Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl++ +) sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid

The potency of thrombin inhibition by 4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfony l]- L-arginyl]-2-piperidinecarboxylic acid (MQPA) depended on the stereoconformation of the 2-piperidinecarboxylic acid moiety. Ki values for bovine alpha-thrombin were 0.019 microM with (2R,4R)-MQPA, 0.24 microM with (2R,4S)-MQPA, 1.9 microM with (2S,4R)-MQPA, and 280 microM with (2S,4S)-MQPA. (2R,4R)-MQPA of the four stereoisomers of MQPA was also the most potent inhibitor for other trypsin-like serine proteases with Ki values of 5.0 microM for trypsin, 210 microM for factor Xa, 800 microM for plasmin, and 1500 microM for plasma kallikrein. Examination of the potency of thrombin inhibition by arginine derivatives related to MQPA in structure suggested the presence of a specific binding site for the carboxamide portion (C-terminal side). The relative inhibitory potency of the four stereoisomers of MQPA for trypsin was nearly identical with that for thrombin, suggesting that the specific binding site for the carboxamide portion is present in both enzymes. Modification of thrombin by phosphopyridoxylation or the presence of heparin did not significantly alter the binding of MQPA.     

A Decreased Level of Serum Soluble Klotho Is an Independent Biomarker Associated with Arterial Stiffness in Patients with Chronic Kidney Disease

Background

Klotho was originally identified in a mutant mouse strain unable to express the gene that consequently showed shortened life spans. In humans, low serum Klotho levels are related to the prevalence of cardiovascular diseases in community-dwelling adults. However, it is unclear whether the serum Klotho levels are associated with signs of vascular dysfunction such as arterial stiffness, a major determinant of prognosis, in human subjects with chronic kidney disease (CKD).

Methods

We determined the levels of serum soluble Klotho in 114 patients with CKD using ELISA and investigated the relationship between the level of Klotho and markers of CKD-mineral and bone disorder (CKD-MBD) and various types of vascular dysfunction, including flow-mediated dilatation, a marker of endothelial dysfunction, ankle-brachial pulse wave velocity (baPWV), a marker of arterial stiffness, intima-media thickness (IMT), a marker of atherosclerosis, and the aortic calcification index (ACI), a marker of vascular calcification.

Results

The serum Klotho level significantly correlated with the 1,25-dihydroxyvitamin D level and inversely correlated with the parathyroid hormone level and the fractional excretion of phosphate. There were significant decreases in serum Klotho in patients with arterial stiffness defined as baPWV≥1400 cm/sec, atherosclerosis defined as maximum IMT≥1.1 mm and vascular calcification scores of ACI>0%. The serum Klotho level was a significant determinant of arterial stiffness, but not endothelial dysfunction, atherosclerosis or vascular calcification, in the multivariate analysis in either metabolic model, the CKD model or the CKD-MBD model. The adjusted odds ratio of serum Klotho for the baPWV was 0.60 (p = 0.0075).

Conclusions

Decreases in the serum soluble Klotho levels are independently associated with signs of vascular dysfunction such as arterial stiffness in patients with CKD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could improve arterial stiffness in CKD patients is warranted.     

Calcitonin receptor-stimulating peptide-1 regulates ion transport and growth of renal epithelial cell line LLC-PK1

Calcitonin receptor-stimulating peptide-1 (CRSP-1) is a peptide recently identified from porcine brain by monitoring the cAMP production through an endogenous calcitonin (CT) receptor in the renal epithelial cell line LLC-PK(1). Here we investigated the effects of CRSP-1 on the ion transport and growth of LLC-PK(1) cells. CRSP-1 inhibited the growth of LLC-PK(1) cells with a higher potency than porcine CT. CRSP-1 enhanced the uptake of (22)Na(+) into LLC-PK(1) cells more strongly than did CT and slightly reduced the (45)Ca(2+) uptake. The enhancement of the (22)Na(+) uptake was abolished by 5-(N-ethyl-N-isopropyl) amiloride, a strong Na(+)/H(+) exchanger (NHE) inhibitor for NHE1, even at a concentration of 1x10(-8)M, although other ion transporter inhibitors did not affect the (22)Na(+) uptake. These results indicate that CRSP-1 enhances the (22)Na(+) uptake by the specific activation of NHE1. Taken together, CRSP-1 is considered to be a new regulator for the urinary ion excretion and renal epithelial cell growth.     

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).     

Purification and characterization of recombinant human interleukin-1 beta produced in Escherichia coli

Recombinant human interleukin-1 beta (rIL-1 beta) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography. The purified rIL-1 beta had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing. These values were almost same as those of natural interleukin-1 beta. The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence. In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC. Besides rIL-1 beta with amino terminal Ala, two molecular species, [Met0] rIL-1 beta and [desAla1] rIL-1 beta, were also obtained. Biological and physicochemical properties of the three species of rIL-1 beta were compared.     

N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.     

An improved assay method for amylase activity using an amylodextrin fraction as substrate

A rapid and simple method for determination of amylase activity, by using a fraction of an amylo-oligosaccharide (amylodextrin, 1) as substrate, is described. The sample solution is incubated with a solution of 1 and the substrate consumed is estimated by measuring the difference in absorbance at 460 nm. The K_m value of 1 is about half that of starch. The homogeneity of 1 in its chemical structure and molecular weight facilitated the specification of amylase units according to the international definition. This procedure was applied for assay of human-serum amylase with excellent reproducibility. This method did not require a large dilution factor, as the standard curve showed a linear relationship over a wide range of amylase concentrations.     

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.     

Both VH and VL regions contribute to the antigenicity of anti-idiotypic antibody that mimics melanoma associated ganglioside GM3

Previously we developed a murine monoclonal anti-idiotype (antiid) antibody (4C10) that mimics the melanoma-associated ganglioside antigen GM₃, that is, it carries the internal image of GM₃. 4C10 was made against the human monoclonal antibody (HuMAb) L612, which reacts with several types of human cancer cells, including melanoma and breast cancer. To reduce mouse components of 4C10, the constant region was replaced by a human constant domain to form the murine/human chimeric anti-id antibody TVE-1. In the present study, we sought to determine which chain (V_H or V_L) of the anti-id is responsible for the antigenicity of GM₃. The TVE-1 V_H and V_L expression vectors were simultaneously transfected with either the V_H or V_L expression vector of a murine-human chimeric IgG antidansyl haptenic antibody, resulting in the construction of three different combinations of V_H and V_L chimeric antibodies. These IgG molecules were produced from the transfectomas, and their reactivity to HuMAb L612 was tested. Neither of the IgG proteins that had cross-combined the V_H-V_L pair showed positive results, suggesting that both heavy and light chains are required to express the antigenicity. The in vivo antigenicity of this chimeric anti-id was confirmed by skin tests in melanoma patients receiving active specific immuntherapy.