Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2-dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.     

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.     

Collagen and mature elastic fibre organisation as a function of depth in the human cornea and limbus

A network of circumferentially oriented collagen fibrils exists in the periphery of the human cornea, and is thought to be pivotal in maintaining corneal biomechanical stability and curvature. However, it is unknown whether or not this key structural arrangement predominates throughout the entire corneal thickness or exists as a discrete feature at a particular tissue depth; or if it incorporates any elastic fibres and how, with respect to tissue depth, the circumcorneal annulus integrates with the orthogonally arranged collagen of the central cornea. To address these issues we performed a three-dimensional investigation of fibrous collagen and elastin architecture in the peripheral and central human cornea using synchrotron X-ray scattering and non-linear microscopy. This showed that the network of collagen fibrils circumscribing the human cornea is located in the posterior one-third of the tissue and is interlaced with significant numbers of mature elastic fibres which mirror the alignment of the collagen. The orthogonal arrangement of collagen in the central cornea is also mainly restricted to the posterior stromal layers. This information will aid the development of corneal biomechanical models aimed at explaining how normal corneal curvature is sustained and further predicting the outcome of surgical procedures.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Biological roles of APP in the epidermis

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimer's disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.     

Development of restoration techniques for Hawaiian thrushes: Collection of wild eggs,artificial incubation,hand‐rearing,captive‐breeding,and re‐introduction to the wild

From 1995 to 1999, two species of endemic Hawaiian thrushes, `Oma`o (Myadestes obscurus) and Puaiohi (M. palmeri), were captive‐reared and re‐introduced into their historic range in Hawai`i by The Peregrine Fund, in collaboration with the U.S. Geological Survey–Biological Resources Division (BRD) and the Hawai`i State Department of Land and Natural Resources. This paper describes the management techniques that were developed (collection of wild eggs, artificial incubation, hand‐rearing, captive propagation, and release) with the non‐endangered surrogate species, the `Oma`o; techniques that are now being used for recovery of the endangered Puaiohi. In 1995 and 1996, 29 viable `Oma`o eggs were collected from the wild. Of 27 chicks hatched, 25 were hand‐reared and released into Pu`u Wa`awa`a Wildlife Reserve. Using the techniques developed for the `Oma`o, a captive propagation and release program was initiated in 1996 to aid the recovery of the endangered Puaiohi. Fifteen viable Puaiohi eggs were collected from the wild (1996–1997) to establish a captive breeding flock to produce birds for re‐introduction. These Puaiohi reproduced for the first time in captivity in 1998 (total Puaiohi chicks reared in captivity 1996–1998 = 41). In 1999, 14 captive‐bred Puaiohi were re‐introduced into the Alaka`i Swamp, Kaua`i. These captive‐bred birds reproduced and fledged seven chicks in the wild after release. This is the first endangered passerine recovery program using this broad spectrum of management techniques (collection of wild eggs, artificial incubation, hand‐rearing, captive‐breeding, and release) in which re‐introduced birds survived and bred in the wild. Long‐term population monitoring will be published separately [BRD, in preparation]. Zoo Biol 19:263–277, 2000. © 2000 Wiley‐Liss, Inc.