Nucleomorph genome diversity and its phylogenetic implications in cryptomonad algae

The relationship between phylogeny and nucleomorph genome size was examined in 16 strains of cryptomonad algae using pulsed‐field gel electrophoresis, Southern hybridization and phylogenetic analyses. Our results suggest that all cryptomonads examined in this study contain three nucleomorph chromosomes and their total genome size ranges from 495 to 750 kb. In addition, we estimated the plastid genome size of the respective organisms. The plastid genomes of photosynthetic strains were approximately 120–160 kb in size, whereas the non‐photosynthetic Cryptomonas paramecium NIES715 possesses a genome of approximately 70 kb. Phylogenetic analysis of the nuclear small subunit ribosomal DNA (SSU rDNA) gene showed that nucleomorph genome size varies considerably within closely related strains. This result indicates that the reduction of nucleomorph genomes is a rapid phenomenon that occurred multiple times independently during cryptomonad evolution. The nucleomorph genome sizes of Cryptomonas rostratiformis NIES277 appeared to be approximately 495 kb. This is smaller than that of Guillardia theta CCMP327, which until now was thought to have the smallest known nucleomorph genome size among photosynthetic cryptomonads.     

Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat

We previously found hydroperoxide-responsive proteins (HPRPs), which are comprised of peroxiredoxin I(Prx I), Prx II, Prx III, Prx VI, HSP27, G3PDH and two unidentified proteins (HPRP-2' and HPRP-5'), in human umbilical vein endothelial cells. It was demonstrated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) that most HPRPs are converted into variants with lower pI upon exposure to hydroperoxides. In this study, we examined the HPRP response on 2D gels upon exposure of human endothelial cells (ECV304) to paraquat (PQ²⁺), which generates reactive oxygen species (ROS) within cells. PQ²⁺ exerted cytotoxic effects in a dose- (10μM-10mM) and time- (24-168h) dependent manner. Two-dimensional PAGE analysis revealed that HPRP-2', and oxidized forms of Prx I, Prx II and Prx III were clearly increased upon exposure of cells to sublethal levels of PQ²⁺. Microsequence analysis revealed that both HPRP-2 and -2' were identical with human DJ-1. Moreover immunoblot analysis confirmed the increase of oxidized forms of Prx II, Prx III and DJ-1 in response to sublethal levels of PQ²⁺. PQ²⁺ treatment failed to increase fluorescence intensity derived from DCF, which is believed to be an indicator for intracellular levels of hydroperoxide. Although pentachlorophenol (PCP), an uncoupler of the mitochondrial respiratory chain, clearly elevated the fluorescence, PCP had no effect on HPRP response. These observations indicated that DCF-derived fluorescence is not correlated with HPRP response. We consider that the response of Prxs and DJ-1 on 2D gels could reflect endogenous production of ROS in PQ²⁺-treated cells, and might be a sensitive indicator of oxidative stress status.     

Novel plastocyanin containing phenylalanine-83 from the gymnosperm Ginkgo biloba

Plastocyanin was purified from the gymnosperm Ginkgo biloba L., and its complete amino acid sequence was determined. The protein was shown to contain Phe-83 instead of Tyr-83 conserved in other land plant plastocyanins. This is the first report of the characterization and complete amino acid sequence of a gymnosperm plastocyanin.     

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.     

Two novel oligosaccharides formed by 1F-fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.)

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-beta-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose, O-beta-D-fructofuranosyl-(2-->1)-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside and 1F(1-beta-D-fructofuranosyl)2-4G-beta-D-galactopyranosylsucrose, [O-beta-D-fructofuranosyl-(2-->1)]2-beta-D-fructofuranosyl-O-[beta-D-galactopyranosyl-(1-->4)]-alpha-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-beta-D-fructofuranosyl-4G-beta-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcusfaecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.     

