γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.     

Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: D_eff, the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. D_eff for PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability.     

Downstream processing in the biotechnology industry

The biotechnology industry today employs recombinant bacteria, mammalian cells, and transgenic animals for the production of high-value therapeutic proteins. This article reviews the techniques employed in this industry for the recovery of these products. The methods reviewed extend from the centrifugation and membrane filtration for the initial clarification of crude culture media to the final purification of the products by a variety of membrane-based and chromatographic methods. The subject of process validation including validation of the removal of bacterial and viral contaminants from the final products is also discussed with special reference to the latest regulatory guidelines.     

Curcumin modulates free radical quenching in myocardial ischaemia in rats

This study was designed to investigate the protective effect of curcumin (CUR) against isoprenaline induced myocardial ischaemia in rat myocardium. The effect of single oral dose of curcumin (15 mg kg(-1)), administered 30 min before and/or after the onset of ischaemia, was investigated by assessing oxidative stress related biochemical parameters in rat myocardium. Curcumin pre and post-treatment (PPT) was shown to decrease the levels of xanthine oxidase, superoxide anion, lipid peroxides (LPs) and myeloperoxidase while the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities were significantly increased after curcumin PPT. Histopathological and transmission electron microscopical studies also confirmed the severe myocardial damage occurring as a consequence of isoprenaline induced ischaemia and they also showed the significant improvement effected by curcumin PPT. These findings provided evidence that curcumin was found to protect rat myocardium against ischaemic insult and the protective effect could be attributed to its antioxidant properties as well as its inhibitory effects on xanthine dehydrogenase/xanthine oxidase (XD/XO) conversion and resultant superoxide anion production.     

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid

Damage-associated molecular pattern molecules (DAMPs) signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3) is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i) MAPK activation, ii) defense-related gene expression, iii) callose deposition, and iv) enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast). Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA), which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.     

Molecular characterization of a two-domain form of the neuronal voltage-gated P/Q-type calcium channel alpha(1)2.1 subunit

We characterized the neuronal two-domain (95kD-alpha(1)2.1) form of the alpha(1)2.1 subunit of the voltage-gated calcium channels using genetic and molecular analysis. The 95kD-alpha(1)2.1 is absent in neuronal preparations from CACNA1A null mouse demonstrating that alpha(1)2.1 and 95kD-alpha(1)2.1 arise from the same gene. A recombinant two-domain form (alpha(1AI-II)) of alpha(1)2.1 associates with the beta subunit and is trafficked to the plasma membrane. Translocation of the alpha(1AI-II) to the plasma membrane requires association with the beta subunit, since a mutation in the alpha(1AI-II) that inhibits beta subunit association reduces membrane trafficking. Though the alpha(1AI-II) protein does not conduct any voltage-gated currents, we have previously shown that it generates a high density of non-linear charge movements [Ahern et al., Proc. Natl. Acad. Sci. USA 98 (2001) 6935-6940]. In this study, we demonstrate that co-expression of the alpha(1AI-II) significantly reduces the current amplitude of alpha(1)2.1/beta(1a)/alpha(2)delta channels, via competition for the beta subunit. Taken together, our results demonstrate a dual functional role for the alpha(1AI-II) protein, both as a voltage sensor and modulator of P/Q-type currents in recombinant systems. These studies suggest an in vivo role for the 95kD-alpha(1)2.1 in altering synaptic activity via protein-protein interactions and/or regulation of P/Q-type currents.