GM1 ganglioside-bound amyloid beta-protein (A beta): a possible form of preamyloid in Alzheimer's disease.

The earliest event so far known that occurs in the brain affected with Alzheimer's disease (AD) is the deposition and fibril formation of amyloid beta-protein (A beta). A beta is cleaved from a glycosylated membrane protein, called beta-amyloid protein precursor, and normally secreted into the extracellular space. Here we report on the presence of membrane-bound A beta that tightly binds GM1 ganglioside. This suggests that this novel A beta species, rather than secreted A beta, may act as a 'seed' for amyloid and further that intracellular abnormalities in the membrane recycling already exist at the stage of amyloidogenesis.

An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants.

Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete increased amounts of long A beta, thereby fostering amyloid deposition.

G protein-mediated neuronal DNA fragmentation induced by familial Alzheimer's disease-associated mutants of APP.

Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation required the cytoplasmic domain of the mutants and appeared to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins).

Visualization of A beta 42(43) and A beta 40 in senile plaques with end-specific A beta monoclonals: evidence that an initially deposited species is A beta 42(43).

To learn about the carboxy-terminal extent of amyloid beta-protein (A beta) composition of senile plaques (SPs) in the brain affected with Alzheimer's disease (AD), we employed two end-specific monoclonal antibodies as immunocytochemical probes: one is specific for A beta 40, the carboxyl terminus of A beta 1-40, while the other is specific for A beta 42(43). In the AD cortex, all SPs that were labeled with an authentic antibody were A beta 42(43) positive, while only one-third of which, on the average, were A beta 40 positive. There was a strong correlation between A beta 40 positivity and mature plaques. Two familial AD cortices with the mutation of beta-amyloid protein precursor 717 (beta APP717) (Val to Ile) showed a remarkable predominance of A beta 42(43)-positive, A beta 40-negative plaques. Diffuse plaques, representing the earliest stage of A beta deposition, were exclusively positive for A beta 42(43), but completely negative for A beta 40.

The beta APP717 Alzheimer mutation increases the percentage of plasma amyloid-beta protein ending at A beta42(43).

We measured plasma levels of amyloid beta protein (A beta) ending at positions 40 (A beta40) and 42(43) [A beta42(43)] in six carriers of beta APP717 (Val to Ile) mutation linked to familial Alzheimer's disease (FAD) as well as in patients with sporadic AD (sAD) and controls. The percentage and the level of A beta42(43) were significantly higher in carriers of beta APP717 mutation relative to sAD, whereas A beta40 levels were decreased. In contrast, A beta levels and ratios were at similar levels in sAD, regardless of the stage of the disease, compared with non-AD neurologic disease controls and nondemented control individuals. These results suggest that the reported increase in the percentage of A beta42(43) secretion in transfected cells with beta APP717 mutant genes actually takes place in the bodies of carriers of beta APP717 mutation, and that plasma A beta could be used as an indicator of the alterations of beta APP/A beta metabolism in subtypes of AD.

Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties.

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.

Fractionation of amyloid beta-protein (A beta) in Alzheimer's disease and Down's syndrome brains. Presence of membrane-bound A beta.

To investigate the early stage of beta-amyloidogenesis in Alzheimer's disease (AD), we performed sucrose density gradient fractionation of amyloid beta-protein (A beta) deposited in cerebral cortices from AD and Down's syndrome patients and normal aged individuals. Each fraction was subjected to Western blotting with monoclonal antibodies BA27 and BC05, which specifically recognize A beta 40 and A beta 42, respectively. The samples from brains with a large number of diffuse plaques showed strong BC05 immunoreactivity at the 1.0/1.2 M (F1) and 1.2/1.5 M (F2) interfaces. In contrast, in brains with advanced AD pathology, most of the BC05 immunoreactivity was recovered at the 1.5/2.0 M (F3) interface. In all cases, the level of BA27 immunoreactivity was negligible. Although F1A beta was determined to be A beta 1-42, it was only weakly reactive with BAN50 (monoclonal antibody raised against A beta 1-16). Delipidation of F1A beta restored full BAN50 immunoreactivity, indicating that F1A beta is bound to membrane. The present results suggest that diffuse plaques are associated with this membrane-bound A beta and thus that this novel A beta species is an initially deposited one.

