Spatiotemporal heterogeneity of plankton communities in Lake Donghu, China, as revealed by PCR-denaturing gradient gel electrophoresis and its relation to biotic and abiotic factors

The 16S and 18S rRNA genes of planktonic organisms derived from five stations with nutrient gradients in Lake Donghu, China, were studied by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, and the relationships between the genetic diversity of the plankton community and biotic/abiotic factors are discussed. The concentrations of total nitrogen (TN), total phosphorus (TP), NH4-N and As were found to be significantly related (P < 0.05) to morphological composition of the plankton community. Both chemical and morphological analyses suggested that temporal heterogeneity was comparatively higher than spatial heterogeneity in Lake Donghu. Although the morphological composition was not identical to the DGGE fingerprints in characterizing habitat similarity, the two strongest eutrophic stations (I and II) were always initially grouped into one cluster. Canonical correspondence analysis suggested that the factors strongly correlated with the first two ordination axes were seasonally different. The concentrations of TN and TP and the densities of rotifers and crustaceans were generally the main factors related to the DGGE patterns of the plankton communities. The study suggested that genetic diversity as depicted by metagenomic techniques (such as PCR-DGGE fingerprinting) is a promising tool for ecological study of plankton communities and that such techniques are likely to play an increasingly important role in assessing the environmental conditions of aquatic habitats.

An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation

To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca2+](i)) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca2+](i) signal can be propagated to the neighboring cells. No [Ca2+](i) response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks. Published by Elsevier Ltd.

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Application of matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in determining arginine esterase from snake venom

Using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The homogeneities and molecular weights of three arginine esterases from snake venom, which possessing therapeutic use in myocardial infarction, were determined and compared, MALDI-TOF-MS is possessed of high accuracy, high sensitivity and rapidity. MALDI-TOF-MS and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can provide complementary and confirmatory results information. MALDI-TOF-MS can be directly used as an important method for the purification of snake venom complexes successfully.

Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement.

We studied the pattern of BCR involvement in 52 patients with chronic myeloid leukemia by Southern blotting. Of 33 Philadelphia (Ph)-positive patients, 30 had evidence of M-BCR rearrangement, two cases were difficult to interpret, and one clearly lacked evidence of M-BCR rearrangement. Of 19 Ph-negative patients, nine showed M-BCR rearrangement, nine showed no rearrangement, and one result was uncertain. We selected for more detailed study eight patients (three Ph-positive and five Ph-negative). Two of the Ph-positive patients, whose Southern blots were difficult to interpret, had rearranged bands when the BCR gene was studied by pulsed field gel electrophoresis (PFGE). Results of PFGE studies and in situ hybridization to metaphase chromosomes in the third Ph-positive patient, whose DNA clearly lacked M-BCR rearrangement on Southern analysis, were consistent with a breakpoint on chromosome 22 located 3' of all known exons of the BCR gene. However, mRNA studied with the polymerase chain reaction showed evidence of a classical b2-a2 linkage. The findings in this patient may be explained by an unusual genomic breakpoint downstream of the BCR gene associated with long range splicing that excluded all of the 3' BCR exons. Of the five patients with Ph-negative M-BCR non-rearranged CML studied by PFGE for BCR gene rearrangement, none had evidence of rearranged bands. We conclude that PFGE is a valuable adjunct to standard molecular techniques for the study of atypical cases of CML. Occasional patients with Ph-positive CML have breakpoints outside M-BCR. The BCR gene is probably not involved in patients with Ph-negative, M-BCR non-rearranged CML.

Pulsating tandem microbubble for localized and directional single-cell membrane poration.

The interaction of laser-generated tandem microbubble (maximum diameter of about 50  μm) with single (rat mammary carcinoma) cells is investigated in a 25-μm liquid layer. Antiphase and coupled oscillation of the tandem microbubble leads to the formation of alternating, directional microjets (with max microstreaming velocity of 10  m/s) and vortices (max vorticity of 350 000  s{-1}) in opposite directions. Localized and directional membrane poration (200 nm to 2  μm in pore size) can be produced by the tandem microbubble in an orientation and proximity-dependent manner, which is absent from a single oscillating microbubble of comparable size and at the same stand-off distance.