Integrated pulsed amperometric detection of glufosinate, bialaphos and glyphosate at gold electrodes in anion-exchange chromatography.

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml(-1), respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1-45, 0.3-32 and 0.1-50 microg ml(-1), respectively. The relative standard deviations (n=5) were 1.7-3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.

Nickel-titanium alloy electrodes for stable amperometric detection of underivatized amino acids in anion-exchange chromatography.

Nickel-titanium (Ni-Ti) alloy electrode was used as an electrochemical detector for the analysis of underivatized amino acids in flow systems. In strong alkaline solution, an oxide film on the Ni-Ti alloy electrode surface exhibited a high catalytic activity toward the oxidation of amino acids. Cyclic voltammetry experiments confirmed that electrogenerated Ni(III)O(OH) functioned as the key redox mediator associated with the oxidation of the amine group in amino acids. The electrochemical behavior of the Ni-Ti electrode in alkaline medium was very similar to the Ni electrode. However, the oxide film was found to be much stable on Ni-Ti than on Ni. Consequently, the Ni-Ti alloy electrode exhibited an excellent stability for constant-potential amperometric detection of amino acids in flow systems. For example, the relative standard deviation (R.S.D.) for the repetitive 100 injections of 50 muM (1.2 nmol) glycine over 10 h was less than 1%. It was postulated that the presence of Ti in the alloy stabilizes the microstructure of oxide layer on the electrode surface. The sensitivities of amino acids at the electrode were different, depending on their chemical structures. The detection limits obtained in a range from 0.9 pmol for arginine to 90.2 pmol for leucine and isoleucine. The Ni-Ti alloy electrodes have been demonstrated to be very suitable for the amperometric detection of underivatized amino acids in anion-exchange chromatography.

Indirect amperometric detection of underivatized amino acids in microcolumn liquid chromatography with carbon film based ring-disk electrodes.

An indirect amperometric detection of underivatized amino acids has been developed using a carbon film based ring-disk electrode (CFBRDE) in microcolumn liquid chromatography (LC). Bromide present in the mobile phase could be efficiently oxidized to bromine at the upstream (disk) electrode, and was subsequently detected at the downstream (ring) electrode. Most of the underivatized amino acids that are electroinactive under conventional amperometric conditions react rapidly with the electrogenerated bromine, the concentration of amino acids can therefore be indirectly determined by continuously monitoring the reduction current of bromine. The signal monitored at the downstream electrode was largely dependent on the bromide concentration in the mobile phase. Under optimized conditions, the response linearly increased with the concentration for most of the amino acids over a concentration range of 1-100 microM, with a correlation coefficient of 0.990-0.993. The detection limits for most of the amino acids were below 1 microM (0.2 pmol). It was demonstrated that detection with a ring-disk electrode offers the advantages of achieving a much higher collection efficiency caused by a decrease in flow rate in the microcolumn LC.

Exon 9 mutations in the WT1 gene, without influencing KTS splice isoforms, are also responsible for Frasier syndrome.

We report new mutations in exon 9 of the WT1 gene that did not alter the ratio of +/- KTS splice isoforms in two unrelated patients with Frasier syndrome (FS). The mutation of intron 9 inducing defective alternative splicing was reported to be responsible for this syndrome. The mutations found in our cases occurred in the same exon of the WT1 gene as detected in Denys-Drash syndrome (DDS) and could not be explained by the previously proposed mechanism. The results suggest that the two syndromes originate from the same WT1 gene abnormality. From a molecular biological point of view, we concluded that the two diseases were not separable, and that FS should be included as an atypical form of DDS.

A controlled trial of combined therapy for newly diagnosed severe childhood IgA nephropathy. The Japanese Pediatric IgA Nephropathy Treatment Study Group.

The most appropriate treatment for patients with IgA nephropathy is controversial. Treatment with prednisolone, azathioprine, heparin-warfarin, and dipyridamole early in the course of disease may prevent immunologic renal injury in children with severe IgA nephropathy. To determine whether similar results can be obtained with a combination of just heparin-warfarin and dipyridamole, the effects of such treatment were compared to those of treatment with prednisolone, azathioprine, heparin-warfarin, and dipyridamole in 78 children with newly diagnosed IgA nephropathy showing diffuse mesangial proliferation. The patients were randomly assigned to receive either prednisolone, azathioprine, heparin-warfarin, and dipyridamole for 2 yr (group 1) or heparin-warfarin and dipyridamole for 2 yr (group 2). All of the 40 patients in group 1 and 34 of the 38 patients in group 2 completed the trial. The mean urinary protein excretion fell in group 1 patients (P < 0.0001), but remained unchanged in group 2 patients. The mean serum IgA concentration was reduced in group 1 patients (P = 0.0002), but was unchanged in group 2 patients. BP and creatinine clearance were normal at the end of the trial in all but one group 2 patient, who developed chronic renal insufficiency. The percentage of glomeruli showing sclerosis was unchanged in group 1 patients, but increased in group 2 patients (P = 0.006). The intensity of mesangial IgA deposits decreased in group 1 patients (P = 0.02), but remained unchanged in group 2 patients. In conclusion, the present study shows that treatment of children with severe IgA nephropathy with prednisolone, azathioprine, heparin-warfarin, and dipyridamole for 2 yr early in the course of disease reduces immunologic renal injury and prevents increase of sclerosed glomeruli.

Analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin G.

To characterize patients with mumps vaccine failure, avidity testing was performed with the Enzygnost Anti-Parotitis Virus/IgG kit using a single-dilution-6 M urea denaturation method. Five groups of patients were tested. Group 1 consisted of 29 patients with primary mumps infections; group 2 was 20 children and adults with a definite history of natural infection; group 3 was 7 patients with a recent mumps vaccination, 1 of whom developed parotid gland swelling and aseptic meningitis; group 4 was 14 patients with mumps vaccine failure; and group 5 was 6 patients with recurrent episodes of parotitis in addition to a history of vaccination. On the basis of the results of groups 1 and 2, an avidity of /=32% was determined to be high. Avidity maturation from low to high appears to occur around 180 days after the acute illness. The results of group 3 showed that the vaccine-induced immunoglobulin G (IgG) had very low avidity. Among the 14 patients in group 4, 12 patients, including 7 with a positive IgM response, were diagnosed as having secondary vaccine failures. The results of group 5 suggested the possibility that the avidity of the mumps vaccine-induced IgG remains low or borderline. These results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in our study population), and therefore, vaccinees are prone to be exposed to wild-type viruses. Avidity testing should provide information useful for the analysis of mumps virus infections.

4q33-qter deletion and absorptive hypercalciuria: report of two unrelated girls.

We report on two unrelated girls with multiple malformations, each of whom had a der(4)t(4;?)(q33;?) chromosome--an unbalanced translocation chromosome with deletion of the 4q33-qter segment and addition of a segment of an unknown chromosome. One of the two girls had asymptomatic kidney stones. Both had excess urinary calcium excretion (0.53 and 0.84 mg/mg creatinine, respectively), exaggerated excretion on oral calcium load, and reduced but excessive excretion on restricted calcium intake. The urinary calcium excretion of their parents was normal. Both girls were thus diagnosed to have sporadic absorptive hypercalciuria. It was deduced that the 4q33-qter segment contains the putative gene for absorptive hypercalciuria.

Oral-facial-digital syndrome type IX in a patient with Dandy-Walker malformation.

We report a girl with oral, facial, and digital anomalies including multiple alveolar frenula, lobulated tongue with nodules, a posterior cleft palate, hypertelorism, a prominent forehead with a large anterior fontanelle, and postaxial polydactyly in both hands and the right foot, features compatible with the oral-facial-digital syndrome (OFDS). In addition, she had bilateral microphthalmia, optic disc coloboma, and retinal degeneration with partial detachment, thus establishing a diagnosis of OFDS type IX. Dandy-Walker malformation and retrobulbar cysts were observed on MRI. These additional malformations have not been reported in OFDS type IX. The frequent apnoeic spells which occurred immediately after birth were relieved after cystoperitoneal shunt implantation for hydrocephalus. Considering our case and previous reports of OFDS type IX, including two male sibs, a boy born to consanguineous parents, and three females, inheritance is probably autosomal recessive.

[Standard versus long-term prednisolone with sairei-to for initial therapy in childhood steroid-responsive nephrotic syndrome: a prospective controlled study].

The most appropriate initial treatment for children with steroid-responsive nephrotic syndrome is controversial. Initial treatment with 18-week prednisolone and the Chinese herbal medicine. Sairei-to, may prevent subsequent relapse. To determine whether similar results can be obtained with a combination of just initial 8-week prednisolone and Sairei-to, we compared the effects of such treatment with those of treatment with 18-week prednisolone and Sairei-to in 196 children with steroid-responsive nephrotic syndrome. The patients were randomly assigned to receive 8-week (group 1) or 18-week (group 2) prednisolone for the initial therapy. All patients received Sairei-to for 2 years in addition to prednisolone. Eighty-eight of the 98 patients in group 1 and 83 of the 98 patients in group 2 completed their trial. At entry, the two groups of patients did not differ in their clinical and laboratory findings. During the 2-year trial, 62 group 1 patients (70%) and 54 group 2 patients (65%) had relapses, and 19 group 1 patients (21%) and 20 group 2 patients (24%) had frequent relapses. The present study demonstrates that a combination of initial 8-week prednisolone and 2-year Sairei-to is effective in children with steroid-responsive nephrotic syndrome.