Amyloid beta protein 1-42/43 (A beta 1-42/43) in cerebellar diffuse plaques: enzyme-linked immunosorbent assay and immunocytochemical study.

Diffuse plaques are immature and amorphous senile plaques and believed to be in the initial phase of plaque formation. In contrast to amyloid angiopathy and the plaque core amyloid, diffuse plaques failed to be purified in preserved forms from the brain. Here, we studied the diffuse plaques in the cerebellar region of the Alzheimer's disease brain based on immunocytochemistry and ELISA using two different monoclonal antibodies specifically recognizing the carboxyl termini of A beta molecules (BA27 for A beta 1-40 and BC05 for A beta 1-42/43). We found that the amount of A beta 1-40 was in proportion to the staining degree on amyloid angiopathy by immunohistochemistry. We found that A beta 1-42/43 comprised diffuse plaques as the major component in the cerebella of AD brains. Taking these findings into consideration, diffuse plaques, the earliest pathological change in the brain with AD, are concluded to be composed mainly of A beta 1-42/43, implicating the critical importance of this kind of A beta species deposition in the pathogenesis of AD.

The extent of amyloid deposition in brain in patients with Down's syndrome does not depend upon the apolipoprotein E genotype.

The extent of deposition of amyloid beta protein (A beta) was investigated in 20 elderly patients with Down's syndrome, using the end-specific monoclonal antibodies BC05 and BA27 to detect the presence of A beta 42(43) and A beta 40 (respectively), and related to apolipoprotein E (ApoE) genotype. No significant differences in the amount of A beta deposited in the brain, either as A beta 42(43) or A beta 40, were noted in patients possessing an ApoE E4 allele, compared to those without. Patients with an ApoE E4 allele in general died at an earlier age than those with only ApoE E3 alleles, the latter in turn being outlived by those with an ApoE E2 allele. In Down's syndrome therefore, ApoE may influence the timing of onset, or the rate of progression, of disease but without affecting the type or total amount of pathology accumulated.

Laparoscopic gastropexy for acute gastric volvulus: a case report.

A 7-year-old girl presented with an acute gastric volvulus that was reduced with a nasogastric catheter. An anterior gastropexy was undertaken laparoscopically. The gastrocolic omentum was deficient along most of the greater curvature, which had allowed organoaxial volvulus. Two years later, gastric volvulus has not recurred. Laparoscopy is an acceptable approach for the evaluation and treatment of children with acute gastric volvulus.

[Effect of beta-glycyrrhizinate on eye mucosa absorption of drugs].

Dipotassium beta-glycyrrhizinate (GK2) is a saponin originated from "kanzo" plant. We studied such two functions of GK2 as a drug carrier and a penetration enhancer on the eye mucosa. The interactions of GK2 with various drugs were investigated using the 1-octanol/water partition method. GK2 increased the partition of cationic drugs, i.e., antihistamines (chlorpheniramine maleate (CM), diphenhydramine hydrochloride (DH)), as well as decongestants (naphazoline hydrochloride (NA), tetrahydrozoline hydrochloride (TH)). The carrier effect of GK2 is remarkable at its concentration above critical miceller concentration (CMC) and, especially at its pH value of between 4 to 5, resulting that transfers of drugs increased 2 to 10 times as compared to drugs without GK2. In vivo experiments were carried out using rats and rabbits. The effect of GK2 on the inhibition efficiency by CM was evaluated using experimental conjunctivitis formed by injection of histamine on rat upper eyelid. The Inhibition efficiency of edema by CM with GK2 was 4 times stronger than that without GK2. Cornea permeability of TH increased 1.8 times by the addition of GK2. These results indicated that GK2 is applicable as a carrier of cationic drugs on the eye mucosa and cornea.

