Anti-idiotypic antibody mimics proteolytic function of parent antigen

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.     

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique^1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components^3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation^6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin α-subunit knockout (Tα^-/-) mice which lack rod signaling⁸.     

Modulation of P2X3 receptors by spider toxins

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.     

Use of inert gas jets to measure the forces required for mechanical gene transfection

Background

Abnormal blood glucose (BG) concentrations have been associated with increased morbidity and mortality in both critically ill adults and infants. Furthermore, hypoglycaemia and glycaemic variability have both been independently linked to mortality in these patients. Continuous Glucose Monitoring (CGM) devices have the potential to improve detection and diagnosis of these glycaemic abnormalities. However, sensor noise is a trade-off of the high measurement rate and must be managed effectively if CGMs are going to be used to monitor, diagnose and potentially help treat glycaemic abnormalities.

Aim

To develop a tool that will aid clinicians in identifying unusual CGM behaviour and highlight CGM data that potentially need to be interpreted with care.

Methods

CGM data and BG measurements from 50 infants at risk of hypoglycaemia were used. Unusual CGM measurements were classified using a stochastic model based on the kernel density method and historical CGM measurements from the cohort. CGM traces were colour coded with very unusual measurements coloured red, highlighting areas to be interpreted with care. A 5-fold validation of the model was Monte Carlo simulated 25 times to ensure an adequate model fit.

Results

The stochastic model was generated using ~67,000 CGM measurements, spread across the glycaemic range ~2-10?mmol/L. A 5-fold validation showed a good model fit: the model 80% confidence interval (CI) captured 83% of clinical CGM data, the model 90% CI captured 91% of clinical CGM data, and the model 99% CI captured 99% of clinical CGM data. Three patient examples show the stochastic classification method in use with 1) A stable, low variability patient which shows no unusual CGM measurements, 2) A patient with a very sudden, short hypoglycaemic event (classified as unusual), and, 3) A patient with very high, potentially un-physiological, glycaemic variability after day 3 of monitoring (classified as very unusual).

Conclusions

This study has produced a stochastic model and classification method capable of highlighting unusual CGM behaviour. This method has the potential to classify important glycaemic events (e.g. hypoglycaemia) as true clinical events or sensor noise, and to help identify possible sensor degradation. Colour coded CGM traces convey the information quickly and efficiently, while remaining computationally light enough to be used retrospectively or in real-time.     

Voltage Dependence of ATP Secretion in Mammalian Taste Cells

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca²⁺ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.     

The stability of enzyme activity and the content of microflora of different soils of the South of Russia to influence of a variable magnetic field of industrial frequency

In modelling experiments of the influence of variable magnetic field of industrial frequency (50 Hz) by induction of 1500 and 6000 mkTl during 5 days on microflora and on enzyme activity of soils the South of Russia of different genesis and properties is investigated.     

Different mechanisms of ion homeostasis are dominant in the recretohalophyte <Emphasis Type="Italic">Tamarix ramosissima</Emphasis> under different soil salinity

Total ion (Na⁺, K⁺, Ca²⁺, SO₄ ^2? and Cl^?) accumulation by plants, ion contents in plant tissues and ion secretion by salt glands on the surface of shoots of Tamarix ramosissima adapted to different soil salinity, namely low (0.06 mmol Na⁺/g soil), moderate (3.14–4.85 mmol Na⁺/g soil) and strong (7.56 mmol Na⁺/g soil) were analyzed. There are two stages of interrelated and complementary regulation of ion homeostasis in whole T. ramosissima plants: (1) regulation of ion influx into the plant from the soil and (2) changing the secretion efficiency of salt glands on shoots. The secretion efficiency of salt glands was appraised by the ratio of ion secretion to tissue ion content. Independent of soil salinity, the accumulation of K⁺ and Ca²⁺ was higher than the contents of these ions in the soil. Furthermore, the accumulation of K⁺, Ca²⁺ and SO₄ ^2? ions by plants was maintained within a narrow range of values. Under low soil salinity, Na⁺ was accumulated, whereas under moderate and strong salinity, the influxes of Na⁺ were limited. However, under strong salinity, the accumulation of Na⁺ was threefold higher than that under low soil salinity. This led to a change in the Na⁺/K⁺ ratio (tenfold), an increase in the activity of salt glands (tenfold) and a reduction in plant growth (fivefold). An apparently high Na⁺/K⁺ ratio was the main factor determining over-active functioning of salt glands under strong salinity. Principal component analysis showed that K⁺ ions played a key role in ion homeostasis at all levels of salinity. Ca²⁺ played a significant role at low salinity, whereas Cl^? and interrelated regulatory components (K⁺ and proline) played a role under strong salinity. Proline, despite its low concentration under strong salinity, was involved in the regulation of secretion by salt glands. Different stages and mechanisms of ion homeostasis were dominant in T. ramosissima plants adapted to different levels of salinity. These mechanisms facilitated the accumulation of Na⁺ in plants under low soil salinity, the limitation of Na⁺ under moderate salinity and the over-activation of Na⁺ secretion by salt glands under strong salinity, which are all necessary for maintaining ion homeostasis and water potential in the whole plant.