Experimental model for stimulation of cultured human osteoblast-like cells by high frequency vibration

Reliable and reproducible experimental methods for studying enhancement of osteoblast proliferation and metabolic activity in vitro provide invaluable tools for the research of biochemical processes involved in bone turnover in vivo. Some of the current methods used for this purpose are based on the ability of the osteoblasts to react metabolically to mechanical stimulation. These methods are based on the hypothesis that intracellular metabolic pathways could be influenced by the excitation of cytoskeletal components by mechanical cell deformation. Based on the same assumptions we developed a new experimental approach of biomechanical stimulation of cultured osteoblast-like cells by vibration. This method is based on the use of a specially designed vibration device that consists of an electric shaker with horizontally mounted well plate containing cell cultures. We used a first passage explant outgrowth of human osteoblast-like cell cultures, originating from samples of cancelous bone, collected from femoral necks of six donors during surgical arthroplasties of osteoarthritic hips. Well plates with replicates of cultured cells were exposed to a sine shaped vibration protocol in a frequency range of 20–60 Hz with displacement amplitude of 25 (±5) μm. We found that vibration at a distinct set of mechanical parameters of 20 Hz frequency and peak to peak acceleration of 0.5 ± 0.1 m/sec² is optimal for cell proliferation, and at 60 Hz frequency with peak to peak acceleration of 1.3 ± 0.1 m/sec² for metabolic activity. The presented easily reproducible experimental model should improve and simplify further research on the interactions between mechanical stimuli and intracellular biochemical pathways in osteoblasts. This revised version was published online in September 2006 with corrections to the Cover Date.     

In Vitro Selection of a Deoxyribozyme That Can Utilize Multiple Substrates

Deoxyribozymes that could catalyze the formation of an internucleotide phosphorothioester linkage were selected from a random sequence pool. During the course of the selection, the pool was successively challenged with five oligonucleotide substrates, each of which terminated in the same hexanucleotide sequence. Selected deoxyribozyme ligases could use all five substrates, albeit to different degrees, and appeared to form secondary structures that allow differential pairing between the deoxyribozyme and each substrate. These results suggest that early replicases may have been able to bind a variety of oligonucleotide substrates while catalyzing ligation via a common junction. Received: 12 December 2000 / Accepted: 28 June 2001     

Reciprocal regulation of expression of pore‐forming KATP channel genes by hypoxia

The ATPsensitive potassium (K_ATP) channel is thought to play an important role in the protection of heart and brain against tissue hypoxia. The genetic regulation of the components of the channel by hypoxia has not been previously described. Here, we investigated the regulation of the two poreforming channel proteins, Kir6.1 and Kir6.2, in response to hypoxia in vivo and in vitro. We find that these two structurallyrelated inwardly-rectifying potassium channel proteins are reciprocally regulated by hypoxia in vivo, with upregulation of Kir6.1 and downregulation of Kir6.2, thereby resulting in a significant change in the composition of the channel complex in response to hypoxia. In vitro we describe neuronal and cardiac cell lines in which Kir6.1 is upregulated by hypoxia, demonstrating that Kir6.1 is a hypoxiainducible gene. We conclude that the heart and brain display genetic plasticity in response to hypoxic stress through specific genetic reprograming of cytoprotective channel genes.     

Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines

Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic features, Caco-2 cells seeded on plastic progressively increased L-FABP quantities, whereas I-FABP was not detectable even very late in the maturation process. On permeable filters that improved differentiation markers (sucrase, alkaline phosphatase, transepithelial resistance), Caco-2 cells furthered their L-FABP content and expressed I-FABP. Western blot analysis showed a significant increase in I- and L-FABP expression following an 8-hour incubation period with butyric acid, oleic acid, and phosphatidylcholine. However, in all cases, I-FABP levels were higher than L-FABP concentrations regardless of the lipid substrates added. Similarly, hydrocortisone and insulin enhanced the cellular content of I- and L-FABP whereas leptin triggered I-FABP expression only after an 8-hour incubation. Finally, tumor necrosis factor-alpha was more effective in increasing the cytosolic amount of I-FABP levels. In conclusion, our data demonstrate that I-FABP expression is limited to fully differentiated Caco-2 cells and can be more easily regulated than L-FABP by lipids, hormones, and cytokines.     

Gene transfer technology in aquaculture

The gene transfer technique, transgenesis, has permitted the transfer of genes from one organism to another to create new lineages of organisms with improvement in traits important to aquaculture. Genetically modified organisms (GMOs), therefore, hold promise for producing genetic improvements, such as enhanced growth rate, increased production and efficiency, disease resistance and expanded ecological ranges. The basic procedure to generate transgenic fish for aquaculture includes: (1) design and construction of transgenic DNA; (2) transfer of the gene construct into fish germ cells; (3) screening for transgenic fish; (4) determination of transgene expression and phenotype; (5) study of inheritance; and (6) selection of stable lines of transgenics.GMOs offer economic benefits, but also pose environmental threats. Optimising the mix of benefits and risks is of fundamental importance. The potential economic benefits of transgenic technology to aquaculture are obvious. Transgenic fish production has the goal of producing food for human consumption; thus the design of genetic constructs must take into consideration the potential risks to consumer health, as well as marketing strategies and product acceptance in the market.     

Experimental fragmentation reduces sexual reproductive output by the reef-building coral Pocillopora damicornis

Natural and anthropogenic disturbances may fragment stony reef corals, but few quantitative data exist on the impacts of skeletal fragmentation on sexual reproduction in corals. We experimentally fragmented colonies of the branching coral Pocillopora damicornis and determined the number and size of planula larvae released during one lunar reproductive cycle. Partially fragmented colonies significantly delayed both the onset and peak period of planula release compared with intact control colonies. Most fragments removed from the corals died within 11–18 days, and released few planulae. The total number of planulae released per coral colony varied exponentially with remaining tissue volume, and was significantly lower in damaged versus undamaged colonies. However, the number of planulae produced per unit tissue volume, and planula size, did not vary with damage treatment. We conclude that even partial fragmentation of P. damicornis colonies (<25% of tissue removed) decreases their larval output by reducing reproductive tissue volume. Repeated breakage of corals, such as caused by intensive diving tourism or frequent storms, may lead to substantially reduced sexual reproduction. Therefore, reef management should limit human activities that fracture stony corals and lead to decreases in colony size and reproductive output. Accepted: 2 February 2000