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)     

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.     

Hypoxanthine guanine phosphoribosyltransferase deficiency: nucleotide substitution causing Lesch-Nyhan syndrome identified for the first time among Japanese

Summary A previously undescribed nucleotide substitution at codon 51 (CGA to TGA) has been identified using the polymerase chain reaction technique in hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA; this is the first molecular evidence for a point mutation in a Japanese patient with Lesch-Nyhan syndrome. The present mutation is the 19th nucleotide substitution identified as a germ-line mutation at this locus and the second mutation generating a stop codon. The position of the nucelotide substitution is exactly the same as a previously described mutation HPRT_Toronto, indicating for the first time that nucleotide substitutions at the same position in the sequence of HPRT can generate different mutant alleles, one causing a partial deficiency and the other a complete deficiency. Although the type of nucleotide substitution is different between the two cases, a single base position has twice become the target of a mutation. However, the calculation of the probability of finding substitution mutations at the same base position in the coding region of hprt indicates that there is no evidence for the presence of a hot spot for substitution mutations in the human hprt germ line.     

Statistical models of the overdispersed molecular clock

The most commonly used statistical model to describe the rate constancy of molecular evolution (molecular clock) is a simple Poisson process in which the variance of the number of amino acid or nucleotide substitutions in a particular gene should be equal to the mean and henceforth the dispersion index, the ratio of the variance to the mean, should be equal to one. Recent sequence data, however, have shown that the substitutional process in molecular evolution is often considerably overdispersed and have called into question the generality of using a simple Poisson process. Several efforts have been made to develop more realistic models of molecular evolution. In this paper, I will show that the spatial (site-specific) variation in the rate of molecular evolution is an improbable cause of the overdispersion and then review various statistical models which take the temporal variation into account. Although these models do not immediately specify what the mechanisms of molecular evolution might be, they do make qualitatively different predictions and give some insight into their inference. One way to distinguish them is suggested. In addition, effects of selected substitutions that presumably occur after a major change in a molecule are quasi-quantitatively examined. It is most likely that the overdispersion of molecular clock is due either to a major molecular reconfiguration (fluctuating neutral space) led by a series of subliminal neutral changes or to selected substitutions fine-tuning a molecule after a major molecular change. Although the latter possibility, of course, violates the simplest neutrality assumption, it would not impair the neutral theory as a whole.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

Analyses of constituents of feces and the effect of a vitamin B12 fortified diet on coprophagy in the mouse

In order to investigate coprophagy from the viewpoint of nutrition, fecal constituents were analyzed in freeze-dried samples. Feces were collected from 7:00 to 11:00 and from 19:00 to 23:00. Inorganic elements and crude fibers per unit weight were 3-4 times more concentrated in feces than in basal diet, whereas, crude proteins, crude fats and nitrogen-free extract showed various degrees of reduction. There were no differences in these tendencies with sampling time. As for some B vitamins, feces collected from 7:00 to 11:00 contained 22-92% more vitamins than feces collected from 19:00 to 23:00. In comparison with the dietary concentration, vitamin B12 was increased by 124-197 times (520-730 micrograms/100 g) in feces collected between 7:00 and 11:00. Folic acid in feces collected between 7:00 and 11:00 was 10 times greater than that in the diet. On the basis of the findings on vitamins, the effect of a vitamin B12 fortified diet (1,350 micrograms/100 g) on coprophagy was examined. Mean frequency of coprophagy per animal per day was 9.6 when animals were fed on the basal diet, whereas the frequency was immediately and significantly (p less than 0.05 approximately p less than 0.01) reduced to 4.7 after the diet had been replaced by the fortified one. However, coprophagy was not completely inhibited by vitamin B12 fortification. This indicates that some nutrient(s) in feces other than vitamin B12 might be of use to the host, and that otherwise, coprophagy might be a basically habitual form of behavior. Furthermore, under the fortified diet, the frequency of coprophagy increased gradually.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations and origin of oxytocin in breast milk

Samples of human milk obtained from lactating women in the early postpartum period were assayed for oxytocin concentrations by specific RIA, following extraction procedures with Florisil. Mean oxytocin concentrations in human milk at postpartum day 1 to 5 were 4.5 +/- 1.1, 4.7 +/- 1.1, 4.0 +/- 1.3, 3.2 +/- 0.4, 3.3 +/- 0.6 microunits/ml (+/- SE), respectively. Oxytocin levels in milk were significantly increased by nursing (3.1 +/- 0.6, 5.3 +/- 1.0 microunits/ml, respectively). 3H-oxytocin in human milk was stable even after incubation at 37 degrees C for 2 hours. The dilution curve for milk was parallel to the curve for the standard oxytocin. The chromatographic fraction of immunoreactive oxytocin was identical to that of 3H-oxytocin. 3H-oxytocin was administered to lactating rats. Radioactivity in the neonatal gastric contents and plasma were 12.8% and 4.4% of the counts in the maternal plasma. It was made clear that oxytocin is stable in milk and that oxytocin in maternal blood can be transferred to mik and then to neonates.     

OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by X-irradiation of N2O-saturated aqueous solutions of purine nucleosides (2'-deoxyadenosine, adenosine, 2'-deoxyguanosine, 3'-deoxyadenosine, guanosine and inosine) and the corresponding homopolymers (poly A and poly I) have been investigated by the technique of spin-trapping and e.s.r. spectroscopy. 2-Methyl-2-nitrosopropane was used as a spin-trap. For 2'-deoxyadenosine and 2'-deoxyguanosine, the resulting spin-adducts were separated by Bio-Gel P-2 column chromatography and analysed by e.s.r. spectroscopy. For homopolymers, e.s.r. spectra were recorded at 50 degrees C after enzymatic digestion to obtain signals with narrower line width. The e.s.r. signal consisting of only a primary triplet without further splittings, which is consistent with assignment to the trapping of an H-abstraction radical at the C4' position of the sugar moiety, was observed in all cases. For 2'-deoxyguanosine an e.s.r. signal consisting of a secondary triplet was observed. Examinations using other spin-trapping reagents such as PBN, 4-PyOBN and DMPO provided no positive evidence supporting the proposal that this was due to an alpha-nitrogen. The e.s.r. signal consisting of a secondary doublet which further splits into a doublet was observed for 2'-deoxyadenosine, adenosine, 3'-deoxyguanosine, 2'-deoxyguanosine, and inosine, and tentatively associated with a radical centered in the sugar moiety.