[Effect of peroral doxifluridine plus hepatic arterial infusion for synchronous liver metastasis of colorectal cancer--correlation with the expression of thymidine phosphorylase and dihydropyrimidine dehydrogenase in primary colorectal cancer lesions].

We investigated whether the efficacy of peroral doxifluridine and hepatic arterial 5-FU infusion on synchronous liver metastasis of colorectal cancer could be predicted based on the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in the primary colorectal lesions. Ten patients with synchronous liver metastasis of colorectal cancer were given doxifluridine (600-800 mg/body/day) orally and 5-FU (500 mg/body, once or twice a week) through the hepatic artery following resection of the primary lesions between June 1996 and July 2001. The levels of TP and DPD in the primary lesions were determined by an enzyme-linked immunosorbent assay. The level of TP, DPD, and the ratio of TP/DPD in patients with partial response (n = 4) were 89.8 +/- 30.0 U/mg protein, 23.5 +/- 25.7 U/mg protein, and 3.8 +/- 1.4, respectively, while those in patients with no response or progressive disease (n = 6) were 41.8 +/- 9.7 U/mg protein, 25.8 +/- 15.8 U/mg protein, and 2.2 +/- 1.6, showing significant difference (p < 0.01) in the level of TP between the groups. These results indicate that determining the level of TS in primary colorectal lesions may be useful for predicting the efficacy of this regimen for patients with synchronous liver metastasis of colorectal cancer.

[Combination chemotherapy with irinotecan hydrochloride plus carboplatin for patients with advanced or recurrent colorectal cancer--a pilot study].

A pilot study was performed to evaluate the feasibility and efficacy of irinotecan hydrochloride (CPT-11) plus carboplatin (CBDCA) for treatment of advanced or recurrent colorectal cancer. Fifteen patients with colorectal cancer (nonresectable, 1; noncurative resection, 5; recurrent disease, 9) were treated with CPT-11 (40-50 mg/m2) plus CBDCA (70-100 mg/m2) once a week for 2-3 weeks followed by a one-week rest. This treatment was repeated until disease progression or severe toxic effects were found. The total dose of CPT-11 ranged from 135 to 1,214 (median, 467) mg/m2 and that of CBDCA ranged from 267 to 2,022 (median, 933) mg/m2. Adverse effects included nausea (grade 2) in 2 (13.3%) diarrhea (grade 2) in 2 (13.3%), leukopenia (grade 3) in 2 (13.3%), thrombocytopenia (grade 1) in one (6.7%), and hair falling (grade 3) in one (6.7%). The response rate of 14 evaluable patients was 14.3% (CR, 1; PR,1; NC,7; PD,5). The median survival time of all patients was 405 days from the start of chemotherapy. The survival time of patients with CR, PR, and NC (n = 9) tended to be longer than that of those with PD (n = 5) (p = 0.06). The median time to disease progression was 105 days. These results suggest that this combination chemotherapy is feasible and effective in the treatment of advanced or recurrent colorectal cancer.

[Clinical trial of prophylactic hepatic arterial chemotherapy for liver metastases in patients with Dukes' C colorectal cancer].

