Particulate cyclic 3',5'-nucleotide phosphodiesterase and calmodulin of cardiac muscle

Cyclic AMP and cyclic GMP phosphodiesterase and calmodulin were measured in purified subcellular fractions of cardiac muscle. Phosphodiesterase activity solubilized by sonication of the nuclear fraction yielded a major 6.6 S form which was calcium-sensitive and cyclic GMP-specific. Phosphodiesterase activity occurring in the nuclear fraction could be further enriched by subfractionation on sucrose density gradients in the presence of MgCl2.     

Precambrian–Cambrian boundary interval occurrence and form of the enigmatic tubular body fossil Shaanxilithes ningqiangensis from the Lesser Himalaya of India

The affinity of the Ediacaran fossil Shaanxilithes ningqiangensis and putatively related forms has long been enigmatic; over the past few decades, interpretations ranging from trace fossils to algae to metazoans of uncertain phylogenetic placement have been proposed. Combined morphological and geochemical evidence from a new occurrence of S. ningqiangensis in the Krol and Tal groups of the Lesser Himalaya of India indicates that S. ningqiangensis is not a trace fossil, but rather an organic‐walled tubular body fossil of unknown taxonomic affinity. Specimens consist of compressed organic cylindrical structures, characterized by extended, overlapping or fragmented iterated units. Where specimens intersect, overlapping rather than branching or intraplanar crossing is observed. Lithologic comparisons and sequence stratigraphic data all suggest a late Ediacaran age for the uppermost Krol Group and basalmost Tal Group. By extending the biogeographical distribution of S. ningqiangensis, hitherto confined to the Ediacaran of China and potentially Siberia, to the Precambrian–Cambrian boundary interval of India, this new occurrence of S. ningqiangensis expands the biostratigraphic utility of this enigmatic fossil to the inter‐regional and intercontinental scale. Moreover, study of these new and exceptionally preserved samples may help to significantly constrain the long‐debated problem of Shaanxilithes' affinity, elucidating its ‘problematic’ status and shedding new light upon the ecology and taphonomy of one of the most significant intervals in early life history.     

C-type natriuretic peptide: new candidate for endothelium-derived hyperpolarising factor

Endothelium-derived hyperpolarising factor (EDHF) is an important regulator of vascular tone; however, its identity is still unclear. Several different molecules have been suggested, the most recent of which is the 22-amino acid peptide C-type natriuretic peptide (CNP). CNP induces hyperpolarisation and relaxation of rat mesenteric resistance artery vascular smooth muscle through activation of natriuretic peptide receptor subtype C (NPR-C) and the same potassium channels as EDHF. In addition, this peptide is released from endothelial cells of the perfused rat mesenteric bed in response to endothelium-dependent vasodilators. Thus, CNP is likely to play a vital role in regulation of vascular tone. In addition, since there is evidence that up-regulation of EDHF occurs where normal endothelium function has been compromised, modulation of this pathway represents a novel target for therapeutics in the treatment of inflammatory cardiovascular pathologies characterised by endothelial dysfunction.     

A low shear stress modular bioreactor for connected cell culture under high flow rates

A generic “system on a plate” modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using “milli‐molding,” a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers can increase or maintain cell viability and function when the flow rates are below 500 µL/min, corresponding to wall shear stresses of 10^?5 Pa or less at the cell culture surface. Biotechnol. Bioeng. 2010; 106: 127–137. © 2010 Wiley Periodicals, Inc.     

Variation in Trans-3′-Hydroxycotinine Glucuronidation Does Not Alter the Nicotine Metabolite Ratio or Nicotine Intake

Background

CYP2A6 metabolizes nicotine to its primary metabolite cotinine and also mediates the metabolism of cotinine to trans-3′-hydroxycotinine (3HC). The ratio of 3HC to cotinine (the “nicotine metabolite ratio”, NMR) is an in vivo marker for the rate of CYP2A6 mediated nicotine metabolism, and total nicotine clearance, and has been associated with differences in numerous smoking behaviors. The clearance of 3HC, which affects the NMR, occurs via renal excretion and metabolism by UGT2B17, and possibly UGT2B10, to 3HC-glucuronide. We investigated whether slower 3HC glucuronidation alters NMR, altering its ability to predict CYP2A6 activity and reducing its clinical utility.