[A prospective controlled study of sairei-to in childhood IgA nephropathy with focal/minimal mesangial proliferation. Japanese Pediatric IgA Nephropathy Treatment Study Group].

To determine the effect of the Chinese herbal medicine, Sairei-to (TJ-114) in children with newly diagnosed IgA nephropathy showing focal/minimal mesangial proliferation, we undertook a prospective controlled study. One hundred and one patients were randomly assigned to receive Sairei-to for 2 years (group 1) or no drug for 2 years (group 2). Forty-six of the 50 patients in group 1 and 48 of the 51 patients in group 2 completed their trial. At entry, the two groups of patients did not differ in the clinical, laboratory and pathologic findings. At the end of the trial, urinary protein excretion and hematuria were significantly reduced in group 1, but were unchanged in group 2. Twenty-one group 1 patients (46%) had normal urine, but only 5 group 2 patients (10%) had normal urine at the end of the trial (p < 0.001). Blood pressure and creatinine clearance were normal at the end of the trial in all but one group 2 patient, who developed chronic renal failure. The present study demonstrates that 2-year Sairei-to treatment early in the course of disease is effective in children with IgA nephropathy showing focal/minimal mesangial proliferation.

Analysis of wool fiber by alkali-catalyzed pyrolysis gas chromatography.

Alkali-catalyzed pyrolysis gas chromatography (PyGC) has been used to identify minute samples of wool fiber. The wool sample to which aqueous sodium hydroxide was added was pyrolyzed in a Curie-point pyrolyzer attached to a gas chromatograph or a gas chromatograph-mass spectrometer. The addition of an aqueous solution of sodium hydroxide increased the production of specific volatile pyrolysis products from the constitutive amino acid residues of wool protein, i.e. acetaldehyde from alanine or proline, isobutyronitrile from valine, 2-methylbutyronitrile from isoleucine, isovaleronitrile from leucine and toluene from phenylalanine. Compared with conventional non-catalyzed PyGC, the alkali-catalyzed PyGC was found to greatly improve the detection limit of wool fiber and make it possible to analyze very minute samples. The alkali-catalyzed PyGC presented here has been shown to be applicable to minute thermally-denatured samples of wool fiber which cannot be identified successfully by morphological inspection using a microscope or by using Fourier-transform infrared microspectroscopy. Furthermore, the present PyGC method was successfully used for several protein samples and was shown to be useful for analysis of proteins other than wool fibers by using different special pyrograms reflecting different amino acid compositions.

Identification and immunohistochemical localization of macrophage migration inhibitory factor in human kidney.

Macrophage migration inhibitory factor (MIF) was the first lymphokine identified in activated T-lymphocytes. MIF can induce proinflammatory cytokines, such as interleukin-1 and tumor necrosis factor-alpha. In this study, we identified MIF expression in a tissue specimen of a normal portion of a nephrectomized human kidney by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Furthermore, immunohistochemical study using an anti-human MIF polyclonal antibody demonstrated that MIF was mostly present in the renal tubule epithelial cells and, to a lesser extent, in Bowman's capsular epithelial cells. We also carried out immunohistochemistry on cultured human renal proximal tubule epithelial cells, which showed that MIF was present in the cytoplasm of the epithelial cells. These results suggest the possibility that MIF takes part in the mechanism of inflammation and immunological events in the human kidney.

Proto-oncogene expression in human glomerular diseases.

The expression of the protein products and mRNA of c-fos, c-myc, p53, and c-raf was examined in normal renal tissues and biopsy specimens from 73 patients with various glomerular diseases. Immunofluorescent staining showed that there were cell nuclei stained for c-Fos, c-Myc, and p53, and cytoplasm positive for c-Raf, in the glomeruli of patients with proliferative types of glomerulonephritis, including IgA nephritis and lupus nephritis, and in patients with focal glomerular sclerosis. Glomerular expression of c-fos and c-myc mRNA was detected by in situ hybridization. The number of proto-oncogene-positive glomerular cells was significantly higher in lupus nephritis, IgA nephritis, and focal segmental sclerosis, as compared with minimal change nephrotic syndrome and normal specimens. In IgA nephritis, the population of glomerular cells positive for c-Fos and c-Myc and the grade of c-Raf immunoreactivity were significantly correlated with the proportion of proliferating cell nuclear antigen (PCNA)-positive glomerular cells, with histological grading of mesangial hypercellularity and matrix increase, and with the magnitude of proteinuria. These data indicate that proto-oncogene expression is associated with mesangial proliferation and matrix expansion in proliferative types of glomerulonephritis and in focal glomerular sclerosis.

