Development of technologies aiding large-tissue engineering

Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.     

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.     

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

Maternal Exposure to Dibutyl Phthalate (DBP) or Diethylstilbestrol (DES) Leads to Long-Term Changes in Hypothalamic Gene Expression and Sexual Behavior

Xenobiotic exposure during pregnancy and lactation has been linked to perinatal changes in male reproductive outcomes and other endocrine parameters. This pilot study wished to assess whether brief maternal exposure of rats to xenobiotics dibutyl phthalate (DBP) or diethylstilbestrol (DES) might also cause long-term changes in hypothalamic gene expression or in reproductive behavior of the resulting offspring. Time-mated female Sprague Dawley rats were given either DBP (500 mg/kg body weight, every second day from GD14.5 to PND6), DES (125 µg/kg body weight at GD14.5 and GD16.5 only), or vehicle (n = 8–12 per group) and mild endocrine disruption was confirmed by monitoring postnatal anogenital distance. Hypothalamic RNA from male and female offspring at PND10, PND24 and PND90 was analyzed by qRT-PCR for expression of aromatase, oxytocin, vasopressin, ER-alpha, ER-beta, kisspeptin, and GnRH genes. Reproductive behavior was monitored in male and female offspring from PND60 to PND90. Particularly, DES treatment led to significant changes in hypothalamic gene expression, which for the oxytocin gene was still evident at PND90, as well as in sexual behavior. In conclusion, maternal xenobiotic exposure may not only alter endocrine systems in offspring but, by impacting on brain development at a critical time, can have long-term effects on male or female sexual behavior.     

Catalytic oxidation of methanol over molybdenum oxide–tungsten oxide

The vapour phase oxidation of methanol to formaldehyde over molybdenum oxide, tungsten oxide and their mixtures has been investigated in an integral flow reactor at atmospheric pressure. The effect of several process variables on the conversion and yield were determined. A high conversion of 95.6% methanol with nearly 95% selectivity was obtained at 430°C. A rate expression has been derived from the kinetics of the reaction.     

In-Plane Cracking Behavior and Ultimate Strength for 2D Woven and Braided Melt-Infiltrated SiC/SiC Composites Tensile Loaded in Off-Axis Fiber Directions

The tensile mechanical properties of ceramic matrix composites (CMC) in directions off the primary axes of the reinforcing fibers are important for the architectural design of CMC components that are subjected to multiaxial stress states. In this study, two-dimensional (2D)-woven melt-infiltrated (MI) SiC/SiC composite panels with balanced fiber content in the 0° and 90° directions were tensile loaded in-plane in the 0° direction and at 45° to this direction. In addition, a 2D triaxially braided MI SiC/SiC composite panel with a higher fiber content in the ±67° bias directions compared with the axial direction was tensile loaded perpendicular to the axial direction tows (i.e., 23° from the bias fibers). Stress–strain behavior, acoustic emission, and optical microscopy were used to quantify stress-dependent matrix cracking and ultimate strength in the panels. It was observed that both off-axis-loaded panels displayed higher composite onset stresses for through-thickness matrix cracking than the 2D-woven 0/90 panels loaded in the primary 0° direction. These improvements for off-axis cracking strength can in part be attributed to higher effective fiber fractions in the loading direction, which in turn reduces internal stresses on weak regions in the architecture, e.g., minicomposite tows oriented normal to the loading direction and/or critical flaws in the matrix for a given composite stress. Both off-axis-oriented panels also showed relatively good ultimate tensile strength when compared with other off-axis-oriented composites in the literature, both on an absolute strength basis as well as when normalized by the average fiber strength within the composites. Initial implications are discussed for constituent and architecture design to improve the directional cracking of SiC/SiC CMC components with MI matrices.     

Dynamic gas disengagement: A new technique for assessing the behaviour of bubble columns

A precise measure of the liquid motion and distribution of bubble sizes is essential for designing gas-liquid reactors for complex reactions in which product distribution may be a function of bubble size. A theoretical approach is presented which shows how the interaction of bubble size distribution and bubble rise velocity functions leads to predictions of the overall steady state hold-up in a bubble column within which the liquid flow is understood. Since this approach is based on a physical understanding of how bubble flow at a given superficial velocity must relate to the static hold-up, the theory can be immediately extended to describe the disengagement of gas bubbles if the gas feed is cut off. Thus the dynamic gas hold-up during gas disengagement can be used to provide new insights into the fundamentals of bubble column behaviour. In this way it becomes possible in principle to inter-relate macroscopic properties such as surface area, gas phase residence time distribution and intensity of mixing in the liquid phase.A new experimental technique is described which measures the dynamic gas hold-up during gas disengagement Experimental results at a nominal 20 mm/s gas superficial velocity are compared with various approaches based upon the theory. The effects of the accompanying induced liquid movements are represented by a simplified core-annulus circulation model. Bubble size distributions and liquid circulation can then be related to both the static and dynamic hold-up behaviour. It is shown that ambiguity due to uncertainty about the relative differences in bubble size distribution in upflow and downflow regions can be resolved from a knowledge of the surface area.     

Fluid catalytic cracking: some recent developments in catalyst particle design and unit hardware

Catalytic cracking is an old process, but is still subject to new pressures for change and improvement. In the 1990s, these pressures arise from the need to crack components drawn from deeper down the barrel as well as to reformulate fuel products which can meet more stringent environmental requirements. Recent efforts at correlating catalyst formulation and cracking performance still rely on largely empirical understanding of the role of catalyst composition and zeolite/matrix pore architecture. However, new tests measuring cracking performance at constant surface area as the zeolite/ matrix ratio varies have indicated that the pore architecture should be designed to facilitate a staged cracking. The whole range of pore sizes from micro-pores (< 20 Å) to giant macro-pores (1000 Å) are now known to be influential in determining cracking yield and activity and these pores should be assembled into an optimal architecture. Unfortunately, the configuration of pores in a typical spray dried FCC catalyst is usually far from such an optimal design. The use of stochastic pore networks to characterise macro-pore structures is described in conjunction with some experimental results employing a visualised porosimetry based on low melting point alloy impregnation. Improved designs of catalyst will impose new requirements on the fluidised bed hardware. Some recent improvements in the chemical engineering of riser reactor/regenerator units are described which centre on atomised injection, segregated feeding, closed cyclones, staged stripping and catalyst cooling.