Enhanced expression of cellular prion protein gene by insulin or nerve growth factor in immortalized mouse neuronal precursor cell lines

In order to understand the fundamental and putative roles of PrP(c) in the central nervous system, neuronal cell lines were established. Cells were immortalized by recombinant retrovirus vector-mediated transduction of SV40 T-antigen gene. Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology. These cell lines showed the properties of neuronal progenitor cells in antigenicity, morphology and responses to differentiating agents. Expression of PrP(c) was detected by immunocytochemical analysis. These cell lines responded to differentiating agents such as dibutyl cyclic AMP (dcAMP) and phorbol 12-myristate 13-acetate (PMA) before developing into neuronal-like cells. Neurite extensions were observed 20 min after incubation with the differentiating agents. Treatment with nerve growth factor (NGF) and insulin induced cell differentiation and enhanced expression of PrP gene (Prnp) mRNA and protein. The latter phenomenon was not inhibited by wortmannin, which is a specific inhibitor of phosphatidylinositol 3-kinase. These results suggest that PrP(c) plays an important role in the differentiation-mediated classic signaling pathway of neuronal cell.     

Effects of salinomycin and vitamin B(6) on in vitro metabolism of phenylalanine and its related compounds by ruminal bacteria,protozoa and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analyzed by HPLC. When PPY was used as a substrate, it completely disappeared without additives and converted mainly to Phe and PAA on the average by 396 and 178, 440 and 189, and 439 and 147 &mgr;M in B, P and BP, respectively, during the 12 h incubation period. The rate of disappearance showed no significant differences between the microbial suspensions with and without SL and B(6) during the incubation period. The production of Phe from PPY with SL was enhanced (p<0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production from PPY with SL was inhibited (p<0.05) by 35, 37 and 38% in B, P and BP, respectively, during the 12 h incubation period. On the other hand, with B(6), the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and 11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation period. When PAA added as a substrate was incubated in the incubation medium without any additives, it disappeared by 483, 462 and 507 &mgr;M and converted mainly to Phe on the average by 231, 244 and 248 &mgr;M in B, P and BP, respectively. The disappearance of PAA with SL was inhibited (p<0.05) by 16, 15 and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6 was almost the same as that without B(6) in B and BP suspensions but tended to be enhanced by more than 9% in P suspensions during the 12 h incubation period. The production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B, P and BP, respectively, during the 6 h incubation period, but the inhibition was weakened during the 12 h incubation period, whereas Phe production from PAA with B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When Phe was added as a substrate, the net Phe disappearance without additives was 549, 365 and 842 &mgr;M and converted mainly to PAA on the average by 254, 205 and 461 &mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL was inhibited (p<0.05) by 38, 28 and 46%, whereas the net disappearance of Phe with B(6) was enhanced (p<0.05) by 9, 8 and 7% in B, P and BP, respectively. The production of PAA from Phe with SL was inhibited (p<0.05) by 73, 54 and 76% in B, P and BP, respectively. On the other hand, with B(6), PAA production from Phe was enhanced (p<0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these results, it seems that SL inhibited Phe disappearance and enhanced the synthesis of Phe from PPY, though not from PAA, and accumulated free Phe in the medium, whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could provide additional amino N for animals.     

Recombination events across the atpA-associated repeated sequences in the mitochondrial genomes of beets

 The mitochondrial atpA gene sequence of the normal fertile sugarbeet (cv ‘TK81-0’) exists in one full-length version and one truncated version, both of which are present in normal stoichiometry and have a 406-bp segment in common. The PCR approach as well as prolonged exposure of Southern blots indicates that the products of the recombination across the 406-bp repeat are present in substoichiometric amounts in the ‘TK81-0’ genome. Intriguingly, one of these substoichiometric sequence arrangements was revealed to be preferentially amplified in an evolutionary lineage that led to a cytoplasmic male-sterile variant [I-12CMS(2)] in wild beets. We also found the 406-bp repeat to be part of a 6.5-kb repeat in the mitochondrial genome of I-12CMS(2). This 6.5-kb duplication is likely to involve recombination between two sets of repeats (the above-mentioned 406-bp repeat and a 7-bp repeat) in an ancestral beet mitochondria. Received: 4 October 1997 / Accepted: 31 October 1997