Shuichiro Maeda

Cloning and sequence analysis of cDNA for human prealbumin.

A cDNA coding for human prealbumin has been cloned from a cDNA library prepared from human liver. The DNA sequence codes for a polypeptide which consists of 147 amino acids including a whole human prealbumin sequence and a putative signal sequence. The elucidation of this prealbumin cDNA sequence is expected to facilitate a prenatal diagnosis of familial amyloidotic polyneuropathy.

Studies on the Metabolism of Retinol and Retinol-binding Protein in Transthyretin-deficient Mice Produced by Homologous Recombination

Tissue needs for retinoids are believed to be satisfied through the delivery in the circulation of retinol by its specific plasma transport protein, retinol-binding protein (RBP), which circulates as a 1-to-1 protein complex with transthyretin (TTR). The binding of RBP to TTR is thought to prevent filtration of retinol-RBP in the kidney and to play a role in secretion of RBP from hepatocytes. Recently a strain of mice (TTR-) that totally lacks immunoreactive TTR was produced by targeted mutagenesis. We have explored the effects of TTR deficiency on retinol and RBP metabolism in this mutant strain. In pooled plasma from the TTR- mice retinol levels averaged 6% of those of wild type animals. Similarly, plasma RBP in the TTR- mice was found to be 5% of wild type levels. Hepatic retinol and retinyl ester levels were similar for mutant and wild type mice, suggesting that the mutation affects neither the uptake nor storage of dietary retinol. Levels of retinol and retinyl esters in testis, kidney, spleen, and eye cups from TTR- mice were normal. Plasma all-trans-retinoic acid levels for the TTR- mice were 2.3-fold higher than those of wild type (425 versus 190 ng/dl). Kidney RBP levels were similar for the mutant and wild type mice and we were unable to detect intact RBP in urine from TTR- mice. Hepatic RBP levels in the TTR- mice were 60% higher than those of wild type mice (39.8 versus 25.0 micrograms of RBP/g of tissue). These data may suggest that there is a partial blockage in RBP secretion from TTR- hepatocytes that leads to lessened plasma levels of retinol-RBP.

Presence of mitochondrial-DNA-like sequences in the human nuclear DNA

Two lambda phage clones carrying contiguous human nuclear DNA sequences with extensive homology to non-contiguous human mitochondrial 16S ribosomal RNA sequences were isolated from a human gene library. The one clone carried mitochondrial-DNA(mtDNA)-like sequences flanked with two kinds of repetitive nuclear DNA sequences and the other carried mtDNA-like sequences, between unique nuclear DNA sequences and repetitive DNA sequences of Alu-family. These results demonstrate that mtDNA-like sequences are present in human nuclear DNA.

Targeted conversion of the transthyretin gene in vitro and in vivo

Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.

Familial amyloidotic polyneuropathy diagnosed by cloned human prealbumin cDNA

A diagnosis of familial amyloidotic polyneuropathy (FAP) can be made by use of restriction endonuclease Nsi I, a cloned human prealbumin cDNA and Southern blot procedures. Digests of DNAs from 10 disease-free individuals showed two bands (6.6 kb and 3.2 kb) complementary to a human prealbumin cDNA, whereas digests from 11 individuals with FAP exhibited two additional bands (5.1 kb and 1.5 kb). We interpret these changes in pattern to be the result of a restriction site for NsiI located in the altered codon and associated with the mutant prealbumin gene. All these individuals with FAP were heterozygous for the prealbumin gene, carrying one normal and one mutant gene.

Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant

The mechanism of amyloid formation in familial amyloidotic polyneuropathy (FAP), a hereditary disorder associated with mutant transthyretin (TTR), is still unknown. It is generally believed that altered conformations exposing cryptic regions are intermediary steps in this mechanism. A TTR mutant—Y78F (transthyretin mutant with phenylalanine replacing tyrosine at position 78)—designed to destabilize the native structure has been shown to expose a cryptic epitope recognized by a monoclonal antibody that reacts only with highly amyloidogenic mutants presenting the amyloid fold or with amyloid fibrils. To test whether TTR deposition in FAP can be counteracted by antibodies for cryptic epitopes, we immunized with TTR Y78F, transgenic mice carrying the most common FAP-associated TTR mutant—V30M (transthyretin mutant with methionine replacing valine at position 30)—at selected ages that present normally with either nonfibrillar or TTR amyloid deposition. Compared to age-matched control nonimmunized mice, Y78F-immunized mice had a significant reduction in TTR deposition usually found in this strain, in particular in stomach and intestine; by contrast, animals immunized with V30M did not show differences in deposition in comparison with nonimmunized mice. Immunohistochemical analyses of tissues revealed that immunization with Y78F lead to infiltration by lymphocytes and macrophages at common deposition sites, but not in tissues such as liver, choroid plexus, and Langerhans islets, in which TTR is produced. These results suggest that Y78F induced production of an antibody that reacts specifically with deposits and leads to an immune response effective in removing/preventing TTR deposition. Therefore, TTR immunization with selected TTR mutants has potential application in immune therapy for FAP.

A novel transthyretin mutation associated with familial amyloidotic polyneuropathy.

We characterized the mutation associated with familial amyloidotic polyneuropathy in a Japanese patient. Sequence analysis of polymerase chain reaction-amplified exons of the transthyretin gene revealed a novel point mutation resulting in a substitution of arginine for glycine at position 47. The mutation was confirmed using allele-specific olgonucleotide hybridization procedures. This most likely represents a de novo mutation since neither parent carries the mutant allele.

Transthyretin Accelerates Vascular Aβ Deposition in a Mouse Model of Alzheimer's Disease

Transthyretin (TTR) binds amyloid‐β (Aβ) and prevents Aβ fibril formation in vitro. It was reported that the lack of neurodegeneration in a transgenic mouse model of Alzheimers disease (AD) (Tg2576 mouse) was associated with increased TTR level in the hippocampus, and that chronic infusion of anti‐TTR antibody into the hippocampus of Tg2576 mice led to increased local Aβ deposits, tau hyperphosphorylation and apoptosis. TTR is, therefore, speculated to prevent Aβ pathology in AD. However, a role for TTR in Aβ deposition is not yet known. To investigate the relationship between TTR and Aβ deposition, we generated a mouse line carrying a null mutation at the endogenous TTR locus and the human mutant amyloid precursor protein cDNA responsible for familial AD (Tg2576/TTR−/− mouse) by crossing Tg2576 mice with TTR‐deficient mice. We asked whether Aβ deposition was accelerated in Tg2576/TTR−/− mice relative to the heterozygous mutant Tg2576 (Tg2576/TTR+/−) mice. Contrary to our expectations, the degree of total and vascular Aβ burdens in the aged Tg2576/TTR−/− mice was significantly reduced relative to the age‐matched Tg2576/TTR+/− mice. Our experiments present, for the first time, compelling evidence that TTR does not suppress but rather accelerates vascular Aβ deposition in the mouse model of AD.

Cloning and sequence analysis of a cDNA for lymphocyte proliferation potentiating factor of rabbit polymorphonuclear leukocytes: Identification as rabbit interleukin 1β

Abstract Complementary DNA for a rabbit PMN-derived lymphocyte proliferation potentiating factor (PMN factor) was cloned from a cDNA library constructed from poly(A)+RNA of early inflammatory exudate PMN. Oligodeoxyribonucleotides as probes for cloning were synthesized according to the previously reported amino acid sequence of the purified PMN factor. This cDNA encodes a 268-residue protein which is homologous to the established structures of human IL 1 β (74%) or murine IL 1 β (71%). The NH2-terminal amino acid sequence of the endogenously produced PMN factor indicates that the mature molecule is made up of carboxy terminal 152 amino acids of the precursor molecule. Taking into consideration the previous studies on biological activities, this cloned PMN factor is therefore considered to be rabbit IL 1β.

Prealbumin gene expression during mouse development studied by in situ hybridization

Localization of prealbumin mRNA in tissues from mice at various stages of gestation was investigated using in situ hybridization procedures. Prealbumin mRNA was detected as early as the 10th day of gestation. It was specifically localized in endodermal cells of the visceral yolk sac, tela choroidea, and hepatocytes. In the adult mice, prealbumin mRNA was localized in the hepatocytes and choroid plexus epithelial cells. These observations indicate that synthesis of prealbumin mRNA is initiated in several different types of cells at early stages of fetal development.