Influence of liquid medium and surface morphology on the response of QCM during immobilization and hybridization of short oligonucleotides

With the goal of developing a quartz crystal microbalance (QCM)-based DNA sensor, we have conducted an in situ QCM study along with fluorescence measurements using oligonucleotides (15-mer) as a model single-stranded DNA (ss-DNA) in two different aqueous buffer solutions; the sequence of 15-mer is a part of iduronate-2-sulphate exon whose mutation is known to cause Hunter syndrome, and the 15-mer is thiolated to be immobilized on the Au-coated quartz substrate. The fluorescence data indicate that the initial immobilization as well as the subsequent hybridization with a complementary strand is hardly dependent on the kind of buffer solution. In contrast, the mass increases deducible from the decrease of QCM frequency via the Sauerbrey equation are 2.7-6.2 and 3.0-4.4 times larger than the actual mass increases, as reflected in the fluorescence measurements, for the immobilization and the subsequent hybridization processes, respectively. Such an overestimation is attributed to the trapping of solvent as well as the formation of quite a rigid hydration layer associated with the higher viscosities and/or densities of the buffer solutions. Another noteworthy observation is the excessively large frequency change that occurs when the gold electrode is deposited in advance with Au nanoparticles. This clearly illustrates that the QCM detection of DNA hybridization is also affected greatly by the surface morphology of the electrode. These enlarged signals are altogether presumed to be advantageous when using a QCM system as an in situ probing device in DNA sensors.     

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.     

Mining Microarrays for Metabolic Meaning: Nutritional Regulation of Hypothalamic Gene Expression

DNA microarray analysis has been used to investigate relative changes in the level of gene expression in the CNS, including changes that are associated with disease, injury, psychiatric disorders, drug exposure or withdrawal, and memory formation. We have used oligonucleotide microarrays to identify hypothalamic genes that respond to nutritional manipulation. In addition to commonly used microarray analysis based on criteria such as fold-regulation, we have also found that simply carrying out multiple t tests then sorting by P value constitutes a highly reliable method to detect true regulation, as assessed by real-time polymerase chain reaction (PCR), even for relatively low abundance genes or relatively low magnitude of regulation. Such analyses directly suggested novel mechanisms that mediate effects of nutritional state on neuroendocrine function and are being used to identify regulated gene products that may elucidate the metabolic pathology of obese ob/ob, lean Vgf-/Vgf-, and other models with profound metabolic impairments.     

Role of primary sensorimotor cortex and supplementary motor area in volitional swallowing: a movement-related cortical potential study

We investigated the role of the cerebral cortex, particularly the face/tongue area of the primary sensorimotor (SMI) cortex (face/tongue) and supplementary motor area (SMA), in volitional swallowing by recording movement-related cortical potentials (MRCPs). MRCPs with swallowing and tongue protrusion were recorded from scalp electrodes in eight normal right-handed subjects and from implanted subdural electrodes in six epilepsy patients. The experiment by scalp EEG in normal subjects revealed that premovement Bereitschaftspotentials (BP) activity for swallowing was largest at the vertex and lateralized to either hemisphere in the central area. The experiment by epicortical EEG in patients confirmed that face/tongue SMI and SMA were commonly involved in swallowing and tongue protrusion with overlapping distribution and interindividual variability. BP amplitude showed no difference between swallowing and tongue movements, either at face/tongue SMI or at SMA, whereas postmovement potential (PMP) was significantly larger in tongue protrusion than in swallowing only at face/tongue SMI. BP occurred earlier in swallowing than in tongue protrusion. Comparison between face/tongue SMI and SMA did not show any difference with regard to BP and PMP amplitude or BP onset time in either task. The preparatory role of the cerebral cortex in swallowing was similar to that in tongue movement, except for earlier activation in swallowing. Postmovement processing of swallowing was lesser than that of tongue movement in face/tongue SMI; probably suggesting that the cerebral cortex does not play a significant role in postmovement processing of swallowing. SMA plays a supplementary role to face/tongue SMI both in swallowing and tongue movements.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).     

Mesenchymal stem/progenitor cells developed in cultures from UC blood

Background Whether umbilical cord blood (UCB) serves as a source of mesenchymal stem/progenitor cells (MSPC) is controversial. MSPC are the best candidates for cellular therapy of orthopedic skeletal tissues. In order to explore the possibility of UCB as a useful source of MSPC, we identified, expanded in culture, and characterized MSPC from UCB harvests on a large scale. Methods Mononuclear cells isolated from UCB harvests (n=411) were cultured in media supplemented with 10% FBS. MSPC-like cells cultured from each UCB harvest were expanded ex vivo by successive subcultivation. UCB harvests with a more than 1000-fold expanding capacity (n=9) were examined for surface Ag phenotypes and in vitro differentiation potentials into osteogenic, chondrogenic and adipogenic lineages. Results Ninety-five out of a total of 411 UCB units (23.1%) generated MSPC-like cells during cultivation. Nine UCB units (2.2%) yielded MSPC with more than 1000-fold expansion capacity. These cells positively expressed MSPC-related Ag, but did not express myeloid, histocompatibility or endothelial Ag. These cells also possessed multiple capacities for osteogenic, chondrogenic and adipogenic differentiation. Discussion Although the incidence of UCB harvests producing MSPC in culture was low, some of them showed a more than 1000-fold expanding capacity, which is enough in cell numbers to be an allogeneic source for cellular therapy. Our results may encourage the use of UCB as an attractive target for allogeneic cellular therapeutic options in tissue engineering.