Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.     

Role of dietary phosphorus in the progression of renal failure

Dietary phosphorus is thought to be a factor that impairs the residual renal function in patients with chronic renal failure. To determine the effect of dietary phosphorus on the prognosis of chronic renal failure, low-phosphorus milk was prepared from normal cow's milk using boehmite, a synthetic phosphate-ion absorbent. Regular diet, normal cow's milk, and low-phosphorus milk were then given to 5/6-nephrectomized rats and the serum levels of inorganic phosphorus, calcium, creatinine, and blood urine nitrogen in the rats in each group were compared. The serum levels of inorganic phosphorus and calcium were not different among the groups, despite a significant difference in phosphorus intakes. On the other hand, serum levels of creatinine (Cr) and blood urine nitrogen (BUN) in the rats fed low-phosphorus milk were significantly lower (Cr, 0.54+/-0.054mg/dl; BUN, 29.2+/-3.90mg/dl) than those in the rats fed a regular diet (Cr, 0.64+/-0.057mg/dl; BUN, 37.4+/-3.55mg/dl) or normal milk (Cr, 0.61+/-0.040mg/dl; BUN, 34.5+/-3.59mg/dl). No beneficial effect of protein restriction was observed when residual renal functions in rats fed a regular diet and those fed normal milk were compared. The results suggest that dietary phosphorus plays a major role in the progression of renal failure.     

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals

It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.     

Production of manganese peroxidase by pellet culture of the lignin-degrading basidiomycete, Pleurotus ostreatus

Pleurotus ostreatus No. 42 produced the ligninolytic enzymes, manganese peroxidase (MnP) and laccase, in agitation culture in glucose/peptone/wheat-bran medium. Formation of mycelial pellets 1-2 mm in diameter was essential for the production of MnP; and the concentration of dissolved oxygen in the culture medium greatly influenced the production of MnP, a concentration over 5 ppm being necessary for MnP production. The maximal activity of MnP was obtained on days 7-9 of culture, after the consumption of nutrient glucose. Introduction of oxygen from the start of the cultivation caused large pellet formation, which resulted in a low MnP activity level. P. ostreatus No. 42 produced two MnP isozymes in agitation culture. The major isozyme, F-2, was 36.4 kDa and had a pI of 3.95. The MnP characteristics, Km values, dependence on Mn2+ and optimum pH showed the similarity between this isozyme and MnP 3, which was produced under different culture conditions. Analysis of the N-terminal amino acid sequence indicated the close similarity of F-2 to MnP 3.     

Synthesis of southern (C1'-C11') and eastern (C8-C18) fragments of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger

Synthesis of the southern C1'-C11' and eastern C8-C18 fragments of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger, was achieved. The southern fragment was synthesized by using the Evans aldol reaction and cis-selective iodoetherification as the key steps in a 9.6% overall yield (7 steps). The eastern fragment was constructed via the Julia coupling reaction and cis-selective iodoetherification in a 3.0% overall yield (8 steps from the known epoxide).     

The acylation of L-glycerol-3-phosphate by microsomes from rat brain: effects arising from the properties of the reaction medium

Abstract— The velocity of the reaction catalysed by acyl-CoA: l -giycerol-3-phosphate acyltransferase (EC 2.3.1.15) of microsomes from rat brain was affected by the nature of the buffering agent, the ionic strength and the sucrose concentration of the reaction medium. The enzyme was inhibited by buffers based on trimethyl-pyridine, diethyl barbituric acid, and boric acid. Buffers based on N-ethyl morpholine, potassium phosphate, sodium arsenate, imidazole, tris and triethanolamine were not inhibitory. Dithiothreitol protected the enzyme and produced maximal activity at levels in the reaction medium between 0.2 and 2.8 mM. Optimum ionic strength was determined by varying the concentration of a potassium phosphate buffer and in this medium the optimum ionic strength was about 0.2 M. In other studies with sodium formate, potassium acetate and other salts there was a broad plateau of activity in a range about 0.2 M. A study of pH vs. activity with two different buffering agents at constant ionic strength showed a broad maximum of activity from pH 7.2 to pH 7.8. The velocity of the reaction could be further increased by the inclusion of 0.25 M-sucrose in the reaction medium in the presence of 0.2 M salts. The sucrose effect produced maximum velocities at sucrose concentrations ranging from 0.2 to 0.6 M. The studies reported here indicate that the activity of the enzyme is dependent upon the state of hydration of the microsomal membranes and in part on the ability of the enzyme or membrane to cope with large micelles of S-palmityl-CoA.