Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Effect of alveolar hypoxia on pulmonary fluid filtration in in situ dog lungs

We have studied the effect of alveolar hypoxia on fluid filtration characteristics of the pulmonary microcirculation in an in situ left upper lobe preparation with near static flow conditions (20 ml/min). In six dogs (group 1), rate of edema formation (delta W/delta t, where W is weight and t is time) was assessed over a wide range of vascular pressures under two inspired O2 fraction (FIO2) conditions (0.95 and 0.0 with 5% CO2-balance N2 in both cases). delta W/delta t was plotted against vascular pressure, and the best-fit linear regression was obtained. There was no significant difference (paired t test) in either threshold pressure for edema formation [18.3 +/- 1.8 and 17.1 +/- 1.2 (SE) mmHg, respectively] or the slopes (0.067 +/- 0.008 and 0.073 +/- 0.017 g.min-1. mmHg-1.100g-1, respectively). In another seven dogs (group 2), delta W/delta t was obtained at a constant vascular pressure of 40 mmHg under four FIO2 conditions (0.95, 0.21, 0.05, and 0.0, with 5% CO2-balance N2). Delta W/delta t for the four conditions averaged 0.60 +/- 0.11, 0.61 +/- 0.11, 0.61 +/- 0.10, and 0.61 +/- 0.10 (SE) g.min-1.mmHg-1.100g-1, respectively. No significant differences (ANOVA for repeated measures) were noted. We conclude that alveolar hypoxia does not alter the threshold for edema formation or delta W/delta t at a given microvascular pressure.     

Steady-state response of quadriplegic subjects to inspiratory resistive load

Normal subjects preserve tidal volume (VT) in the face of added inspiratory resistance by increasing maximal amplitude and duration of the rising phase of respiratory driving pressure (DP) and by changing the shape of this phase to one that is more concave to the time axis. To explore the possible role of chest wall afferents in mediating these responses, we determined averaged DP in eight quadriplegic subjects during steady-state unloaded breathing and while breathing through an inspiratory resistance (8.5 cmH2O X 1(-1) X s). As with normal subjects, quadriplegics preserved VT (loaded VT = 106% control) by utilizing all three mechanisms. However, prolongation of the inspiratory duration derived from the DP waveform (+22% vs. +42%) and shape response were significantly less in the quadriplegic subjects. Shape response was completely absent in subjects with C4 lesions. The results provide strong evidence that respiratory muscle spindles are responsible for shape response and that changes in afferent feedback from the chest wall play an important role in mediating inspiratory prolongation.     

Central adaptation to inspiratory-inhibiting expiratory-prolonging vagal input

We reported earlier on the changes in excitability of central respiratory switching mechanisms in the course of a brief inspiratory-inhibiting vagal stimulus (J. Appl. Physiol. 50: 1183-1192, 1981). To further define the dynamics of central processing of such input we studied the changes in the excitability of timing mechanisms in the immediate (less than 1.0 s) and late (1-20 s) periods after stimulus removal. We also examined the changes in respiratory timing in the course of protracted (greater than 20 s) stimulation. Studies were done using pentobarbital-anesthetized cats. For studies involving long-term stimulation or late off responses, cats were paralyzed, vagotomized, carotid denervated, and artificially ventilated. We found that the inspiratory inhibitory influence of a brief stimulus continues, in a declining fashion, for 0.3-10 s after removal of the stimulus. This was followed by a paradoxical response, inspirations were prolonged and expirations were shortened, which was maximal 1-2 s after stimulus removal and which declined gradually over a period of 6-16 s. There was progressive decline in inspiratory-shortening expiratory-prolonging influence in the course of sustained stimuli. These results indicate substantial adaptation in the course of even brief stimuli and provide an explanation for inspiratory-expiratory duration and expiratory-inspiratory duration linkages.     

Temporal changes in effectiveness of an inspiratory inhibitory electrical pontine stimulus

We determined the temporal changes in effectiveness of inspiratory-shortening expiratory-prolonging stimulus trains delivered in the region of the nucleus parabrachialis medialis and compared the responses to those observed during trains delivered to the vagus in the same animals (pentobarbital, sodium-anesthetized paralyzed cats). The inspiratory inhibitory effect of the pontine stimulus was assessed from the effect the stimulus has on threshold for terminating inspiration. Stimulus effect increased gradually, reached a peak at 0.2-0.4 s, and declined thereafter. The time of occurrence of peak effect was different from that observed in the course of vagal stimulus trains. With long stimulus trains (19-40 s), the initial effect on inspiratory duration (TI) (i.e., shortening) rapidly subsided and, in six of eight animals, was replaced by TI prolongation. The initial effect on expiratory duration (TE) (i.e., prolongation) also gradually declined with time but TE remained above control throughout. The time constant of adaptation was very similar with vagal and pontine stimulus trains (12.2 and 11.0 s, respectively), but the gain of the adapting response was much more pronounced with pontine stimuli, resulting in a paradoxical effect while stimulation continued. We conclude that the response to pontine stimuli, as with vagal stimuli, displays both integrative and adaptive characteristics. The similarity of the time constants for vagal and pontine adaptation responses suggests that these two inputs share common processing pathways.     

Effect of slowly increasing elastic load on breathing in conscious humans

Because the detection of added loads is a function of the step change in load relative to background level of load (Weber's law), we reasoned that detection may be delayed if the load is increased in very small steps over a protracted period. This would permit the study of subliminal load responses over a greater range than would otherwise be possible. In 13 healthy males, an external elastic load (delta E) was added in very small steps (load increased every few seconds) such that delta E increased from zero to 6.0 cmH2O/l in 18 min. Each subject underwent a control study in which an identical protocol was followed but no load was added. Five subjects sensed the load after a variable period of load application. Their results were discarded. In the remaining eight subjects, there was no perception of increased load throughout the 18 min. By comparison with the control studies in the same subjects, there was a progressive reduction in tidal volume and breath duration with loading. To assess the response to an increase in load associated with perception, in five of the eight "nonsensers" the load was suddenly doubled (delta E = 6 cmH2O/l) at the end of the 18 min of progressive nonsensed loading. This evoked perception in all subjects and was associated with highly variable responses in tidal volume and breath duration. We conclude that tachypnea associated with elastic loading in conscious humans is a reflex response that is facilitated by consciousness but does not require perception.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Pierce into the Native Structure of Ata,a Trimeric Autotransporter of Acinetobacter baumannii ATCC 17978

International Journal of Peptide Research and Therapeutics - Acinetobacter baumannii is an important pathogen responsible for nosocomial infections worldwide. Trimeric autotransporters, the...