RP-HPLC binding domains of proteins

Procedures have been developed to identify the chromatographic binding domains of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) during their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained when these proteins are adsorbed to the chromatographic sorbent revealed significant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific regions on the surface of both Cyt c and bGH are inaccessible to tryptic cleavage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorbent. Based on the assumption that the region of the protein surface that is in contact with the sorbent remains intact and bound to the sorbent during the digestion step, while the protein surface that is exposed to the solvent is accessible to proteolysis, the regions that were inaccessible to tryptic digestion were found to correspond to hydrophobic domains on the protein surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface.     

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.     

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Thrombolytic therapy in Missouri hospital emergency departments: compliance with the National Heart Attack Alert Program guidelines

Adhesive interactions between leukocytes and endothelium are required for subsequent leukocyte extravasation toward inflammatory sites. Understanding the possible kinetic expression of vascular cell adhesion molecule-1 (VCAM-1) in the middle ear cavity during an inflammatory cascade in vivo may be important for clarifying local immunological responses in otitis media. Two inflammatory models were produced in the rat and involved acute middle ear mucosal and cutaneous inflammation induced after inoculation or intradermal injection of lipopolysaccharide (LPS). After intravenous injection of both 125I-labeled anti-VCAM-1 and 131I-labeled control monoclonal antibody (mAb), the kinetic expression of VCAM-1 in the middle ear and skin was assessed by local radionuclide uptake. The biodistribution of an 125I-labeled anti-VCAM-1 mAb as a potential detector of focal inflammation was examined in normal rats. Both inflammatory lesions were characterized by early and sustained (up to 24 h) expression of VCAM-1, with maximal expression at 4 h after LPS stimulation. The kinetics of VCAM-1 expression was similar among the middle ear mucosa or skin specimens studied and different stimulation methods. A similar biodistribution and clearance of radioactivity between 125I-labeled anti-VCAM-1 mAb and 131I- or 99mTc-labeled control mAb were observed. The present result suggest that functional VCAM-1 induced by LPS is expressed in both middle ear tissue and skin lesions and may play a role in the initial stage of inflammatory response produced. Although VCAM-1 upregulation is a very early event in the inflammatory cascade, 125I-labeled anti-VCAM-1 mAb may be useful for the early detection of focal inflammation in the middle ear.     

A glutamate-sensitizing activity in conditioned media derived from rat cerebellar granule cells

When cerebellar granule cells that had been cultured in vitro for 8 days were subjected to a cytotoxic glutamate pulse (100 microM, 30 min incubation), the response varied according to cell density and the volume of medium in which cells were grown. Thus, lowering the cell density by a factor of 4 compared with usual conditions (2.6 x 10(5) cells/cm2) or increasing the volume by an identical 4-fold factor reduced cell death from 90-95% to 20-30%. Addition of a conditioned medium derived from high-density to low-density cultures or to high-volume cultures markedly increased the sensitivity of the cells to glutamate. This glutamate-sensitizing activity, which accelerated by several days the onset of the response of cerebellar cultures to glutamate, was inhibited by actinomycin D and was not detectable in conditioned medium derived from confluent cultures of cerebellar astroglia, or from cell lines such as PC12, GT1-7, 3T3 and CHP 100. Glutamate-sensitizing activity was not mimicked by trilodo-L-thyronine, insulin-like growth factor-I (IGF-I), truncated IGF-I, GPE [a tripeptide (gly-pro-glu) derived from IGF-I], brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor or tumour necrosis factor-alpha. However, IGF-I added to cultures of granule cells plated at high density and grown in basal medium Eagle's without serum or any other constituent of chemically defined media was capable of supporting production of glutamate-sensitizing activity to an extent similar to that shown by whole fetal calf serum. Under the same conditions triiodo-L-thyronine and BDNF did not support the production of glutamate-sensitizing activity. Glutamate-sensitizing activity was not mimicked by glutamate, NMDA, glycine or lactate, and was not inhibited by glucose, haemoglobin or N-omega-nitro-L-arginine methyl ester. At variance with the response of granule cells, the response to glutamate of GABAergic cells present in the same culture was not affected by cell density or by glutamate-sensitizing activity.     

