Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during andeven after purification. We developed a method to stabilizePPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethyleneglycol at pH 6.5. The protein slowly degraded by itself whenthe stabilizing reagents were removed. Ascorbate and/or H₂O₂accelerated the degradation. The ascorbate-induced degradationwas inhibited by catalase, suggesting that H₂O₂ is generatedthrough reduction of PPO by ascorbate. It is likely that dissolvedoxygen is converted to peroxide through two-electron reductionby the reaction center of PPO, binuclear Cu site, and a Fenton-typereaction occurred on it. This understanding was supported bythe finding that the H₂O₂-induced degradation was inhibitedby metal-chelators as well as by polyphenolic substrate of PPO.Considering the postulated mechanism of the self-degradationof PPO, we re-examined the degradation of the 23-kDa proteinof PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38:179]. The obtained results suggested that the 23-kDa proteintriggers the active oxygen production by the binuclear Cu site,probably as reductant, and receives the radical species preferentiallyto the polypeptide moiety of PPO. (Received April 15, 1999; Accepted July 21, 1999)     

Pr-SNTX,a short-chain three-finger toxin from Papuan pigmy mulga snake,is an antagonist of muscle-type nicotinic acetylcholine receptor (α2βδε)

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis. Pr-SNTX protein inhibited muscle-type (α₂βδε), but not neuronal α7 nicotinic acetylcholine receptor (nAChR) activity.     

Repeated streptozotocin injections cause early onset of glomerulosclerosis in mice.

Diabetic nephropathy (DN), a major cause of end-stage chronic renal failure, is histologically characterized by glomerulosclerosis. To investigate the molecular mechanisms of DN, it is important to establish a stable model of glomerulosclerosis in mice, because genomic manipulation techniques (such as gene destruction or transgene insertion) are well established in rodent species. In this study, we found that repeated administrations of streptozotocin led to early onset of glomerular sclerotic lesions in C57BL/6 mice, accompanied with renal dysfunction. During the natural course of DN, glomerular endothelial cells decreased at 10 weeks after the start of streptozotocin-injections, whereas myofibroblastic mesangial cells became evident. Our results provide an animal tool to elucidate the molecular mechanisms of DN, for example to investigate vascular pathology in diabetic glomerular diseases.     

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

Coprophagy in the germfree mouse

Coprophagy was observed in germfree (GF) ICR mice of both sexes, and the results were compared with those of conventional mice. Frequency of coprophagy per animal per day in GF mice was 5.1 in males and 5.8 in females. In conventional (CV) mice, the frequencies were 6.2 in males and 5.3 in females (data from Zoological Science 2:249-255, 1985), with no significant differences compared with GF mice. Coprophagy in CV mice was frequently observed during 6-8 hr after lighting, whereas such close time relationships tended to weaken in GF animals. In a comparison of levels of constituents per unit weight between feces and diet, fecal crude protein and crude fat exhibited lower values than those in the diet. Levels of fecal crude ash and crude fiber were higher than those in the diet, and nitrogen-free extract was almost equal to that in the diet. No essential difference in these tendencies was found compared with CV mice. Levels of fecal vitamin B1, B2, B12 and folic acid were lower than those in the diet. In CV mice, except for vitamin B1, these vitamins exhibited either almost equal or much higher levels compared with those in the diet (data from Experimental Animals 35: 381-386, 1986). From the fact that coprophagy was observed in GF mice, it is suggested that the behavior is inherent in the mouse.     

Expression and processing of cyanobacterial Mn-stabilizing protein in Escherichia coli

The woxA gene of cyanobacterium Anacystis nidulans R2, which encodes the precursor of the Mn-stabilizing protein involved in photosynthetic water oxidation, was found to be expressed in Escherichia coli. The 30-kDa expression product was indistinguishable from the authentic mature protein on SDS/PAGE. Upon fractionation of E. coli cells, the expression product was co-precipitated with the membrane fraction, which is consistent with the water-insoluble nature of the authentic mature protein. Analysis of the amino-terminal amino acid sequence of the product revealed that it is identical to the sequence from the 28th residue of the precursor, indicating that the precursor is processed in E. coli. The expression product was digested by trypsinization of E. coli spheroplasts, but not by that of intact cells. This observation suggests that the product is secreted from the cytoplasmic membrane, but not from the outer membrane. The occurrence of both processing and secretion suggests that a signal peptidase of E. coli can recognize the structure for translocation across the thylakoid membrane. Comparison of the signal sequence and the presequence of sweet potato sporamin A suggests that the processing enzymes of the thylakoid membrane and endoplasmic reticulum possess a common substrate specificity.     

Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families

Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Construction of protocellular structures under simulated primitive earth conditions

We have developed experimental approaches for the construction of protocellular structures under simulated primitive earth conditions and studied their formation and characteristics. Three types of envelopes; protein envelopes, lipid envelopes, and lipid-protein envelopes are considered as candidates for protocellular structures. Simple protein envelopes and lipid envelopes are presumed to have originated at an early stage of chemical evolution, interaction mutually and then evolved into more complex envelopes composed of both lipids and proteins.Three kinds of protein envelopes were constructedin situ from amino acids under simulated primitive earth conditions such as a fresh water tide pool, a warm sea, and a submarine hydrothermal vent. One protein envelope was formed from a mixture of amino acid amides at 80 °C using multiple hydration-dehydration cycles. Marigranules, protein envelope structures, were produced from mixtures of glycine and acidic, basic and aromatic amino acids at 105 °C in a modified sea medium enriched with essential transition elements. Thermostable microspheres were also formed from a mixture of glycine, alanine, valine, and aspartic acid at 250 °C and above. The microspheres did not form at lower temperatures and consist of silicates and peptide-like polymers containing imide bonds and amino acid residues enriched in valine. Amphiphilic proteins with molecular weights of 2000 were necessary for the formation of the protein envelopes.Stable lipid envelopes were formed from different dialkyl phospholipids and fatty acids.Large, stable, lipid-protein envelopes were formed from egg lecithin and the solubilized marigranules. Polycations such as polylysine and polyhistidine, or basic proteins such as lysozyme and cytochromec also stabilized lipid-protein envelopes.     

Effect of guanidinoethanesulfonic acid on brain monoamines in the mouse

Guanidinoethanesulfonic acid (GES) is known to induce convulsive seizures when administered intracisternally to rabbits and cats. The effects of GES on behavior, electroencephalographic recording and brain monoamine levels were examined in mice. When GES (900 nmol) was intraventricularly injected into mice, focal clonic movements of the face, vibrissae and ears together with twitching of the limbs were observed 0.5–1 min after the injection. Hypersensitivity was observed up to 7 min after the injection, after which the mice behaved normally. GES also induced sporadic spike discharges on electrocorticogram. The levels of 5-hydroxytryptamine (5-HT) of the GES-injected mice were lower than those of the saline-injected mice in the hippocampus, diencephalon, pons-medulla oblongata and cerebellum 5 min after the injection. No changes in the norepinephrine or dopamine levels were found after the GES injection. The level of 5-hydroxyindoleacetic acid increased in the striatum and cerebellum 5 min after the GES injection. These results suggest that GES-induced convulsive activities enhance the serotonergic neuroactivity in order to suppress the convulsions.