Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: Large scale production of the E1 protein

The two envelope glycoproteins of rubella virus (RV), El of 58 kDa and E2 of 42–47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10−1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.     

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters

Summary A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active. It was further shown that the composite plasrnid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh. Neither pBR322 nor mini-F alone produced these inhibitory effects. Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla. Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect. Introduction of a deletion and a frameshift mutation into bla abolished the inhibition. Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, -lactamase.     

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

Summary Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA ⁺ gene. When the dnaA gene was induced from either the plac or the pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.     

Reversibility of DnaA protein activity in the'irreversible'dnaA204 mutant of Escherichia coli

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.     

Female preference for large males in the bushcricketRequena sp. 5 (Orthoptera: Tettigoniidae)

Receptive females of the bushcricketRequena sp. 5 (Orthoptera: Tettigoniidae) are attracted to male calls. In this experiment we investigate whether females discriminate between males on the basis of their calls. When virgin females were presented with two males of different size, they preferred the larger male. Larger males produce calls with a lower carrier frequency compared to smaller males, suggesting that females may use male carrier frequency as a predictor of male size. Furthermore, larger males produce heavier spermatophores. This suggests that females may prefer to mate with larger males to receive large nuptial gifts.     

Explorative Multivariate Analyses of 16S rRNA Gene Data from Microbial Communities in Modified-Atmosphere-Packed Salmon and Coalfish

Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with Photobacterium phosphoreum was observed. Brochothrix spp. and Carnobacterium spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C. piscicola had distinct substrate requirements, while the requirements of B. thermosphacta and C. piscicola were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.     

3,4,5-Trisubstituted isoxazoles as novel PPARdelta agonists. Part 2

A series of PPARdelta-selective agonists was investigated and optimized for a favorable in vivo pharmacokinetic profile. Isoxazole LCI765 (17d) was found to be a potent and selective PPARdelta agonist with good in vivo PK properties in mouse (C(max)=5.1 microM, t(1/2)=3.1 h). LCI765 regulated expression of genes involved in energy homeostasis in relevant tissues when dosed orally in C57BL6 mice. A co-crystal structure of compound LCI765 and the LBD of PPARdelta is discussed.     

Evolution and maintenance of pollen-colour dimorphisms in Nigella degenii: habitat-correlated variation and morph-by-environment interactions

Dimorphism in pollen colour is rare among flowering plants, but occurs in two geographically and morphologically distinct subspecies of Nigella degenii (Ranunculaceae). We evaluated the role of genotype-by-environment interactions in the maintenance of two pollen morphs within each of these subspecies. Morph frequencies in a number of populations were related to current habitat conditions, and an extensive common-garden experiment involving both optimal and stressful conditions (drought and nutrient deficiency) was carried out. The putatively derived (dark) pollen morph of N. degenii ssp. barbro has a higher frequency on slopes facing north or east than on slopes facing south or west. Plants of the dark morph also have a higher mortality under drought stress or nutrient deficiency. Data available for N. degenii ssp. jenny provide little evidence for habitat-correlated variation in morph frequency or morph-specific differences in fitness under optimal and stressful growth conditions. Our results suggest that morph-by-environment interactions in mortality could contribute to the maintenance of pollen-colour dimorphisms in N. degenii ssp. barbro.     

Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression

Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-beta (TGF-beta) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-beta in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-beta on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-beta was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-beta during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-beta was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-beta was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-beta, we examined RANK expression and p38 MAPK activation. We found the TGF-beta strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-beta to monocytes down-regulated RANK expression. The current results suggest that TGF-beta promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-beta abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.