Fine structures of wing scales in Sasakia charonda butterflies as photonic crystals.

We investigate the microstructure of scales in the wings of male Sasakia charonda charonda butterflies by scanning electron microscopy with the aid of optical microscopy. Six types of scales are identified: B1, W1 and R1 in brown background yellow spots and red spots, respectively; B2 in iridescent purple-blue and W2 in white pearl, both of which characterize the male and B3 in the wing edges. The B1, W1 and R1 scales are almost the same in structure and the B2 and W2 scales are almost the same. The difference among the B, W and R scales is in species and content of pigment. The B1, W1 and R1 scales have only two layers of cuticle lapped on the ridges. In contrast with them, the B2 and W2 scales have seven multilayers of cuticle piled on the ridge. The multiple interference of light that occurs among these cuticle layers, spaced with air layers, generates the significant iridescence of the B2 and W2 scales. Thus, the characteristic purple-blue of the male wings is ascribed to the combination of the structural and chemical colouration in the B2 scales with melanin. The photonic crystals of these scales may be applicable to fine light manipulators such as reflection elements in laser diodes. B3 has many holes between the ridges and no multilayers of cuticle on the ridges. These structures may play any role in aerodynamically easy flight and/or in drainage of wet wings.

Identification, characterization, and cloning of TIP-B1, a novel protein inhibitor of tumor necrosis factor-induced lysis.

Some cancer cells evade elimination by virtue of their insensitivity to agents that induce apoptosis. Conversely, the side effects of anticancer agents could be diminished if normal cells were more resistant. To further elucidate the factors that contribute to the susceptibility of a cell to apoptosis, these investigations were designed to identify proteins isolated from cells exposed to low concentrations of tumor necrosis factor (TNF) that, when incubated with normally TNF-sensitive cells, protect these cells from TNF-induced cytotoxicity. TIP-B1, a novel protein, has been identified, purified, and characterized from cytosolic extracts of TNF-treated human fibroblasts. The approximately 27 kDa pI-4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids) and the nucleotide sequence of the corresponding 783-bp cDNA partial clone. Western blot analyses using polyclonal antisera raised against both the purified native TIP-B1 and the approximately 14 kDa product of the cDNA partial TIP-B1 clone, as well as Northern blot analyses using the cDNA insert as a probe, indicate that TIP-B1 may belong to a family of proteins that are expressed in a number of cell lines from diverse tissues. TNF-sensitive cells, when exposed to 4-10 microg/ml concentrations of TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. Preincubation of TIP-B1 with TNF does not affect the ability of TNF to induce lysis. Moreover, TIP-B1 does not seem to interfere with the interactions between TNF and the TNF receptors, based on a preliminary flow cytometric analysis of the cellular binding of biotinylated TNF. On the basis of these characteristics, TIP-B1 is not a soluble TNF receptor, an anti-TNF antibody, nor a protease that degrades TNF; yet TIP-B1 functions when added exogenously to cells. These characteristics, its novel sequence, and its function when added exogenously to cells indicate that TIP-B1 is unique and is not one of the other proteins reported previously to be involved in resistance to TNF. The ability of TIP-B1 to function after exogenous incubation with target cells makes TIP-B1 a likely candidate for therapeutic manipulation of TNF-induced effects.

Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I.

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.

Age-related changes in the proportion of amyloid precursor protein mRNAs in Alzheimer's disease and other neurological disorders.

In the human brain, alternative splicing of amyloid precursor protein (APP) gene transcript generates at least three types of mRNA coding for APP770, APP751 and APP695. The former two types harbor, but the latter one lacks a domain of Kunitz-type serine protease inhibitor (KPI). We studied, by using the RNase protection technique, the expression of APP mRNAs in brains of Alzheimer's disease (AD) and other neurological disorders with special reference to aging. We found that the ratio of (APP770 mRNA+APP751 mRNA)/APP695 mRNA in the frontal cortex increased approximately 1.5-fold in AD compared with other neurodegenerative or cerebrovascular disorders. The ratio in other neurological disorders did not change significantly from control even in their affected brain regions. On the other hand, we found a positive correlation between the ratio and age; the ratio (y) increased gradually with the advance of age (x) as expressed by y = 0.005x + 0.014 (r = 0.372) for the AD group, and y = 0.004x -0.037 (r = 0.486) for the non-AD group. These correlations indicate that the AD brain reached the same ratio of KPI-harboring to lacking APP mRNAs a few decades earlier than the non-AD brain in senescence. This finding of AD-specific and age-related change led us to the idea that a relative increase in KPI-harboring APPs over a KPI-lacking APP may perturb normal degradation of APPs, thereby leading to deposition of beta A4 protein as amyloid.

Structure and expression of a cloned cDNA for human (2'-5')oligoadenylate synthetase.

