The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate, or H2O2 alone. Employing Western blotting with anti-P-Tyr antibodies, we have identified in Fao cells at least four proteins (pp180, 150, 114, and 100) whose P-Tyr content is rapidly increased upon treatment of the cells with 3 mM H2O2. Tyrosine phosphorylation of these and additional proteins was markedly potentiated (6-10-fold) when 100 microM sodium orthovanadate was added together with H2O2. The effects of H2O2 and vanadate on protein tyrosine phosphorylation were rapid and specific. The enhanced tyrosine phosphorylation was accompanied by a concomitant inhibition of a cytosolic protein tyrosine phosphatase activity. The latter was inhibited by 50% in 3 mM H2O2-treated cells. The inhibitory effect was augmented in the combined presence of H2O2 and vanadate. Half- and maximal effects of vanadate were obtained at 15 microM and 1 mM, respectively. Vanadate (1 mM) alone, added to the cells, had only a trivial effect on protein tyrosine phosphatase activity. A 45-s challenge with insulin (10(-7) M) of cells pretreated with H2O2 largely mimicked the potentiating effects of vanadate on protein tyrosine phosphorylation but not on protein tyrosine phosphatase activity. Our results suggest the involvement of multiple tyrosine-phosphorylation proteins in mediating the biological effects of H2O2/vanadate. Their enhanced phosphorylation can be attributed at least in part, to the inhibitory effects exerted by H2O2 alone, or in combination with vanadate, on protein tyrosine phosphatase activity. The similarity between proteins phosphorylated in Fao cells in response to H2O2/vanadate or H2O2/insulin, suggests that either treatment stimulates protein tyrosine kinases having similar substrate specificities. The insulin receptor kinase is a likely candidate as its activity is markedly enhanced either by insulin (plus H2O2) or by H2O2/vanadate.     

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.      

A New Method of Assessing Lipid Mixtures by 31P Magic-Angle Spinning NMR

A variety of lipids that differ by their chains and headgroups are found in biomembranes. In addition to studying the overall membrane phase, determination of the structure, dynamics, and headgroup conformation of individual lipids in the mixture would be of great interest. We have thus developed, to our knowledge, a new approach using solid-state ³¹P NMR, magic-angle spinning, and chemical-shift anisotropy (CSA) recoupling, using an altered version of the recoupling of chemical shift anisotropy (ROCSA) pulse sequence, here penned PROCSA. The resulting two-dimensional spectra allowed the simultaneous measurement of the isotropic chemical shift and CSA of each lipid headgroup, thus providing a valuable measure of its dynamics and structure. PROCSA was applied to mixtures of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) in various relative proportions, to mimic bacterial membranes and assess the respective roles of lipids in shaping these bilayers. The results were interpreted in terms of membrane topology, lipid propensity to adopt various phases or conformations, and lipid-lipid miscibility. Our results showed that PG dictates the lipid behavior when present in a proportion of 20 mol % or more. A small proportion of PG is thus able to impose a bilayer structure to the hexagonal phase forming PE. We discuss the requirement for lipids, such as PE, to be able to adopt non-bilayer phases in a membrane.     

Whole cell solid-state NMR study of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> microalgae

In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically ¹³C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically ¹³C labelled micro-organisms.     

Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor.

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.     

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HC_ABD/LC_ABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LC_ABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.     

Experimental and observational studies of radiolarian physiological ecology: 2. Trophic activity and symbiont primary productivity ofSpongaster tetras tetras with comparative data on predatory activity of some Nassellarida

The prey ofSpongaster tetras tetras, determined by transmission electron microscopic examination of ultrathin sections through food and digestive vacuoles, was predominantly picoplankton (1 to 3 μm, largely monerans) and only occasional vacuoles contained masses of larger digested organic matter. By contrast, Nassellarian specimens collected in the same location and fixed and examined identically contained nanoplankton prey (5–10 μm) including small phytoplankton cells. The differences in prey may reduce competition for nutritional resources and account for the co-existence of these two groups of radiolaria in the same water mass. The primary productivity of the symbionts in intactS. tetras tetras was assessed at varying light levels to more fully document potential sources of carbon compounds for nutrition. Thirty-five μg of carbon were fixed per radiolarian per hour at an intensity as low as 20 μE/m²/s which is approximately one-third the maximum productivity in the intensity range of 170 to 260 μE/m²/s. The mean abundance ofS. tetras tetras, density of potential prey, and physico-chemical characteristics of the water are presented for early spring (March–April), mid-summer (June and July), and late summer (August). The mean density ofS. tetras tetras varied from c. 12 individuals/m³ during the low productivity periods (spring and late summer) to 37.8 individuals/m³ in July. The concentration of cyanobacteria was at a peak during July while larger autotrophic plankton was at a minimun further suggesting that cyanobacteria can be a significant food source forS. tetras tetras.     

Neuronal growth via hybrid system of self-growing and diffusion based grammar rules: I

The formation of neuronal networks requires axonal growth towards target neutons. A simple set of grammar rules is introduced to describe axonal growth towards target cells situated both at short and long distances from the growing neuron. Growth for short distances is descrbed by growth following the highest gradient of a chemical compound (which is spread by diffusion from the targets). This approach fails to describe long-distance growth, which is addressed by adopting a graph grammar theory for growing trees. With these rules a flexible tool to draw network of neurons by computer can be developed.     

The MORPH Algorithm: Ranking Candidate Genes for Membership in Arabidopsis and Tomato Pathways

Closing gaps in our current knowledge about biological pathways is a fundamental challenge. The development of novel computational methods along with high-throughput experimental data carries the promise to help in the challenge. We present an algorithm called MORPH (for module-guided ranking of candidate pathway genes) for revealing unknown genes in biological pathways. The method receives as input a set of known genes from the target pathway, a collection of expression profiles, and interaction and metabolic networks. Using machine learning techniques, MORPH selects the best combination of data and analysis method and outputs a ranking of candidate genes predicted to belong to the target pathway. We tested MORPH on 230 known pathways in Arabidopsis thaliana and 93 known pathways in tomato (Solanum lycopersicum) and obtained high-quality cross-validation results. In the photosynthesis light reactions, homogalacturonan biosynthesis, and chlorophyll biosynthetic pathways of Arabidopsis, genes ranked highly by MORPH were recently verified to be associated with these pathways. MORPH candidates ranked for the carotenoid pathway from Arabidopsis and tomato are derived from pathways that compete for common precursors or from pathways that are coregulated with or regulate the carotenoid biosynthetic pathway.     

Regulation of output from the plant circadian clock

Plants, like many other organisms, have endogenous biological clocks that enable them to organize their physiological, metabolic and developmental processes so that they occur at optimal times. The best studied of these biological clocks are the circadian systems that regulate daily (approximately 24 h) rhythms. At the core of the circadian system in every organism are oscillators responsible for generating circadian rhythms. These oscillators can be entrained (set) by cues from the environment, such as daily changes in light and temperature. Completing the circadian clock model are the output pathways that provide a link between the oscillator and the various biological processes whose rhythms it controls. Over the past few years there has been a tremendous increase in our understanding of the mechanisms of the oscillator and entrainment pathways in plants and many useful reviews on the subject. In this review we focus on the output pathways by which the oscillator regulates rhythmic plant processes. In the first part of the review we describe the role of the circadian system in regulation at all stages of a plant's development, from germination and growth to reproductive development as well as in multiple cellular processes. Indeed, the importance of a circadian clock for plants can be gauged by the fact that so many facets of plant development are under its control. In the second part of the review we describe what is known about the mechanisms by which the circadian system regulates these output processes.