Modulation of the p27kip1 cyclin-dependent kinase inhibitor expression during IL-4-mediated human B cell activation

IL-4 activates resting B cells and, in conjunction with cosignals such as anti-IgM (anti-mu) Ab or CD40 ligand, modulates progression of B cells through the cell cycle, leading to proliferation. In this study, we show that the mitogenic combination of IL-4 and anti-mu Ab triggered induction of cyclin D3 and up-regulated cyclin-dependent kinase (cdk) 6 expression, whereas such regulation was not observed in B cells activated by IL-4 or anti-mu Ab alone. Furthermore, cyclin D3 immunoprecipitated fron as associated with cdk6, and the cyclin D3/cdk6 complex was able to phosphorylate recombinant retinoblastoma protein in vitro. In addition, B cells activated with either IL-4 or 1L-13 alone expressed a higher amount of p27kip1 (p27) cdk inhibitor than nonstimulated cells. In contrast, p27 expression was decreased when cells were activated with mitogenic combinations of IL-4 and anti-mu Ab or anti-CD40 mAb. We also observed that the IL-4-mediated inhibition of the proliferation of anti-mu/IL-2- or anti-mu/phorbol 12,13-dibutyrate-activated human leukemic B cells was associated with the maintenance of large amounts of p27 in these cells. These data suggest that IL-4 controls B cell proliferation by action during at least two steps of the regulation of the cell cycle, cyclin D3/cdk6 complex regulation and p27 inhibitor expression.     

Quantitative image analysis of broadband CARS hyperspectral images of polymer blends

We demonstrate that broadband coherent anti-Stokes Raman scattering (CARS) microscopy can be very useful for fast acquisition of quantitative chemical images of multilayer polymer blends. This is challenging because the raw CARS signal results from the coherent interference of resonant Raman and nonresonant background and its intensity is not linearly proportional to the concentration of molecules of interest. Here we have developed a sequence of data-processing steps to retrieve background-free and noise-reduced Raman spectra over the whole frequency range including both the fingerprint and C-H regions. Using a classical least-squares approach, we are able to decompose a Raman hyperspectral image of a tertiary polymer blend into quantitative chemical images of individual components. We use this method to acquire 3-D sectioned quantitative chemical images of a multilayer polymer blend of polystyrene, styrene-ethylene/propylene copolymer, and polypropylene that have overlapping spectral peaks.     

The influence of the visual cortex on the spatiotemporal response properties of lateral geniculate nucleus cells

Previous studies of the cortical input to the mammalian dorsal lateral geniculate nucleus (LGN) have identified a number of possible functions for the corticogeniculate pathway, including alteration of LGN spatial frequency selectivity and facilitation of both binocular interactions and orientation selectivity. These changes may be due to either a tonic or a phasic cortical facilitation or both. The temporal differences between each of these inputs suggests that their impact on LGN cell temporal tuning should be unique. To test this hypothesis, we reversibly blocked the visual cortex (VI) and measured the effects on several indices of the temporal properties of LGN cells, including peak frequency, bandwidth, and response phase. Macaque monkeys were anesthetized and paralyzed during single cell recording from the LGN while area VI was cryogenically deactivated. Single-cell responses were visually evoked with drifting, luminance-modulated, sine-wave gratings and discrete-Fourier analyzed. Cortical cooling produced statistically significant increases or decreases in response amplitude in 64% of cells recorded. In most cases, alterations in response amplitude occurred for stimuli that varied in spatial as well as temporal frequency. For those cells influenced by changes in stimulus temporal frequency, the majority showed changes over a broad range of frequencies. A minority of cells showed changes in either peak temporal tuning or temporal frequency bandwidth. Response phase angles for all temporal frequencies tested were unaffected by cortical cooling. Overall, these results suggest that the cortical input may alter the temporal response properties of LGN cells, perhaps by tonic, but not exclusively excitatory, corticofugal influences.     

Latex hypersensitivity/glove component reaction as a contributing factor to osteomyelitis of the hand: a case report and review of the literature

