Pufferfish Saxitoxin and Tetrodotoxin Binding Protein (PSTBP) Analogues in the Blood Plasma of the Pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca.

Pufferfish saxitoxin and tetrodotoxin (TTX) binding protein (PSTBP) is a glycoprotein that we previously isolated from the blood plasma of the pufferfish Takifugu pardalis; this protein was also detected in seven species of the genus Takifugu. We proposed that PSTBP is a carrier protein for TTX in pufferfish; however, PSTBP had not yet been found in genera other than Takifugu. In this study, we investigated the presence of PSTBP-like proteins in the toxic pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca. On the basis of ultrafiltration experiments, TTX was found to be present and partially bound to proteins in the plasma of these pufferfish, and Western blot analyses with anti-PSTBP antibody revealed one or two bands per species. The observed decreases in molecular mass following deglycosylation with glycopeptidase F suggest that these positive proteins are glycoproteins. The molecular masses of the deglycosylated proteins detected in the three Arothron species were larger than that of PSTBP in the genus Takifugu, whereas the two bands detected in C. patoca had molecular masses similar to that of tributyltin-binding protein-2 (TBT-bp2). The N-terminal amino acid sequences of 23⁻29 residues of these detected proteins were all homologous with those of PSTBP and TBT-bp2.

Distribution of homologous proteins to puffer fish saxitoxin and tetrodotoxin binding protein in the plasma of puffer fish and among the tissues of Fugu pardalis examined by Western blot analysis.

Puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP) is a glycoprotein (200 kDa as a dimer) that we previously isolated from the plasma of Fugu pardalis (Yotsu-Yamashita et al., 2001). For the study on functions of PSTBP, here we examined distribution of homologous proteins to PSTBP in the plasma of seven species of puffer fish, and among the tissues of F. pardalis by Western blot analysis probed with a polyclonal IgG against unglycosylated PSTBP1 expressed in Echelichia coli. One or two major positive broad bands were detected at 105-140 kDa molecular weight range in the plasma (0.5 microg protein) of all species of puffer fish tested, while no band was detected in the plasma (5 microg protein) of fish other than puffer fish. Glycopeptidase F treated plasma of all species of puffer fish tested commonly showed the bands at approximately 42 kDa that was consistent to the molecular weight of unglycosylated PSTBP. These data suggest that puffer fish commonly possess glycoproteins homologous to PSTBP, but the sizes of N-glycan are specific to the species. Among soluble protein extracts (5 microg protein) from the tissues of F. pardalis, PSTBP was detected in all tissues examined, most prominently in heart, skin, and gall.

A family of generalized epilepsy with febrile seizures plus type 2-a new missense mutation of SCN1A found in the pedigree of several patients with complex febrile seizures.

We report a family with complex febrile seizures (FS). The proband is a 15-year-old boy with seizures that persisted beyond 6 years of age. His father, aunt, and cousin also have the histories of FS until 8, 9, and 8 years old, respectively. A base substitution 5569G-->T of voltage-gated sodium channel alpha-1 subunit gene was found in DNA derived from the affected members of this family.

A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

A family with liddle's syndrome caused by a mutation in the beta subunit of the epithelial sodium channel.

Liddle's syndrome is a rare form of autosomal-dominant salt-sensitive hypertension. Constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel (ENaC) is essential for salt-sensitive hypertension. Recently, several DNA analysis studies have indicated that there is a mutation of C-terminus of either the beta or y subunit. We sequenced the C-termini of the beta and -gamma subunits of the ENaC in a Japanese family with hypertension and hypopotassemia without excess minerarocorticoids, clinically diagnosed as Liddle's syndrome. The mutation of the ENaC of this family was beta R564X. Since such case seem to be rare in the literature, detailed data are shown in this report.

Genetic variation of the gamma subunit of the epithelial Na+ channel and essential hypertension. Relationship with salt sensitivity.

We evaluated the association of a common polymorphism in gammaENaC, consisting in a C to G transversion in codon 649, with essential hypertension and to the pressor response to salt in whites. Two hundred fifteen essential hypertensive patients, and 137 normotensive controls were genotyped for the gamma649 ENaC polymorphism by polymerase chain reaction method and diagnostic restriction enzyme digestion. The genotype distribution of the gamma649 ENaC polymorphism in the hypertensives, 129 CC (60%) and 86 CG/GG (40%) was not significantly different from that of the control group, 84 CC (61%) and 53 CG/GG (39%) (P = .81). Salt sensitivity was assessed in a group of 48 patients by 24-h mean blood pressure response to changes in salt intake. Nineteen patients were diagnosed as salt sensitive, whereas 29 had salt-resistant hypertension. The gamma649 ENaC genotype distribution in salt-sensitive patients was 12 CC (63%) and 7 CG/GG (37%), not significantly different from the distribution in the salt-resistant group, 19 CC (65%) and 10 CG/GG (35%), P = .87. The changes in systolic, diastolic, and mean blood pressure as measured by ambulatory blood pressure monitoring, and in plasma renin activity and plasma aldosterone induced by high salt diet were not different among the gamma649 ENaC genotypes. In the present study we found no association between the gamma649 ENaC polymorphism and essential hypertension or salt sensitivity. Although these data do not support a major causative role for this polymorphism, we cannot exclude that a functional mutation elsewhere in ENaC might be associated with essential hypertension.

Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles.

A tetrodotoxin binding protein has been purified from the plasma of the puffer fish kusafugu, Takifugu niphobles, through DEAE-cellulose treatment, ammonium sulfate fractionation, Sephadex gelfiltrations and Sephacryl S-200 and Cellulofine A-500 column chromatography. Final purification by HPLC on a TSK G-3000 SL column yielded a protein which showed only a single protein peak. The molecular weight of the protein was estimated to be 116,000 and 91,000 by SDS-PAGE and mass spectrometry, respectively. A blast search on the amino-terminal amino acid sequence of the purified protein revealed that the protein had no homology to any other protein on data base.