Growth kinetics and treatment response of the intracerebral rat 9L brain tumor model: a quantitative in vivo study using magnetic resonance imaging

We report the use of magnetic resonance imaging (MRI) for in situ tumor growth rate studies of experimental intracranial 9L tumors. T2-weighted spin-echo coronal magnetic resonance images of rat brains with 9L tumors were obtained every 2 days beginning at 8-11 days postimplantation using a 7 tesla MRI system. Tumors were clearly delineated in the images as a hyperintense region with a relatively well-demarcated border and minimal peritumoral edema. Tumor volumes from individual slices were summed together to yield the total tumor volume. The accuracy of this methodology for volumetric determination was verified by MRI phantom studies. Tumor growth rates determined from sequential MRI measurements of tumor volumes were quantitated in terms of volumetric doubling time. Tumor doubling times were found to range from 50 to 81 h, with an average of 66 +/- 8 h (n = 10). Intracranial 9L tumors were found to grow exponentially over the entire life span of the animal, allowing treated animals to serve as their own controls since the volumetric doubling time could be determined from three to four MRI scans before treatment administration. The intracerebral tumor growth delay following a single injection of 1, 3-bis(2-chloroethyl)-1-nitrosourea (13.3 mg/kg i.p.) allowed for noninvasive determination of in vivo log cell kill. A 2.0 +/- 0.2 (n = 3) log cell kill from 1,3-bis(2-chloroethyl)-1-nitrosourea treatment was found from post-treatment MRI volume measurements. These results demonstrate that MRI provides a powerful and sensitive method for assessing the growth and treatment response of intracranial 9L tumors in the rat.     

Electrooxidation of CO and CO/H2 mixtures on a Pt-Sn catalyst prepared by an implantation method

A new type of ion implantation technique is used to create a non-equilibrium Pt-Sn(IMP) near-surface alloy with ca. 8.6 at% Sn. The surface composition was determined by low-energy ion-scattering (LEIS). The kinetics of the electrooxidation of CO and 2% CO/H₂ mixtures on Pt-Sn(IMP) is essentially identical to that of Pt₃Sn(110). The fact that any non-equilibrium composition can be easily prepared by this implantation method opens an interesting practical opportunity to create a new Pt-Sn alloy fuel cell catalyst having an otherwise unobtainable surface composition of Sn. This method also appears to have general utility in alloy catalysis as a means of exploring compositions in thermodynamically unfavorable regions of the bulk phase diagram. This revised version was published online in July 2006 with corrections to the Cover Date.     

A kinetic and surface study of the thermal decomposition of cellulose powder in inert and oxidizing atmospheres

The thermal decomposition of fibrous cellulose powder from 275° to 340°C has been studied by thermogravimetry, scanning electron microscopy, krypton adsorption, and gas-chromatographic analysis of the gaseous products arising from pyrolysis in various oxidizing and inert atmospheres. The reaction kinetics fit a phase boundary model where the rate is controlled by the movement of an interface through a cylindrical particle and the principal kinetic parameters fit a compensation curve described previously for the decomposition of wood products. An explanation of the physical mechanism of pyrolysis is proposed which is consistent with the observed rate data and the structural changes observed by scanning electron microscopy.     

Reactive Laser Ablation Synthesis of Nanosize Alumina Powder

An aluminum (Al) target was laser ablated in an oxygen (O₂) atmosphere, producing nanosize alumina (Al₂O₃) powder. The powder surface area decreased (and the particle size increased) with both increasing oxygen pressure and laser fluence. All powders produced had surface areas between 135 and 250 m²/g, corresponding to primary particle sizes ranging from 7 to 3 nm in radius. Phase evolution with temperature was studied via X-ray diffraction. These powders showed a direct transformation from γ- to α-alumina at approximately 1200°C, bypassing other transition alumina phases, while still maintaining small particle size ( 30 nm). Despite the nanosize particles, green densities equal to 54% of the skeletal density (i.e., true density of the solid phase) were obtained by uniaxial pressing at 40 MPa.     

A model of ammonia volatilisation from a dairy farm: an examination of abatement strategies

A model (MAST) to calculate the mass flow of NH₃ through amodel dairy farm has been developed. Updated emission factors for UKagriculturewere used to examine different abatement strategies available for a typicaldairy farm. A range of annual NH₃ emissions was calculated for bothslurry and FYM based dairy systems. Emission for the slurry based system ranged between 27 kg NH₃-N ha^–1 yr^–1, achieved using a combination of abatementstrategies, and 107 kg NH₃-N ha^–1 yr^–1, calculated for a worst casescenario. For FYM, this range was between 33 and 86 kg NH₃-Nha^–1 yr^–1. The greatest reductionswereachieved by manipulating options linked to fertiliser usage and manureapplication.