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PubMed2003

Purification, characterization, molecular cloning, and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae).

A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.

Amino Acid Sequence↗

PubMed2026

Chemotactic sensing of extracellular antibiotic resistance genes enables their efficient removal by Stutzerimonas stutzeri.

The dissemination of antibiotic resistance genes (ARGs) in wastewater environments poses a severe threat to public health. Extracellular ARGs (eARGs) persist as free DNA fragments that are refractory to efficient removal by conventional physicochemical treatment technologies. Here, we isolated Stutzerimonas stutzeri CHY07 from municipal sewage and demonstrated that extracellular DNA fragments, including eARGs, can serve as chemoattractants for environmental bacteria. Through genomic mining, molecular docking, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and protein-ligand interaction profiling, we identified the chemoreceptor Mcp16 as the primary sensor of extracellular DNA and revealed that it achieves sequence-independent recognition of the DNA phosphate backbone. We further established the endogenous pentapeptide VRSVR as a methylation substrate for CheR and constructed the engineered strain CHY07-2 (mcp16::VRSVR) using an SSB/CRISPR-Cas9 ribonucleoprotein (RNP) system. This strain exhibited significantly enhanced chemotactic responsiveness, achieving 72-h removal efficiencies of 96.56% and 91.60% for low- and high-molecular-weight eARGs in non-sterile WWTP secondary effluent; conversely, mcp16 deletion markedly attenuated both chemotaxis and removal, whereas in situ complementation restored them. These findings reveal a "chemotaxis-contact-removal" cascade - with a proposed self-reinforcing loop - in eARG-removing bacteria, providing both a theoretical framework and a technical paradigm for enhancing pollutant removal through targeted amplification of microbial chemotaxis.

Chemotaxis↗

PubMed1998

In-situ polymerase chain reaction: foundation of the technology and today's options.

"In situ PCR" is the marriage of two established technologies in molecular genetics, the polymerase chain reaction (PCR) and in situ hybridization (ISH). It is based on the amplification within intact cells or tissue sections of specific gene sequences, or mRNA species, to levels detectable by ISH and/or immunohistochemistry. Methods to achieve in situ PCR, while sharing fundamental steps, have differed between different laboratories. On the basis of our own experience, in situ PCR appears to be best suited for the detection of DNA in single cell preparations, in which fixation and pre-treatments can be optimally controlled. Emphasis is placed on the requirement for appropriate and meaningful controls at the multiple steps involved. It is instructive to the view the emergence of this new technology in perspective. In situ PCR has not developed in isolation and is just one of several creative approaches that have been employed in recent years to study nucleic acids (DNA and RNA) intracellularly. Some approaches are more suitable for detection of mRNA, or viral RNA, while others are more easily applied to chromosomal DNA. Some further techniques, such as the isothermal self-sustained sequence replication (3SR), refined in-situ transcription (PRINS), or high sensitivity histochemical detection systems, will complement or even add to the potential of situ PCR. It is highly probable that tests will emerge, based on investigation of unique genetic markers, with important roles in specialized diagnostic laboratories for the evaluation of viral diseases, as well as hematological and other malignancies.

Humans↗

PubMed2004

Homogeneous detection of single rolling circle replication products.

We describe a simple and straightforward approach for homogeneous and isothermal detection of individual rolling circle replication (RCR) products, which represent individual padlock probe circularization events. The RCR products constitute tens of kilobases long single-stranded tandem repeated copies of the probe sequence, and in solution, they fold into micrometer-sized random coils. The method is based on the local enrichment of fluorescence-labeled probes that hybridize to the coiled RCR products compared to the concentration of free probes in solution. We present a detailed characterization of the fluorescence-labeled products using a highly sensitive and fast microscopy setup. At a 10(4)-fold excess of free label, we were able to detect and follow individual RCR products at a signal-to-background noise ratio of 27. This high signal-to-background noise ratio leaves room for analysis in a simple detection device at higher speeds or at lower labeling ratios.

Animals↗

PubMed1994

Cellular and molecular characterization of Ca2+ currents in acutely isolated, adult rat neostriatal neurons.

Ca2+ currents in acutely isolated, adult rat neostriatal neurons were studied with whole-cell voltage-clamp techniques. In the vast majority of neurons (approximately 90%, n > 250), currents were exclusively of the high-voltage-activated (HVA) type. HVA currents activated near -40 mV and reached their maximum amplitude near 0 mV. Quasi-steady-state inactivation curves in many neurons were well fitted only with a sum of Boltzmann functions, suggesting that the HVA currents were heterogeneous. Although the block of whole-cell current by Cd2+ was well fitted with a single isotherm having an IC50 of near 1 microM, experiments with organic channel antagonists suggested that at least four types of HVA channels were expressed by most cells. On average, the L-channel antagonist nifedipine (5-10 microM) blocked 31 +/- 10% of the whole-cell current (n = 20), the N-channel antagonist omega-conotoxin GVIA (omega-CgTx) (2-5 microM) blocked 27 +/- 11% (n = 20), and the P-channel antagonist omega-agatoxin IVA (100-500 nM) blocked 21 +/- 10% (n = 18). In many neurons, the block by omega-CgTx was partially or completely reversible. In cells tested with a combination of these antagonists, 34 +/- 17% of the peak Ca2+ current remained unblocked (n = 13). Single-cell expression profiling of medium-sized neurons revealed the presence of rbA and rbB Ca2+ channel alpha 1 subunit mRNAs but low or undetectable levels of rbC mRNA (n = 12). These findings suggest that although adult neostriatal projection neurons do not express significant levels of LVA Ca2+ current, they do express a pharmacologically and structurally heterogeneous population of HVA currents.

Animals↗