Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Synthesis and application of the first fluorogenic substrate, N-carbobenzoxyglycylprolyl-4-methylcoumarinyl amide (Z-Gly-Pro-MeCouNH) for the determination of the post-proline cleaving enzyme (EC 3.4.21.-) were reported. Maximal activity of the enzyme purified from lamb kidney for the new substrate was observed at pH 7.0. This substrate showed a higher affinity (Km = 0.02 mM) for the enzyme than the proline containing substrates studied previously and allowed the detection of 10-50 ng post-proline cleaving enzyme activity per ml sample after a 1 min incubation period. Distribution of post-proline cleaving enzyme and other proline specific peptidases in rat tissues was studied using Z-Gly-Pro-MeCouNH and other proline-containing substrates. High post-proline cleaving enzyme activity was observed in testis, liver and skeletal muscle. Inhibition experiments indicated that post-proline cleaving enzyme activity was completely inactivated by 0.1 mM diisopropylphosphofluoridate and Z-Gly-Pro-chloromethylketone, as had been found in the case of the enzyme isolated from lamb kidney. Activity in human body fluids was also tested for levels of post-proline cleaving enzyme activity using Z-Gly-Pro-MeCouNH and semen was found to show the highest cleaving activity.     

Fibroblast growth-stimulating activity of S100A9 (MRP-14).

Fibroblasts play a critical role in chronic inflammation and wound healing. In this study, a fibroblast growth-stimulating factor was purified from the exudate of carrageenan-induced inflammation in rats. The purified protein was a disulfide-linked homodimer. Amino acid sequence analysis of the peptides generated by cleavage with cyanogen bromide and proteinase V8 resulted in identification of the protein as S100A9. Recombinant S100A9 as well as its disulfide-linked homodimer stimulated the proliferation of fibroblasts at a similar concentration of the purified protein. The concentration of S100A9 in the exudate was determined by immunoblot analysis. The total protein concentration in the exudate reached a maximum 4 days after carrageenan injection and then slightly decreased, whereas the concentration of S100A9 reached a maximum at day 3 and then decreased rapidly. These studies show that S100A9 is present at a high concentration in the exudate of carrageenan-induced inflammation in rats, and that S100A9 stimulates proliferation of fibroblasts, suggesting that it plays a role in chronic inflammation.     

The release of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from human erythrocyte membranes lysed by hemolysin of Prevotella oris

We found that a 38-kDa protein was released from erythrocyte membranes lysed by hemolysin of Prevotella oris, although hypotonic hemolysis did not show such a phenomenon. The 38-kDa protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal amino acid sequencing. This study discusses the relationship between GAPDH and hemolysis.     

Intracellular pH and Catecholamine Synthesis in Cultured Bovine Adrenal Medullary Cells: Effect of Extracellular Na+ Removal

Incubation of cultured bovine adrenal medullary cells in Na+-free sucrose medium or in Na+-free Cs+ medium enhanced the synthesis of 14C-catecholamines from [14C]tyrosine about two- to threefold or sixfold, respectively. The increment of 14C-catecholamine synthesis produced by Na+-free medium was partially dependent on the presence of Ca2+ in the medium. Dibutyryl cyclic AMP also stimulated the synthesis of 14C-catecholamines in adrenal medullary cells, and the effects of Na+ removal and dibutyryl cyclic AMP (5 mM) on the synthesis were almost additive. The intracellular pH measured by using a weak acid 5,5-dimethyloxazolidine-2,4-dione was 7.14 in control cells and when Na+ was replaced by sucrose or Cs+, it shifted down to 6.56 or 5.66, respectively. The fall in intracellular pH and the stimulation of 14C-catecholamine synthesis were similarly dependent on the concentration of Na+ in the medium. The optimal pH of soluble tyrosine hydroxylase was 5.5-6.0 both in control cells and in cells incubated in Na+-free medium. These results suggest that removal of extracellular Na+ increases the synthesis of catecholamines, at least in part, by shifting the intracellular pH toward the optimal pH of tyrosine hydroxylase.     

Impacts of dam-regulated flows on channel morphology and riparian vegetation: a longitudinal analysis of Satsunai River, Japan

We examined the impacts of the Satsunai River Dam on the hydrology and development of riparian vegetation along the upper and lower reaches of the Satsunai River downstream from the dam. We estimated frequency curves of the flood discharge during the pre-dam (1976–1996) and post-dam (1997–2006) periods and simulated the flood frequency at sampling points within sites under pre-dam, post-dam and dam-removal (using the pre-dam flood discharge and post-dam cross-sections) scenarios. Changes in channel morphology and land cover were investigated by analyzing aerial photographs. Our results indicate that the 20-year flood at the upper site decreased substantially (from 599 to 271 m³/s) after dam operation, while that of the lower site decreased slightly (from 1025 to 977 m³/s). Within the upper site, the proportion of >20-year return periods increased considerably (from 31.0 to 48.6%) while the proportion of 1- to 20-year return periods decreased (from 30.5 to 8.9%) after dam operation. Flood frequency results for the dam-removal scenario were similar to those for the pre-dam period, suggesting that a return to pre-dam discharge rates would restore the pre-dam distribution of flood frequency at the upper site. Within the lower site, however, the distribution of flood frequency varied little between the pre- and post-dam scenarios, because tributary inflows between the sites mitigated the impacts of dam-regulated flows. Land cover types were associated with flood frequency at both sites. The reduced flood frequency of the upper site resulted in increased area of riparian vegetation and decreased area of active channel.     

Cell movements in a living mammalian tissue: Long-term observation of individual cells in wounded corneal endothelia of cats

Although the cells in tissues are known to be motile under special conditions (e.g., during tissue turnover or wound healing), there are not many reports that polygonal cells covering an area without leaving any gaps are also capable of movement. In the present study, cell movements (cell shifting and rearrangement) in a living mammalian eye tissue were documented by identifying and locating individual cells over intervals as long as 100 days. Cat corneal endothelium, a monolayered cell sheet, was wounded by removing a small number (about 180) of endothelial cells from the internal lining of the cornea. Healing of the wounded tissue was observed with a wide-view specular microscope applied to the outer surface of the cornea, enabling us to identify individual cells for as long as two to three months. Cells surrounding the wound underwent areal enlargement, elongated toward the wound, and shifted to cover the wound surface. During days 4–7, cells became rearranged by changing neighbors in such a way that they retained their enlarged size but recovered their non-elongated, original shape. This pattern of cell rearrangement was interpreted by a computer simulation which assumed that cells shorten their boundary length while maintaining contacts with contiguous cells. After day 7, the enlarged cells adjacent to the wounded area gradually contracted and pulled surrounding cells toward the wounded area. These movements were followed by a temporary halt in cell shifting, then by a recovery of shifting and cell elongation. These movements are interpreted as a result of the contractility of endothelial cell microfilaments.     

Apparent binding activity of [H]glutathione in rat central and peripheral tissues

An apparent binding activity of [³H]glutathione (GSH) was detected in the synaptic membranous preparations obtained from the rat brain. Both methionine- and leucine-enkephalins exhibited a profound diminution of the apparent binding at 100 μM in a naloxone-insensitive fashion. The retina was found to have the highest binding activity amongst various central structures examined, followed by the hypothalamus, striatum, spinal cord, midbrain, hippocampus, medulla-pons, cerebellum and cerebral cortex. In peripheral organs employed, the pituitary possessed an apparent binding activity higher than that in the retina, with progressively lower activities in the adrenal, liver, spleen, skeletal muscle and heart. No significant activity was detected in the kidney. These results suggest that specific binding sites of GSH may be located in the central and peripheral excitable tissues.