Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Antipruritic and antihyperalgesic actions of loperamide and analogs

Loperamide and three of its analogs were evaluated for their ability to inhibit binding to cloned human opioid receptor subtypes and to produce antipruritis and antinociception following local s.c. administration to rodents. All four compounds were fully efficacious agonists with affinities of 2 to 4 nM for the cloned human mu opioid receptor. Local s.c. injection of loperamide, ADL 01-0001 or ADL 01-0002 at the same site as the introduction of the pruritogenic compound 48/80 resulted in antipruritic activity in a mouse model of itch. Similarly, i.paw or i.pl. administration of compounds ADL 01-0001, ADL 01-0002 and ADL 01-0003 to inflamed paws caused potent antinociception, inhibiting late phase formalin-induced flinching, Freund's adjuvant-induced mechanical hyperalgesia and tape stripping-induced mechanical hyperalgesia. Loperamide and its analogs were efficacious in animal models of itch and inflammatory pain, and may have potential therapeutic utility as antipruritic and antihyperalgesic agents.     

Insulin resistance and impaired baroreflex gain during pregnancy

Pregnancy decreases baroreflex gain, but the underlying mechanism is unclear. Insulin resistance, which has been associated with reduced transport of insulin into the brain, is a consistent feature of many conditions exhibiting impaired baroreflex gain, including pregnancy. Therefore, using conscious pregnant and nonpregnant rabbits, we tested the novel hypothesis that the pregnancy-induced impairment in baroreflex gain is due to insulin resistance and reduced brain insulin. Baroreflex gain was determined by quantifying changes in heart rate in response to stepwise steady-state changes in arterial pressure, secondary to infusion of nitroprusside and phenylephrine. We found that insulin sensitivity and baroreflex gain were strongly correlated in nonpregnant and term pregnant rabbits (r2 = 0.59). The decrease in insulin sensitivity and in baroreflex gain exhibited similar time courses throughout pregnancy, reaching significantly lower levels at 3 wk of gestation and remaining reduced at 4 wk (term is 31 days). Treatment of rabbits with the insulin-sensitizing drug rosiglitazone during pregnancy almost completely normalized baroreflex gain. Finally, pregnancy significantly lowered cerebrospinal fluid insulin concentrations. These data identify insulin resistance as a mechanism underlying pregnancy-induced baroreflex impairment and suggest, for the first time in any condition, that decreased brain insulin concentrations may be the link between reductions in peripheral insulin sensitivity and baroreflex gain.     

Amino acid conjugates as kappa opioid receptor agonists

A novel series of kappa (kappa) opioid receptor agonists were synthesized by incorporating the key structural features of known kappa opioid agonists while replacing the aryl acetamide portion with substituted amino acid conjugates. Compounds 3j (Ki = 6.7 nM), 3k (Ki = 3.6 nM), 3l (Ki = 4.6 nM), 3m (Ki = 0.83 nM) and 3o (Ki = 2 nM) possessed potent affinities for the kappa opioid receptor in vitro with reasonable selectivity over other opioid receptors.     

Epigenomic stability assessment during cryopreservation and physiology among various strains of Chromochloris zofingiensis (Chlorophyceae) and their genetic variability revealed by AFLP and MS-AFLP

Chromochloris zofingiensis (Dönz) Fucíková & L.A.Lewis, due to its production of highly valuable carotenoids such as astaxanthin, is a model organism in biotechnology. Since the recognition of this physiological property, many biotechnological applications have only used a single strain (SAG 211-14 = CCAP 211/14 = UTEX 32 = ATCC 30412) to produce biomass and carotenoids. However, multiple acquisitions of strains putatively belonging to the same species raised the question of the conspecificity of those strains and their properties. In this study, the conspecificity of the available strains, which are deposited axenically in SAG, was tested using SSU and ITS rDNA sequencing and AFLP (EcoRI/PstI) analyses. The comparison of SSU and ITS rDNA sequences as well as the AFLP patterns revealed that the investigated strains formed two very similar groups, (1) SAG 211-14, SAG 4.80, SAG 31.80, and SAG 34.80 and (2) SAG 221-2. All strains belonged to one species, C. zofingiensis, and represented one monophyletic lineage within the so-called DO-group of the Chlorophyceae. The robustness to cryopreservation and the subsequent epigenetic variability was detected using the methylation-sensitive AFLP (EcoRI/MspI and EcoRI/HpaII) among the five Chromochloris strains. All strains showed a high rate of survival (54.4–98.1%) during cryopreservation. The methylation patterns varied between precryo and postcryo in all strains detected among three time points (before, shortly after, and 8 weeks after cryopreservation), showing that the MS-AFLP technique has the potential to detect epigenetic effects occurring in response to cryopreservation and other stresses. Finally, the potential of these five strains for usage in biotechnological applications was proven by growing them in aerated cultures with and without additional carbon dioxide supply. The comparison showed that all strains produced high amounts of biomass and carotenoids under aeration with additional CO₂ and were therefore suitable in biotechnology.