Methods

Plasma NMR, three urinary NMRs, three urinary 3HC glucuronidation phenotypes and total nicotine equivalents were examined in 540 African American smokers. The UGT2B17 gene deletion and UGT2B10*2 were genotyped.

Results

The UGT2B17 gene deletion, but not UGT2B10*2 genotype, was associated with slower 3HC glucuronidation (indicated by three 3HC-glucuronidation phenotypes), indicating its role in this glucuronidation pathway. However, neither lower rates of 3HC glucuronidation, nor the presence of a UGT2B17 and UGT2B10 reduced function allele, altered plasma or urinary NMRs or levels of smoking.

Conclusions

Variation in 3HC glucuronidation activity, including these caused by UGT2B17 gene deletions, did not significantly alter NMR and is therefore unlikely to affect the clinical utility of NMR in smoking behavior and cessation studies. This study demonstrates that NMR is not altered by differences in the rate of 3HC glucuronidation, providing further support that NMR is a reliable indicator of CYP2A6 mediated nicotine metabolism.     

Growth inhibitory activity of Millettia pachycarpa (Bentham) extracts against Tobacco Caterpillar,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Abstract

Millettia pachycarpa (Bentham), a perennial climbing tree, found endemic in north-east India used by tribes for treating various ailments. To ascertain the potential of M. pachycarpa for pest management, the present investigation was formulated against a polyphagous pest, Spodoptera litura (Fabricius). Growth inhibitory activity of various extracts and extract fractions was evaluated against third instar larvae of S. litura through topical as well as diet incorporation method. It was observed that dichloromethane extract showed very good growth inhibition in both topical (GI₅₀ 7.77?ppm) and diet incorporation methods (GI₅₀< 5?ppm). The growth inhibitory potential of this plant is also demonstrated by the presence of various abnormalities and reduction in larval and pupal weight.     

An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor

The energy balance in vivo is maintained through inter-organ cross-talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two-way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three-way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L-alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.     

The impact of fabrication parameters and substrate stiffness in direct writing of living constructs

Biomolecules and living cells can be printed in high‐resolution patterns to fabricate living constructs for tissue engineering. To evaluate the impact of processing cells with rapid prototyping (RP) methods, we modeled the printing phase of two RP systems that use biomaterial inks containing living cells: a high‐resolution inkjet system (BioJet) and a lower‐resolution nozzle‐based contact printing system (PAM²). In the first fabrication method, we reasoned that cell damage occurs principally during drop collision on the printing surface, in the second we hypothesize that shear stresses act on cells during extrusion (within the printing nozzle). The two cases were modeled changing the printing conditions: biomaterial substrate stiffness and volumetric flow rate, respectively, in BioJet and PAM². Results show that during inkjet printing impact energies of about 10^?8 J are transmitted to cells, whereas extrusion energies of the order of 10^?11 J are exerted in direct printing. Viability tests of printed cells can be related to those numerical simulations, suggesting a threshold energy of 10^?9 J to avoid permanent cell damage. To obtain well‐defined living constructs, a combination of these methods is proposed for the fabrication of scaffolds with controlled 3D architecture and spatial distribution of biomolecules and cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

A Novel Method to Produce Immobilised Biomolecular Concentration Gradients to Study Cell Activities: Design and Modelling

It is well known that many cell functions are activated by chemical signals with a time and space-dependent profile. To mimic these profiles in vitro, it is necessary to develop a system that is able to generate concentration gradients with a resolution similar to that perceived by cells, which is around nanomolar with a spatial resolution of a few tens of microns. Many devices capable of generating steady-state concentration gradients have been developed using continuous flow micro-fluidic techniques. However, these systems cannot reproduce the immobilised concentration gradients that are present in the extracellular matrix. For this reason, we have developed a new gradient generator to enable precise and reproducible studies on the effects of immobilised concentration gradients on cell behaviour. A well-known gradient of a desired molecule was generated on the bottom surface of a hydrogel, which was then used as a stamp to immobilise the molecule on a functionalised substrate. A concentration gradient was thus obtained using a simple silane-based chemical reaction. To validate the method, image analysis was performed on glass slides printed with fluorescein isothiocyanate (FITC)- collagen and FITC-poly-lysine concentration gradients. Preliminary cell adhesion tests were also carried out by seeding NIH-3T3 and mesencephalic cells on lab-glass slides printed with concentration profiles of collagen and poly-lysine, respectively.