Rapid and accurate measurement of anti-A/B IgG antibody in ABO-unmatched living donor liver transplantation by surface plasmon resonance.

High anti-blood group A or B (anti-A/B) immunoglobulin G (IgG) haemagglutination titres are associated with poor graft survival in ABO-unmatched liver transplantation. We have previously reported that the surface plasmon resonance (SPR) method can be used to measure anti-A/B IgG levels in the plasma very quickly and quantitatively. The aim of this study was to brush up this SPR method. The anti-A/B IgG antibodies (Abs) were purified from the plasma of healthy volunteers by affinity chromatography and used to establish standard curves for the SPR and flow cytometry (FCM) methods. The haemagglutination test tube (TT), FCM and SPR methods were then used to measure the changes over time in the anti-A/B IgG titres of 25 ABO-unmatched living donor liver transplantation (LDLT) recipients. The standard curve permitted the SPR values for the anti-A/B IgG titres to be expressed in microg mL(-1) units. The SPR measurements of the anti-A/B IgG levels in the LDLT recipients correlated very well with the FCM values, whereas the TT values correlated poorly with either method. Furthermore, the SPR method accurately detected the effects of plasma exchange. In conclusion, the SPR method is an accurate, time- and labour-saving method for measuring anti-A/B IgG titres that can be easily standardized.

VPA-induced neural tube defects in mice. I. Altered metabolism of sulfur amino acids and glutathione.

Valproate (VPA) has been shown to induce neural tube defects (NTDs) in humans and mice, but the mechanism of action has not been elucidated. Folate supplementation has been reported to prevent the defect. It was the aim of our experiment to reveal effects of VPA and of folate coadministration on amino acid metabolism in an NTD mouse model. After treating pregnant mice intraperitoneally with 2.1 mmol VPA/kg body weight, plasma homocysteine concentrations were found to be increased. Coadministration of 4 mg/kg folate decreased this level. Plasma methionine levels were reduced under both experimental conditions. Fifteen min after treating mice with 3 mmol VPA/kg body weight, hepatic levels of both S-adenosylmethionine (SAM) and S-adenosylhomocysteine were found to be increased by +175% and +348%, respectively; but the levels had normalized again 30 min after VPA injection. Simultaneously, plasma methionine and serine levels had decreased by -43% and -51%, respectively, while homocysteine and cysteine increased by +71% and +81%, respectively. Reduced glutathione (GSH) decreased by -45%, but total glutathione did not change. These changes were statistically significant, and they occurred dose-dependently. We proposed that VPA induces methionine deficiency inhibition of folate metabolism and homocysteine remethylation, increase in aminothiols, and suppression of the GSH system in maternal blood within 1 h after application. These changes may be responsible for the teratogenic potential of VPA. Folate may prevent NTDs by changing homocysteine catabolism.

hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1.

Proteins of the tolloid/bone morphogenetic protein (BMP)-1 family play important roles in the differentiation of cell fates. Among those proteins are BMP-1, which plays a role in cartilage and bone formation in mammals, the TOLLOID protein, which is required for the establishment of the dorsoventral axis of Drosophila embryos and BP10/SpAN, which are thought to act in the morphogenesis of sea urchins. These proteins have some properties in common. First, they contain the astacin metalloprotease domain, the CUB domain and the epidermal growth factor-like domain. Second, they are expressed in embryos at stages expected for their role in cell differentiation. Third, at least BMP-1 and TOLLOID are thought to interact with proteins of the transforming growth factor-beta family. We report that the hch-1 gene of the nematode Caenorhabditis elegans encodes a tolloid/BMP-1 family protein. The protein has the characteristic domains common to the tolloid/ BMP-1 family. Like other members of the family, it is expressed in embryos. However, the phenotype of hch-1 mutants shows that it is required for normal hatching and normal migration of a post-embryonic neuroblast. Furthermore, in spite of its expression in embryogenesis, it is not required for the viability of embryos. These results show new functions of the tolloid/BMP-1 family proteins and give insight into their evolution.

[A case of Guillain-Barré syndrome with ophthalmoplegia showing high titers of anti-GQ1b and -GD1b antibodies in the serum].

A 24-year-old housewife developed double vision, tingling sensation, and weakness in the legs following a flu-like illness. She then developed dysphagia and difficulty in standing. On admission her eyes were fixed in midline due to ophthalmoplegia. Doll's eye sign was negative. She had severe generalized muscle weakness and no sensory deficits. All tendon reflexes were lost. CSF protein was 58 mg/dl with cell count of 2/mm3. Antibodies to campylobacter jejuni, mycoplasma, EBV, and other microbes were negative. We treated her with plasmapheresis after which she showed rapid clinical recovery, although the ophthalmoplegia improved slightly later. Increased titer of IgG class antibodies to GD1b and to GQ1b, and of IgM antibody to GQ1b, were detected in the serum taken during the acute phase of the illness. In parallel with clinical amelioration, both the anti-GD1b and -GQ1b antibodies decreased in titer, or became negative. Since there is no common epitope to GD1b and GQ1b gangliosides, we speculated that the anti-GD1b and -GQ1b antibodies were induced independently by different antigens in this patient. Moreover, the presence of high titer IgM antibody to GQ1b possible indicates that this patient was at the relatively early stage of infection of unknown microbe, which then induced the IgG antibodies to GD1b and GQ1b by cross sensitization, which might correlate with the tetraplegia and the ophthalmoplegia, respectively.

The role of acidic residues in the "fusion segment" of influenza A virus hemagglutinin in low-pH-dependent membrane fusion.