High-resolution computed tomography (HRCT) can be used to diagnose and quantify emphysema noninvasively, as significant correlations have been found between the histological grade on resected lung specimens and quantified (q) computed tomography (CT). In this study, we performed thin section qHRCT in patients with severe hereditary alpha-1-antitrypsin (AAT) deficiency. AAT deficiency is the most common genetic cause of emphysema in adults, and exercise intolerance is the most disabling, distressing consequence of emphysema for the majority of patients. qHRCT was used to quantify precisely the alterations in the lung parenchyma due to pulmonary emphysema. Up until now, the important relationship between the severity of emphysema and the reduced exercise capacity has received little attention. Therefore the purpose of the study was to investigate the relationship between emphysema as displayed by qHRCT and cardiopulmonary exercise testing (CPX) in patients with severe cardiopulmonary impairment. - qHRCT was performed in 21 patients with homozygous AAT deficiency. CT scans were obtained at three spirometrically standardized levels at the carina and (5 cm above and below the carina). The mean lung density at 50% of vital capacity and a quantitative histogram analysis of the frequencies of CT values were determined. All patients underwent symptom-limited CPX to analyse simultaneously cardiovascular and ventilatory systems responses. - In all patients, qualitative CT assessment demonstrated panlobular emphysema with large and extensive areas of uniform low attenuation, characteristically with a lower-lobe distribution. Mean CT density values of the patients (-845 +/- 6.9 (mean +/- SEM)) were significantly correlated with work capacity (r = 0.55, p <0.01), oxygen-pulse (r = 0.54, p <0.01) and functional dead space ventilation (r = -0.54, p <0.01). Moreover, severe emphysema index (CT values below a threshold value of 950 HU) correlated positively with functional dead space ventilation (r = 0.60, p <0.01) and alveolar-arterial oxygen difference (r = 0.70, p <0.001). - These results clearly demonstrate that CPX parameters, indicating a disturbed pulmonary gas exchange and a ventilation-perfusion-mismatch during exercise, are significantly related to the extent of lung emphysema.     

A column centrifugation method for the reconstitution in liposomes of the mitochondrial F0F1 ATP synthase/ATPase

A method for reconstitution of membrane proteins into unilamellar liposomes is described. The model enzyme was the F0F1 ATP synthase from mitochondria when in complex or free from its inhibitor protein. The enzymes were first solubilized with either of two detergents, i.e., n-dodecyl-beta-D maltoside or lauryldimethylamine oxide. After solubilization, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered from the column were subsequently passed through a centrifuge column of Sephadex G-50 fine. The eluate contained liposomes in which the F0F1 complex (with and without inhibitor protein) had been reconstituted. The reconstituted enzymes were capable of hydrolyzing ATP with formation of electrochemical H+ gradients. They also catalyzed the ATP-Pi exchange reactions. Thus the F0F1 complex which is formed by 18 subunits can be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and its inhibitor protein are not perturbed in the reconstitution procedure.     

Preparation and activity of complexes of transition metals and thiolic heterocyclic ligands

The activities of cell-associated IL-1 (IL-1 alpha) and extracellular IL-1 (IL-1 beta) in alveolar macrophages (AM) from rats with bleomycin-induced pulmonary fibrolosis were measured to determine their role in fibroblast growth. AM were obtained on Days 1, 3, 6, 9 and 12 after a intratracheal injection of bleomycin, and the IL-1 activity and fibroblast growth-stimulating activity in fixed AM and AM supernatants were measured. Higher cell-associated IL-1 activity was detected in AM from bleomycin-treated rats than in those of control on Day 1 through 9. But extracellular IL-1 activity in the supernatant of AM from bleomycin-treated rats significantly higher only on Day 1. Expression of IL-1 alpha mRNA in AM from bleomycin-treated rats was significantly higher than that in AM of control, but there was no significant difference in the mRNA levels of IL-1 beta in AM of these two groups. Fixed AM from bleomycin-treated rats caused growth-inhibition of fibroblasts in a density-dependent manner. The inhibitory activity was decreased by pretreatment of fixed AM with anti IL-1 alpha antibody, but not anti IL-1 beta antibody. These results suggest that cell-associated IL-1 (IL-1 alpha) is produced continuously in AM from rats with bleomycin-induced pulmonary fibrosis and may be important in regulation of this disorder by inhibiting fibroblast growth.     

Use of reversed polarity and a pressure gradient in the analysis of disaccharide composition of heparin by capillary electrophoresis

A capillary electrophoresis method with reversed polarity, combining both the application of a voltage and a pressure gradient between the buffer vials, was developed for the analysis of eight heparin-derived delta-disaccharides obtained by enzymatic depolymerization. A 60 mM formic acid buffer at pH 3.40 was selected as running electrolyte, with an applied voltage of -15 kV and an over-imposed pressure gradient (3.45.10(-3) MPa) for 6 min from inlet to outlet starting at 20 min. Figures of merit such as run-to-run and day-to-day precision, and limits of detection were established. The electrophoretic method was applied to the analysis of depolymerization products of different kinds of heparins. The composition of the depolymerization buffer was selected in order to reduce baseline distortions in the electrophoretic separation, thus a buffer solution containing 20 mM Tris, 50 mM sodium chloride, and 3 mM calcium chloride at pH 7.10 was used. Percentages of molar disaccharide compositions for unfractionated heparins from porcine, bovine and ovine intestinal mucosa, and bovine lung were determined. In addition, low-molecular-mass heparins from bovine and porcine intestinal mucosa were analysed as well.