Expression of heat shock protein 70 in freshwater prawn Macrobrachium malcolmsonii (H. Milne Edwards) following exposure to Hg and Cu

Juveniles of freshwater prawn M. malcolmsonii were exposed to 1/6th concentration of LC50 of Hg and Cu for 48 hr. Sampling was done at 1 8, 12, 16, 24, 30, 36, 42 and 48 hr of exposure. Gill and hepatopancreas were dissected and subjected to one-dimensional electrophoresis. Western blotting was employed to determine the relative concentration of heat shock protein, hsp 70 (stress-70) in each sample. In the gill tissue of the prawn that had been exposed to Hg (0.024 mgHg 1(-1)), stress-70 was detected from the 1st hr till the 16th hr of exposure. But in the gills of Cu exposed prawn, synthesis of stress-70 appeared from the 1st hr till the 24th hr. Synthesis of hsp70 was not recorded after the 24th hr in the gills of exposed prawns. Synthesis of stress-70 was also found to be tissue-specific for both metals in this prawn. When the antibody probe raised against stress-70 was used, synthesis of stress-70 was not observed in hepatopancreas of prawns exposed to Hg or Cu, during the entire period of exposure of 48 hr. The expression of stress-70 in M. malcolmsonii following exposure to Hg and Cu is apparently only transient, and also a differential expression of stress-70 between gill and hepatopancreas was observed for both the metals.     

Control of fermenters – a review

Fermenter control has been an active area of research and has attracted more attention in recent years. This is due to the new developments in other related areas which can be exploited to overcome the inherent difficulties in fermenter control. Beginning with conventional regulatory control of operating variables such as temperature, pH and dissolved oxygen concentration, research in fermenter control has undergone significant changes including the recent neural network based approaches. The objective of the paper is to focus the attention of the researchers to the developments in the control of batch, fed-batch and continuous fermenters over the past few years.     

Molecular modeling of the human serotonin(1A) receptor: role of membrane cholesterol in ligand binding of the receptor

Serotonin(1A) receptors are important neurotransmitter receptors and belong to the superfamily of G-protein coupled receptors (GPCRs). Although it is an important drug target, the crystal structure of the serotonin(1A) receptor has not been solved yet. Earlier homology models of the serotonin(1A) receptor were generated using rhodopsin as a template. We have used two recent crystal structures of the human β(2)-adrenergic receptor, one of which shows specific cholesterol binding site(s), as templates to model the human serotonin(1A) receptor. Since the sequence similarity between the serotonin(1A) receptor and β(2)-adrenergic receptor is considerably higher than the similarity between the serotonin(1A) receptor and rhodopsin, our model is more reliable. Based on these templates, we generated models of the serotonin(1A) receptor in the absence and presence of cholesterol. The receptor model appears more compact in the presence of cholesterol. We validated the stability of 'compactness' using coarse-grain MD simulation. Importantly, all ligands exhibit higher binding energies when docked to the receptor in the presence of cholesterol, thereby implying that membrane cholesterol facilitates ligand binding to the serotonin(1A) receptor. To the best of our knowledge, this is one of the first reports in which lipid-specific receptor conformations have been modeled by homology modeling.     

The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail

Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1A receptor

The serotonin_1A (5-HT_1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT_1A receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT_1A receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT_1A receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT_1A receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT_1A receptor function.     

下游工艺开拓中如何系统地筛选离子交换层析介质

下游层析工艺开拓中,选择层析介质最重要的因素包括纯度、动力载量和凝胶的使用寿命。本文作者示范了介质的动力载量及其回收率的重要区别。     

The PNA-DNA hybrid I-motif: implications for sugar-sugar contacts in i-motif tetramerization

We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA–DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H⁺-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H⁺-C base pairs per heteroduplex. The imino protons are protected from D₂O exchange suggesting intercalation of the heteroduplexes as seen in DNA₄ i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA–DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1′–H1′ NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar–sugar interactions as seen in DNA₄ i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar–sugar contacts in i-motif tetramerization.     

Cation-mediated cross-linking in natural organic matter: a review

Interactions between cations and natural organic matter (NOM) are central for the stability of organic matter, formation of supramolecular NOM structure, formation of organo-mineral associations, soil aggregation and binding of organic contaminants. The effect of multivalent cations on environmental functionalities of NOM strongly depends on the relative importance between intramolecular complexation and intermolecular cross-linking, the degree of which will be determined by the spatial arrangement of the hydrophilic functional groups in NOM. This literature review seeks to evaluate the current state of the art regarding the relevance of intermolecular cross-links via bridges of multivalent cations. Cross-linking has been suggested to explain among others aggregate stability, retarded dissolved organic matter release, reduced organic matter (OM) solubility as well as increase in degree and nonlinearity of sorption or organic chemicals to NOM. Although the cross-linking mechanism has been suggested in numerous studies, it has not yet been verified directly. The dynamics of the intermolecular cross-links, their persistence as well as their interplay with OM and their influence on stability and bioavailability of organic chemicals is up to now unknown. The major challenge in this context is the development of a suitable combination of experimental and instrumental techniques and relating the results to molecular and physicochemical models on the basis of targeted combination of spectroscopic, molecular modelling and thermoanalytical methods.