To clarify the role of acidic amino acid residues in the "fusion segment" of hemagglutinin (HA) of influenza A virus (H1N1) in pH-dependent membrane fusion, we have constructed and expressed five mutant HA cDNAs in CV-1 cells by SV40-HA virus vectors (SVHA). Fusion activities of the five mutant HAs were examined by lipid mixing and polykaryon formation assays. In spite of the substitution of Gly and Lys for the acidic residues, all the mutants were found to retain their low-pH-dependent fusion activity by lipid mixing assay. Although SVHA-G19(HA(2)19D-->G), -K11 (HA(2)11E-->K) and -K19(HA(2)19D-->K) induced polykaryon formation at low pH as wild type HA did, SVHA-G11(HA(2)11E-->G) induced limited polykaryon formation and SVHA-G11,19 (HA(2)11E-->G, 19D-->G) did not. The substitution of Gly for Glu at position 11 inhibited widening of the initial fusion pore. However, Lys mutants induced the formation of an initial fusion pore and widened it at low pH where Lys residues might have positive charges. These results suggest that the neutralization of the charges on acidic residues in the "fusion segment" at low pH is not important for interaction of the "fusion segment" with the target lipid bilayer or for triggering the membrane fusion.

Pheochromocytoma with multiple islet cell carcinoma.

We reported a rare case of a 17-year-old female with pheochromocytoma associated with multiple islet cell carcinoma. Pheochromocytoma was identified in the right adrenal gland. Multiple pancreas tumours were demonstrated unpredictably in the diagnostic imaging of the pheochromocytoma. No other endocrinological neoplasm was observed in the pituitary, thyroid and parathyroid gland. The patient underwent right adrenalectomy and total pancreatectomy. Pheochromocytoma was benign, however, pancreas tumours were non-functioning islet cell tumours and histologically malignant. This combination is assumed to represent a mixed form of multiple endocrine neoplasia (MEN) 1 and MEN 2.

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.

[Rapid recovery of chronic inflammatory demyelinating polyneuropathy induced by steroid pulse therapy--changes in nerve conduction].

A 47-year-old female patient with chronic inflammatory demyelinating polyneuropathy started to recover from her numbness and weakness within a few hours following the commencement of intravenous methylprednisolone 1,000 mg. In parallel with the recovery of muscle strength, a prolonged latency time of the M-wave was normalized within a day by a revival of the new motor units with a normal latency. In many cases with CIDP, it has been recognized that the gradual decrease in latency time over weeks is a later phenomenon following early increase in amplitude of the M-wave during recovery of weakness, which is explained by remyelinating process. On the other hand, the revival of motor units with a normal latency time from demyelinating conduction block is difficult to explain by remyelinating process, because remyelinating fibers usually have a very slow conduction velocity. Some minor morphological changes of paranodes or humoral factors may be partly responsible for development conduction block in CIDP.

In vitro fusion of rabbit liver Golgi membranes with liposomes.

Fusion of Golgi membranes isolated from rabbit liver with liposomes was studied by lipid mixing of fluorescent lipid analogues and internal content mixing and by electron microscopic observation of transfer of horseradish peroxidase from liposomes into Golgi membranes. A monoclonal antibody was used to confirm fusion of Golgi membranes but not other contaminating vesicles. Fusion was rapid and efficient, reaching about 20% of the maximum after a 5-min incubation using small or large unilamellar dioleoylphosphatidylcholine vesicles. The fusion was dependent on temperature, decreasing at lower temperatures, and becoming nearly zero below 10 degrees C. The addition of ATP, GTP, cytosolic factors, or N-ethylmaleimide did not affect fusion. Treatments of Golgi membranes with 0.1 M Na2CO3 or 1 M KCl did not cause any changes in fusion. However, treatment with proteases inhibited fusion. These results suggest that Golgi integral membrane protein(s) are involved in fusion. Changing the medium to an isoosmotic substance, sucrose, in place of KCl or NaCl inhibited fusion. The binding assay of fluorescent liposomes to Golgi membranes showed that lowering the temperature or replacing salts with sucrose did not affect binding. However, treatment of Golgi membranes with proteases inhibited binding. Addition of phosphatidylserine or phosphatidylethanolamine to dioleoylphosphatidylcholine liposomes caused a 2-fold increase in binding and fusion. Fusion between Golgi membranes by themselves did not occur. These results provide some information on the mechanism of intracellular vesicular transport.

Persistent hemichorea associated with thyrotoxicosis.

We describe a case with unilateral chorea associated with thyrotoxicosis. A 23-year-old female with no family history of neurological diseases acutely developed choreic movements of the left extremities during gross thyrotoxicosis. CT scan and MRI study demonstrated no abnormality. Single-photon emission CT with technetium Tc 99m-labeled hexamethylpropyleneamine oxime revealed normal cerebral perfusion. Although the choreic movements were partially improved by dopamine antagonist, they persisted for two months until successful treatment of the thyrotoxicosis finally abolished these movements. Increased sensitivity of dopamine receptors may be responsible for persistent choreic movements in thyrotoxicosis.

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.

Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity.

The amphiphilic anionic peptides E5 and E5L can mimic the fusogenic activity of influenza hemagglutinin(HA). These peptides induced fusion of egg yolk phosphatidylcholine small or large unilamellar vesicles only at acidic pH in a similar manner to viral HA. Acetylation or acetimidylation of the N-terminus of the peptides drastically reduced the fusion activity of the intact peptides, while C-terminal amidation left the activity unchanged. The binding assay suggested that the interaction of the modified peptides with lipid membranes was almost unchanged in comparison with those of the parent peptides, and the CD spectra showed that these peptides were alpha-helical. The results showed the importance of the N-terminus of the peptides on the membrane fusion activity, although why the N-terminal modifications affect the activity is still unclear.