     

Roles of nitric oxide and angiotensin II in the impaired baroreflex gain of pregnancy

The present study tested the hypothesis that nitric oxide (NO) contributes to impaired baroreflex gain of pregnancy and that this action is enhanced by angiotensin II. To test these hypotheses, we quantified baroreflex control of heart rate in nonpregnant and pregnant conscious rabbits before and after: 1) blockade of NO synthase (NOS) with Nomega-nitro-L-arginine (20 mg/kg iv); 2) blockade of the angiotensin II AT1 receptor with L-158,809 (5 microg x kg(-1) x min(-1) iv); 3) infusion of angiotensin II (1 ng x kg(-1) x min(-1) nonpregnant, 1.6-4 ng x kg(-1) x min(-1) pregnant iv); 4) combined blockade of angiotensin II AT(1) receptors and NOS; and 5) combined infusion of angiotensin II and blockade of NOS. To determine the potential role of brain neuronal NOS (nNOS), mRNA and protein levels were measured in the paraventricular nucleus, nucleus of the solitary tract, caudal ventrolateral medulla, and rostral ventrolateral medulla in pregnant and nonpregnant rabbits. The decrease in baroreflex gain observed in pregnant rabbits (from 23.3 +/- 3.6 to 7.1 +/- 0.9 beats x min(-1) x mmHg(-1), P < 0.05) was not reversed by NOS blockade (to 8.3 +/- 2.5 beats x min(-1) x mmHg(-1)), angiotensin II blockade (to 5.0 +/- 1.1 beats x min(-1) x mmHg(-1)), or combined blockade (to 12.3 +/- 4.8 beats x min(-1) x mmHg(-1)). Angiotensin II infusion with (to 5.7 +/- 1.0 beats x min(-1) x mmHg(-1)) or without (to 8.4 +/- 2.4 beats x min(-1) x mmHg(-1)) NOS blockade also failed to improve baroreflex gain in pregnant or nonpregnant rabbits. In addition, nNOS mRNA and protein levels in cardiovascular brain regions were not different between nonpregnant and pregnant rabbits. Therefore, we conclude that NO, either alone or via an interaction with angiotensin II, is not responsible for decrease in baroreflex gain during pregnancy.     

Embedding permanent watermarks in synthetic genes

As synthetic biology advances, labeling of genes or organisms, like other high-value products, will become important not only to pinpoint their identity, origin, or spread, but also for intellectual property, classification, bio-security or legal reasons. Ideally information should be inseparably interlaced into expressed genes. We describe a method for embedding messages within open reading frames of synthetic genes by adapting steganographic algorithms typically used for watermarking digital media files. Text messages are first translated into a binary string, and then represented in the reading frame by synonymous codon choice. To aim for good expression of the labeled gene in its host as well as retain a high degree of codon assignment flexibility for gene optimization, codon usage tables of the target organism are taken into account. Preferably amino acids with 4 or 6 synonymous codons are used to comprise binary digits. Several different messages were embedded into open reading frames of T7 RNA polymerase, GFP, human EMG1 and HIV gag, variously optimized for bacterial, yeast, mammalian or plant expression, without affecting their protein expression or function. We also introduced Vigenère polyalphabetic substitution to cipher text messages, and developed an identifier as a key to deciphering codon usage ranking stored for a specific organism within a sequence of 35 nucleotides.     

Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter

A segment of DNA from the genome of figwort mosaic virus (FMV) strain M3 possesses promoter activity when tested in electroporated protoplasts from, and transgenic plants of, Nicotiana tabacum cv. Xanthi nc. The 1.1 kb DNA segment, designated the 34S promoter, is derived from a position on the FMV genome comparable to the position on the cauliflower mosaic virus (CaMV) genome containing the 35S promoter. The 34S and 35S promoters show approximately 63% nucleotide homology in the TATA, CCACT, and –18 to +1 domains, but in sequences further upstream the homology drops below 50%. Promoter activities were estimated using -glucuronidase and neomycin phosphotransferase II reporter gene systems. The activity of the 34S promoter segment approximates that of the 35S promoter in both protoplast transient expression assays and in stably transformed tobacco plants. Truncation of 5 sequences from the 34S promoter indicates that promoter strength depends upon DNA sequences located several hundred nucleotides upstream from the TATA box. In leaf tissue the 34S promoter is 20-fold more active than the mannopine synthase (MAS) promoter from Agrobacterium tumefaciens T-DNA. The 34S promoter lacks the root-specific and wound-stimulated expression of the MAS promoter, showing relatively uniform root, stem, leaf, and floral activities.     

Mechanical properties of foods used in experimental studies of primate masticatory function

In vivo studies of jaw-muscle behavior have been integral factors in the development of our current understanding of the primate masticatory apparatus. However, even though it has been shown that food textures and mechanical properties influence jaw-muscle activity during mastication, very little effort has been made to quantify the relationship between the elicited masticatory responses of the subject and the mechanical properties of the foods that are eaten. Recent work on human mastication highlights the importance of two mechanical properties-toughness and elastic modulus (i.e., stiffness)-for food breakdown during mastication. Here we provide data on the toughness and elastic modulus of the majority of foods used in experimental studies of the nonhuman primate masticatory apparatus. Food toughness ranges from approximately 56.97 Jm(-2) (apple pulp) to 4355.45 Jm(-2) (prune pit). The elastic modulus of the experimental foods ranges from 0.07 MPa for gummy bears to 346 MPa for popcorn kernels. These data can help researchers studying primate mastication select among several potential foods with broadly similar mechanical properties. Moreover, they provide a framework for understanding how jaw-muscle activity varies with food mechanical properties in these studies.