Three novel AVPR2 mutations in three Japanese families with X-linked nephrogenic diabetes insipidus.

We identified three novel mutations of the arginine vasopressin (AVP) V2 receptor (AVPR2) gene in Japanese families with X-linked congenital nephrogenic diabetes insipidus (NDI). In kindred #1 of siblings, a single base deletion of one out of three guanosines (nucleotides 786-788, 786delG) was detected. This deletion shifts the reading frame with an altered amino acid sequence and introduces a premature stop codon (TGA) at position 270. In kindred #2 of siblings and one unrelated additional patient (patient #3), point mutations that change the same Pro residue at codon 322 in the seventh transmembrane domain to either a Ser or His (P322S or P322H) were detected. This P322 residue is well conserved among rat V1 and V2 receptors, the human oxytocin receptor, and other G protein-coupled receptors, and is thought to be important for proper insertion of the receptor into the membrane. The AVPR2 mutations are heterogeneous both in Japanese and Caucasians populations.

[Gas chromatographic analysis of reduction products of paraquat, diquat and the related compounds: reductive cleavage in the pyridine ring on N-alkylpyridinium derivatives with NaBH4-NiCl2 reduction system, and inhibition of the cleavage].

When N-alkylpyridinium derivatives were reduced with sodium borohydride-nickel (II) chloride reduction system, reductive cleavage occurred at the C-N bond in the pyridine ring of N-alkylpyridinium derivatives to give a small amount of reductive cleavage product along with the major perhydrogenated product. It was presumed in the previous report that this reductive cleavage in the pyridine ring proceeded through a complex of nickel ion and 1,2,3,6-tetrahydropyridine derivatives produced with NaBH4 alone reduction. The abundances of these reductive cleavage products arising from N-alkylpyridinium derivatives, i.e., paraquat, diquat and so on, are capable of giving a bad effect on the accuracy of gas chromatographic analysis. For the purpose of inhibition of the reductive cleavage in this reduction system, a suitable catalyst was examined. In addition, we pursued whether borane-1,2,3,6-tetrahydropyridine derivative complexes arose from N-alkylpyridinium derivatives by NaBH4 alone reduction or not, and whether these borane-amine complexes were the precursors of reductive cleavage products or not. N-Alkyl-1,2,3,6-tetrahydropyridine derivatives (III-I, IV-I, VI-I, VII-I and VIII-I) and the corresponding borane-amine complexes (III-II, IV-II, VI-II, VII-II and VIII-II) were synthesized by NaBH4 reduction in aqueous solution of N-alkylpyridinium salts, i.e. I, II, 1,4-dimethylpyridinium iodide (III), 1-dodecylpyridinium chloride (IV), 1,1'-diethyl-4,4'-dipyridinium dichloride (V), 1-methyl-4-phenylpyridinium iodide (VI), 1-n-propylpyridinium iodide (VII) and 1-n-butylpyridinium iodide (VIII). The structure of the borane-amine complexes were proved by the Mass spectrometry and 1H- and 13C-NMR analysis. The NiCl2-NaBH4 reduction of the borane-amine complexes gave the perhydrogenated products alone, but not reductive cleavage products. In conclusion, it was recognized that the precursors of reductive cleavage products were not borane-amine complexes, but 1,2,3,6-tetrahydropyridine. Furthermore, it was found the reductive cleavage at the C-N bond in the pyridine ring of these 1,2,3,6-tetrahydropyridine derivatives was hindered by applying Amberlite-Ni2B, NaBH4 reduction system.

Type IV collagen alpha 5 chain. Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody.