Preliminary results of a prospective non-randomized trial of prophylactic hepatic arterial chemotherapy for liver metastases from colorectal cancer are presented. Twenty-two colorectal cancer patients (infusion group) in Dukes' C stage were given hepatic arterial infusion of 5-FU (500 mg/body for 1 hr per week, repeated 50 times) and peroral UFT-E (2.0 g/body, daily). Informed consent was obtained from all patients. Adverse effects and postoperative recurrence in the infusion group were compared with those of patients with UFT-E alone (control group). Complications related to hepatic arterial infusion in the infusion group were also demonstrated. There was no adverse effect in the control group, while diarrhea (grade 1) developed in one patient (5%) and pigmentation in five (24%) in the infusion group. Complications related to infusion were found in five patients (5%). Three patients in the infusion group presented with metachronous hepatic lesions, two of which were resected successfully. In the control group, one patient died of marked hepatic metastases, and one developed ovarian metastasis with lymph node involvement. Our regimen appears hematologically safe, however, the high frequency of pigmentation should be kept in mind. The oncological benefit of this chemotherapy would be clarified by a larger series of cases and longer follow-up.

[A case of external supravesical hernia--repair with laparoscopic surgery].

We report a case of external supravesical hernia. A 39-year-old Japanese male with history of bilateral herniorrhaphy visited our hospital with complaint of uncomfortable thumb-tip-sized subcutaneous mass cephalad and lateral to the pubic bone. Hernia port was observed laparoscopically in the right supravesical fossa, establishing the diagnosis of right external supravesical hernia. The patient received laparoscopic herniorrhaphy with fixing of polypropylene mesh on the preperitoneal layer and he was discharged on the third postoperative day. This patient seems the first case of external supravesical hernia ever receiving herniorraphy based on laparoscopic findings from inside the abdominal cavity.

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.

c-jun inhibited the alternative splicing of neuron-specific amyloid precursor protein, but stimulated the non-neuron type one in P19 EC cells.

Three alternative splicing products of amyloid precursor protein (APP), APP770, 751 and 695, were detected in mouse embryonal carcinoma (EC) P19 cells by reverse transcriptase RNA polymerase chain reaction (RT-PCR). Alternative splicing of APP pre-mRNA in P19EC cells was remarkably changed by c-jun transformation. The relative ratio of APP770 encoding exons 7 and 8, non-neuron type, was increased by c-jun transformation, while that of APP 695 not encoding exons 7 and 8, neuron-specific one, was decreased. These results suggested that skipping of exons 7 and 8 was specifically blocked in c-jun transformed cells. APP 695, which increases in P19 EC cells under the culture conditions that induce the neuronal differentiation, did not increase in C2C5 cells under the same conditions, suggesting that c-jun transformed cells were not in the neuronal cell lineage and lost the ability to differentiate into neurons.

Carbon-13 NMR study of switch variant anti-dansyl antibodies: antigen binding and domain-domain interactions.

A 13C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical VH, VL, and CL domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of these antibodies has been selectively labeled with 13C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by following the procedure described previously for the switch variant antibodies labeled with [1-13C]Met [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 270-278]. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, we discuss how 13C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.

Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody.

A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain [Takahashi, H., Igarashi, T., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 2840-2847]. A variety of Fv analogues labeled with 2H in the aromatic rings and with 13C and/or 15N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv in the absence and presence of epsilon-dansyl-L-lysine (DNS-Lys). It has been shown that 1H-15N shift correlation spectra of Fv sensitively reflect the antigen binding and can be used along with 1H and 13C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been followed in the absence and presence of DNS-Lys by using the 1H-15N shift correlation spectra. Use of the beta-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.

High tissue content of soluble beta 1-40 is linked to cerebral amyloid angiopathy.

We developed two highly sensitive enzyme immunoassays for beta-protein with different specificities. One is specific for beta 1-40, while the other is equally sensitive to beta 1-38, beta 1-39, beta 1-40, and beta 1-42. With the enzyme immunoassays we investigated whether the soluble fraction from brain tissue contains beta 1-40 or other species of beta-protein. Aged control and Alzheimer's diseased brains showed highly variable values of beta 1-40, which was found to be the major beta species in their extracts. High tissue content of soluble beta 1-40 was not correlated to the abundance of senile plaques but was invariably associated with cerebral amyloid angiopathy. Thus, the tissue level of soluble beta 1-40 should be useful for the quantification of cerebral amyloid angiopathy.