The effect of dietary magnesium supplementation on the cellular abnormalities of erythrocytes in patients with beta thalassemia intermedia.

BACKGROUND AND OBJECTIVE: Reduced serum or erythrocyte Mg have been reported in human beta thalassemia. These deficiencies may play a role in the cellular abnormalities characteristic of this disorder. We have therefore studied the effect of dietary Mg supplementation in patients with beta thalassemia intermedia in order to establish whether it improves the abnormalities of thalassemic erythrocytes. DESIGN AND METHODS: Plasma and erythrocyte Mg were determined in 11 patients with b thalassemia intermedia, not requiring chronic transfusion therapy, and in 17 normal controls. Inclusion criteria included normal renal and liver function and performance status of 70% or greater. Seven patients were enrolled for the Mg supplementation study, after the appropriate informed consent was obtained. They were given a starting dose of 0.6 mEq/kg/day of magnesium pidolate, divided into two oral daily doses, for four weeks. In a 70-kg subject, a daily Mg dose of 42 mEq corresponds to 504 mg of Mg, with the daily Mg intake of normal subjects being 418 +/- 120 mg for males and 343 +/- 94 mg for females. After 28 days of treatment, five of the patients continued the protocol with a daily dosage increased to 1.2 mEq magnesium pidolate/kg/day, divided into two oral administrations, for an additional four weeks. RESULTS: In patients with untransfused beta thalassemia intermedia we found reduced erythrocyte Mg (in mmol/kg Hb, 6.12 +/- 1.5, n = 11 vs. 8.69 +/- 0.89, n = 17, respectively, p < 0.0001) and normal serum Mg. In the seven patients given oral Mg supplements, at Mg dosages of 0.6 mEq/kg/day we observed significant increases in erythrocyte Mg, and significant improvement in some of the characteristic abnormalities of beta that erythrocytes (increased Na-K pump, KCl cotransport, cell dehydration, increased osmotic resistance). These changes were maintained in the 5 patients who were treated with 1.2 mEq of Mg/kg/day. Follow-up studies showed a return to baseline conditions. There were no signs of Mg toxicity, with the only side effect being diarrhea, which was generally mild, but led to discontinuation for one patient after the first four weeks. INTERPRETATION AND CONCLUSIONS: These data indicate that dietary Mg supplementation improves some of the characteristic cellular function abnormalities of b thalassemia intermedia. The possible therapeutic value of this strategy should be further tested in these patients.

The effects of tert-butyl hydroperoxide on human erythrocyte membrane ion transport and the protective actions of antioxidants.

The oxidising actions of tert-butyl hydroperoxide (tBH) (0-3 mmol/l) on human erythrocyte membrane ion transport have been studied using measurements of 86Rb+ influx. Ouabain and bumetanide were used to distinguish Rb+ flux via the sodium pump (Na,K-ATPase), Na,K,2Cl cotransporter and through residual membrane permeability. The protective actions of antioxidants and related molecules (vitamin E, vitamin E acetate, Trolox, butylated hydroxytoluene (BHT) and dithioerythritol (DTE) were studied. The effects of tBH were concentration dependent and the mean residual (ouabain and bumetanide insensitive) Rb+ permeability was increased by a factor of 8.5 (S.E.M. 2.2, n = 15) by a 5-min exposure to 2 mmol/l tBH. This action was almost completely prevented by co-incubation with Trolox or BHT, and partially prevented by the presence of vitamin E or DTE. Incubation with 2 mmol/l tBH for 5 min increased intracellular Na+ by a factor of 1.8 (S.E.M. 0.1, n = 8) and reduced intracellular K+ by a factor of 0.93 (S.E.M. 0.03, n = 8). These effects were prevented by Trolox and partially prevented by vitamin E, whereas DTE and vitamin E acetate were ineffective. Incubation with 2 mmol/l tBH for 5 min reduced the mean apparent sodium pump Vmax by 43% (S.E.M. 4, n = 8). This effect was completely prevented by Trolox and partially prevented by vitamin E. Vitamin E acetate had no effect. The mean bumetanide-sensitive Rb+ influx via the Na,K,2Cl cotransporter was reduced by 30% (S.E.M. 8.7, n = 25) by a 5-min exposure to 2 mmol/l tBH. This action was variable and no significant actions of the antioxidants studied could be demonstrated. This study suggests that tBH-mediated oxidative damage occurs from a hydrophilic site and involves increased non-selective membrane cation permeability and inhibition of specific transport systems.

Lymphocytic and erythrocytic k+, Na+, Ca++, Mg++ content in patients with congestive heart failure.

The content of lymphocytic and erythrocytic K+, Na+, Ca++, Mg++ changed in patients with congestive heart failure, related to clinical treatment and prognosis. The longer the course of disease, the more significant the changes, with no relation to the etiology of heart disease. The changes were partially improved after treatment of the heart failure. The content of electrolytes is not significantly different in patients in NYHA Class III, compared to those in NYHA Class IV. The metabolic changes of electrolytes occur on the myocardial cells, skeletal muscle cells and peripheral erythrocytes in patients with congestive heart failure. However, serum electrolyte content does not reflect precisely overall electrolyte metabolism in the body. Whether similar changes of electrolyte content occur in the peripheral lymphocytes is rarely reported. We studied the lymphocytic and erythrocytic electrolyte content in patients with congestive heart failure, to provide some reference in the clinical treatment and prognosis of congestive heart failure.