Although the evidence indicates that mutation of the gene for the alpha 5 chain of type IV collagen, alpha 5-(IV), is the primary defect in X-linked Alport syndrome, protein data for the alpha 5(IV) chain with regard to its normal distribution and its distribution in patients with Alport syndrome is lacking. We produced a rat monoclonal antibody (H51) by immunizing rats with a synthetic peptide corresponding to the nonconsensus amino acid sequence of alpha 5(IV) NC1 domain. H51 reacted by Western blotting with 26-kd cationic monomers and associated dimers of human type IV collagen NC1 domain. Immunohistochemical studies demonstrated that in normal human kidney alpha 5(IV) was present in the glomerular basement membrane and basement membranes of the Bowman's capsule and in some tubules (collecting ducts). The alpha 5(IV) chain was also detected in the basement membranes of normal skin, eye, and lung. Male patients with X-linked Alport syndrome revealed no reactivity of renal and epidermal basement membranes with H51, whereas alpha 5(IV) staining was normal in the glomerular basement membrane of patients with other types of glomerular diseases, including benign familial hematuria. The staining was also normal in the skin of nonaffected males in X-linked Alport families. Female heterozygous for Alport syndrome exhibited a discontinuous or mosaic pattern in the immunofluorescent staining of the epidermal basement membrane. These findings confirm that in patients with X-linked Alport syndrome there are abnormalities in alpha 5(IV) in renal and epidermal basement membranes at the protein level. Immunofluorescent staining of skin biopsies with this antibody may be of value in making a diagnosis of Alport syndrome, and, furthermore, may aid in detecting carrier females in whom urinary abnormalities are often mild or silent.

Current topics in childhood lupus nephritis.

Lupus nephritis is a major predictor of the prognosis of systemic lupus erythematosus (SLE). The present paper discusses lupus nephritis from clinical and immunopathological points of view. Although recent advances in diagnosis and treatment improve the prognosis of children with SLE, there remain many unsolved clinical problems. One of the current topics in the treatment for SLE is intermittent intravenous cyclophosphamide therapy which is effective even for the steroid-resistant patients with severe lupus nephritis, at least for short-term observation. Immunopathologically, the following issues are discussed: (i) The C5b-9 terminal complement complex plays an important role in the pathogenesis of lupus nephritis. The possible interaction of vitronectin and SP-40,40 is also mentioned; (ii) A semi-quantitative analysis of the charge barrier of the glomerular basement membrane reveals that the charge barrier dysfunction plays an important role in the pathogenesis of proteinuria in lupus nephritis. This study also demonstrates that the charge of immune deposits is important for the initiation of glomerular injury in lupus nephritis; (iii) It is demonstrated that the histopathological diversity of lupus nephritis is based on biological properties of nephritogenic auto-antibodies in murine lupus models.

IgA nephropathy in patients with congenital C9 deficiency.

The clinical, histologic, and immunopathological findings of three young Japanese males with congenital C9 deficiency and primary IgA nephropathy are reported. The C9 deficiency was discovered either through mass complement screening, or when low hemolytic activity for CH50 and normal C3 levels were detected in plasma. Hematuria and proteinuria were detected at the age of 8 or 9 years as a result of annual urinary screening tests for school children. Renal biopsy showed focal and segmental mesangial proliferation with small epithelial crescents in one patient, and mild, diffuse mesangial proliferation in two. IgA and C3 were deposited predominantly in the mesangial area, and staining for C9 was negative in these patients. Electron microscopy revealed electron dense deposits predominantly in mesangial and paramesangial zones. Immunohistochemical staining in renal biopsy tissues from two patients showed mesangial staining for C5, C8, and S-protein, but staining for C5b-9 neoantigen was completely negative. These results show that the formation of C5b-9 complex is not essential for the induction of human IgA nephropathy, and also for the proliferation of mesangial and even parietal epithelial cells.

Acute serum sickness in normal and C6 deficient rabbits: role of membrane attack complex.

Acute serum sickness was induced in New Zealand White (NZW) and in C6 deficient (C6D) rabbits, to compare the histology, immunofluorescence, especially distribution of poly C9 (MAC), and electron microscopic characteristics of the disease in each strain. Glomerulonephritis and albuminuria of comparable extent occurred in 13/17 NZW and 4/8 C6D rabbits. In NZW rabbits with albuminuria an early intense glomerular infiltration by mononuclear cells was associated with focal small fine granular glomerular basement membrane (GBM) deposits of IgG and BSA and more diffuse and larger deposits of C3 and MAC. After the disappearance of monocytes and decrease in mesangial cell proliferation, development of large subepithelial GBM deposits rich in all immune reactants was observed in NZB rabbits. In C6D rabbits with albuminuria a similar monocytic infiltrate occurred, but no association with IgG and C3 GBM immune deposits was noted. No deposits of MAC and no large subepithelial GBM 'humps' were observed in C6D rabbits. We conclude that the exudative (monocytic) phase of glomerular injury and albuminuria in acute serum sickness nephritis are not dependent upon terminal complement components, but the subsequent formation of large subepithelial GBM deposits does not occur in this model in the absence of MAC.