ear asymmetry, gender and history of ear infection effects on the data. Results indicated a variety of significant effects, particularly in the high frequencies, for DPOAE signal-to-noise ratio. The measurement parameter, DPOAE amplitude (DP-amp), was found to display potentially less clinical applicability due to large standard deviation values. Use of descriptive normative data, as derived in the present investigation, may contribute toward future improvements in the hearing screening of 6-year-old schoolchildren

In vivo DNA electrotransfer

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subeloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.