The Amino Acid Content in Gamma-irradiated Rice†

High phosphate accumulating bacteria were isolated by autoradiography. One isoate, Arthrobacter globiformis PAB-6 accumulated phosphate intracellularly at 20% of dry cell mass in a simple synthetic medium. This amount was 3~7 times higher than type cultures examined. Almost no phosphate was released into the medium after cessation of growth. Fifty percent of total intracellular phosphate was fractionated as nucleic acids, while 20% each was recovered from cold PCA soluble fractions and polyphosphate fractions. The large content of nucleic acids in this bacterium appeared due to increased RNA content, specifically 4 S RNA fraction.     

An immunoelectron-microscopical observation of mouse juxtaglomerular cells in the case of experimental hydronephrosis.

Changes in juxtaglomerular (JG; renin-containing) cells in experimental hydronephrosis 1 month after ureteral ligation were investigated with immunoelectron-microscopical techniques. Two types of granules, electron dense (D) and lucent (L), were observed. D type granules were labeled more intensely with gold particles than those of L type. Granules intermediate between D and L types and exocytosis of D types were observed. In the cells containing D types exclusively, gold particles were restricted to the granules, whereas in the cells containing both D and L type granules, the particles were scattered throughout the cytoplasmic cytosol. The authors discuss the mechanisms of renin release in JG cells.     

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.     

Global and temporal regulation of gene expression in Xenopus kidney cells in response to presumed microgravity generated by 3D clinostats.

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Non-requirement of calcium on protamine phosphorylation by calcium-activated, phospholipid-dependent protein kinase

Phosphorylation of clupeine sulfate by purified rat brain calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was studied. In the absence of Ca2+, phosphatidylserine and diolein markedly stimulated its phosphorylation. However Ca2+ did not stimulate but inhibit this phosphorylation about 30% in the presence of phospholipids. Random polymer (Arg, Ser) 3:1 and (Lys, Ser) 3:1 could be phosphorylated by protein kinase C. In the presence of phospholipids Ca2+ is not needed for the phosphorylation of polymer (Arg, Ser) 3:1, while Ca2+ is necessary for polymer (Lys, Ser) 3:1. Non-requirement of Ca2+ on clupeine phosphorylation by protein kinase C is briefly discussed.     

Effect of Osmotic Stress on Turgor Pressure in Mung Bean Root Cells

Turgor pressure in cells of the elongating region of intactmung bean roots was directly measured by using the pressure-probetechnique. After the external osmotic pressure had been increasedfrom 0 MPa to 0.5 MPa, turgor pressure rapidly decreased byabout 0.5 MPa from 0.65 MPa to 0.14 MPa and root elongationstopped. Subsequent turgor regulation was clearly confirmed,which followed the osmotic adjustment to maintain a constantdifference in the osmotic pressure between root-cell sap andthe external medium ( II). It took at least 6 h for turgor pressureto recover to an adjusted constant level of about 0.5 MPa dueto turgor regulation, but rootelongation resumed within onlyan hour after the osmotic treatment. Therefore, the resumptionof root elongation under osmotic stress could not have beendirectly connected with turgor regulation. Furthermore, sincethe amounts of decrease in turgor pressure just after applicationsof various degrees of osmotic stress could be interpreted inrelation to those in II, hydraulic conductivity between theinside and the outside of root cells must be large enough toattain water potential equilibrium rapidly in response to osmoticstress. We conclude that turgor pressure in the cells of theelongating region of mung bean roots is determined mainly by II because of water potential equilibrium. (Received January 27, 1987; Accepted May 21, 1987)     

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.