Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Prebiotic synthesis of orotic acid parallel to the biosynthetic pathway

By heating an aqueous solution of aspartic acid and urea, carbamylaspartic acid is first formed and then the molecule is cyclized to dihydroorotic acid (DHO) with loss of water. Irradiation of an aqueous solution of DHO with a tungsten lamp yields orotic acid by photo-dehydrogenation of the molecule. This pathway of orotic acid formation is quite similar to that of biosynthesis of the molecule.     

Nonadrenergic inhibitory nerves attenuate neurally mediated contraction in cat bronchi

Effects of nonadrenergic and noncholinergic (NANC) inhibitory nerves on cholinergic neurotransmission were examined in isolated bronchial segments from cats in the presence of propranolol (10(-6) M) and indomethacin (10(-6) M) by use of electrical field stimulation (EFS) techniques. EFS caused contraction alone in tissues at the baseline tension and biphasic responses (contraction and relaxation) in tissues precontracted with 5-hydroxytryptamine. Contraction was abolished by atropine (10(-6) M), and relaxation was abolished by tetrodotoxin (10(-6) M). At the baseline tension, EFS at frequencies greater than 10 Hz inhibited the subsequent (4 min later) contraction induced by EFS at 1-5 Hz. EFS-induced inhibition was stimulus frequency dependent and reached maximum at 20 Hz. However, EFS at 20 Hz did not inhibit the subsequent contractile response to acetylcholine (10(-7) to 10(-3) M). Exogenously applied vasoactive intestinal peptide mimicked EFS-induced inhibitory effects, but substance P and calcitonin gene-related peptide did not. The inhibitory effect of EFS at 20 Hz was not altered by pyrilamine, cimetidine, naloxone, methysergide, phentolamine, BW755C, AF-DX 116, or removal of epithelium. These results imply that the NANC transmitter acts via presynaptic cholinergic receptors.     

Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.