17S poly(A)+RNA, which hybridized to an oligonucleotide complementary to a part of the partial cDNA (E1cDNA) (Merlin et al. (1983) Proc. Natl. Acad. Sci. U.S. 80, 4904) for 2-5A synthetase, was isolated from interferon-treated human KB cells and used for cDNA cloning. Several overlapping cDNAs were cloned by using the oligonucleotide as a probe. Two of them were joined at their overlapping region, resulting in a cDNA (22-1 cDNA) of 1.4 kb containing a long open reading frame. When the cDNA was expressed in COS-7 cells with an eukaryotic promoter, active 2-5A synthetase was produced and localized mainly in the cytoplasm. The 5'-proximal ATG in 22-1 cDNA is followed immediately by another ATG. This second ATG was assumed to work as the initiator codon. If so, this enzyme comprises 363 amino acids.

Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C.

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.

Cloning and expression in Escherichia coli of the gene for mouse tumor necrosis factor.

The mouse tumor necrosis factor (TNF) gene was isolated from a mouse genomic library. The entire sequence of the gene was determined using both an automated DNA sequencer with improved primer extension reaction conditions as well as the standard radioisotopic method. Comparison of the nucleotide sequence of the gene with that of mouse TNF cDNA showed that the mouse gene consists of four exons, like rabbit and human TNF genes. There is strong nucleotide sequence homology in the 5'-flanking region among the mouse, rabbit, and human TNF genes, suggesting that the mechanisms regulating TNF gene expression are highly conserved. Direct expression of mature mouse TNF was achieved using a plasmid constructed by site-directed mutagenesis. Purified mouse TNF produced in Escherichia coli showed cytotoxicity to mouse L cells.

Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity.

Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.

Three types of amyloid protein precursor mRNA in human brain: their differential expression in Alzheimer's disease.

Three types of amyloid protein precursor (APP) mRNA, produced by alternative splicing, were detected by Northern blotting in human brains, both control and Alzheimer's disease. These mRNAs encode APP695 consisting of 695 amino acids, APP751 harboring a 56 amino acid insert homologous to a Kunitz-type trypsin inhibitor inside APP695, and APP770 containing an additional 19 amino acid insert. Another possible APP mRNA which encodes "APP714" containing a 19 amino acid insert was not found in brain samples tested. Quantitative analysis revealed that, although the relative expression levels of the three mRNAs were variable among individuals, there was no remarkable change in expression of APP695 and APP751 mRNAs in Alzheimer's disease compared with control, but that APP770 mRNA level was elevated significantly in Alzheimer's disease.

Tissue-specific expression of three types of beta-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain.

Expression of three types of mRNA encoding amyloid beta-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimer's disease (AD). The total content and the proportion of APP mRNAs were specific to each tissue. Among eight tissues examined, the brain was distinct in that the expression level was highest and APP695 mRNA was expressed in abundance. The ratio of APP770/APP751/APP695 mRNAs was approximately 1:10:20 in the cerebral cortex of control brain. The proportions of APP770 mRNA and APP770-plus-APP751 mRNAs increased up to 2.6- and 1.4-fold, respectively, in various regions of AD brain compared with control. The enhanced expression of protease inhibitor-harboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of beta-protein as amyloid.

Immunohistochemical localization of the proteinase inhibitor region of amyloid precursor proteins in the neocortex of Alzheimer's disease and aged controls.

The immunohistochemical localization of the proteinase inhibitor region of amyloid protein precursors (APPI) in the postmortem human neocortex was studied using a polyclonal antibody raised against a purified recombinant human APPI derivative produced by COS-1 cells. APPI-like immunoreactivity (APPI-LI) was found diffusely in the human neocortex. APPI-LI appeared as irregularly shaped granular structures. The size of the APPI-LI structures was 1-4 microns in diameter. APPI-LI usually formed a cluster of 10- to 20-microns diameter in the cortical gray matter and 20- to 40-microns diameter in the subcortical white matter. Double staining for APPI and glial fibrillary acidic protein indicated that APPI-LI in the white matter and molecular layer was localized exclusively in the fibrillary astrocytes. In contrast, APPI-LI was found in neurons as well as in the fibrillary astrocytes in layers II through to VI. Under fluorescence microscopy, APPI-LI in both neurons and fibrillary astrocytes were found in close association with lipofuscin. The present observations indicate that APPI is localized in neurons and astrocytes in the human neocortex and that APPI may be associated with lipofuscin or lysosome in the human neocortex.

Determination of amyloid beta protein precursors harboring active form of proteinase inhibitor domains in cerebrospinal fluid of Alzheimer's disease patients by trypsin-antibody sandwich ELISA.

beta-Amyloid protein precursors (APP) having proteinase inhibitor domains (APPI) were quantified by a new sandwich enzyme linked immunosorbent assay for detection of active (free) form of proteinase inhibitors by using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microtiterplate. The concentration of APPs having APPI in cerebrospinal fluid of Alzheimer's disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.