Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

Production and properties of a dextransucrase from Leuconostoc mesenteroides IBT-PQ isolated from ‘pulque’, a traditional Aztec alcoholic beverage

Dextransucrase was produced from a Leuconostoc mesenteroides isolated from pulque, a traditional Aztec alcoholic beverage produced from agave juice containing sucrose as the main carbon source. Almost all the dextransucrase activity (87%) was associated with the cells, and was unusually high (1.04 U mg⁻¹ of cells). The culture medium composition was optimized through a Box-Behnken method resulting in a process yielding 2.2 U ml⁻¹ of insoluble glucosyltransferase activity. The enzyme had a molecular weight of 166 kDa. Optimal temperature was 35°C with a half-life of 137 min at the same temperature. As with dextransucrase from the industrial strain L. mesenteroides NRRL B-512F, the enzyme showed Michaelis–Menten kinetic behavior with excess substrate inhibition (K _m and K _i values of 0.026 M and 1.23 M respectively); produced soluble linear dextran with glucose molecules linked mainly in α(1–6) with branching in α(1–3) in a proportion of 4:1 as shown by NMR studies; and produced a high yield of isomalto-oligosaccharides in the presence of maltose. Received 4 February 1998/ Accepted in revised form 25 July 1998     

Distribution of ATPase and ATP-to-ADP phosphate exchange activities in magnesium chelatase subunits of Chlorobium vibrioforme and Synechocystis PCC6803

Insertion of magnesium into protoporphyrin IX is a complex ATP-dependent reaction catalysed by the enzyme Mg-chelatase. Three separate proteins (Mg-chelatase subunits), designated as D, H and I, are involved in the chelation reaction. The genes encoding the Mg-chelatase subunits of the green sulfur bacterium Chlorobium vibrioforme and of the cyanobacterium Synechocystis strain PCC6803 were expressed in Escherichia coli. The recombinant proteins were purified, tested for ATPase and phosphate exchange activities, and compared with the activities of the corresponding subunits of Rhodobacter sphaeroides. The Synechocystis strain PCC6803 I subunit and the C. vibrioforme H and I subunits hydrolysed ATP at the rates of 2.0, 1.8 and 0.16 nmol (mg protein)^–1 min^–1, respectively. The ATPase activity of the C. vibrioforme H subunit was similar to that reported for the R. sphaeroides H subunit. The Synechocystis strain PCC6803 H subunit failed to hydrolyse ATP. The I subunit of Synechocystis strain PCC6803 and C. vibrioforme catalysed a transfer of PO₄ from ATP to ADP (exchange activity) at the rate of 1.75 ± 0.15 nmol (mg protein)^–1 min^–1. This exchange rate was 300-fold lower than that reported for the R. sphaeroides I subunit. The PO₄ exchange activities were correlated with the presence of the sequence GXRGTGKSTXVRALA in the primary structure of the three I subunits. Mg-chelatase activity was reconstituted by combining the three subunits of the same bacterium [rates of 41–89 pmol Mg-deuteroporphyrin (mg protein)^–1 min^–1]. Heterologous subunit combinations resulted in low or no Mg-chelatase activity. Received: 25 May 1998 / Revision received: 24 November 1998 / Accepted: 27 November 1998     

Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10^–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10^–5 m), kinetin (Kn) (5×10^–6 m), 1-indolebutyric acid (IBA) (2×10^–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l^–1), or MS with 2,4-D (1×10^–5 m), 6-benzylaminopurine (BAP) (1×10^–5 m), and soluble PVP (250 mg l^–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10^–6 m), Kn (5×10^–6 m) and soluble PVP (250 mg l^–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10^–6 m) and IBA (2.5×10^–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced. Received: 16 April 1998 / Revision received: 25 September 1998 / Accepted: 10 October 1998     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Kinetic study of DNA binding of cisplatin and of a bicycloalkyl-substituted (ethylenediamine)dichloroplatinum(II) complex

 Salmon sperm DNA platination has been conducted under strictly pseudo-first-order conditions with cisplatin (1) and rac-{(1S,2S,4S)-exo-2-(aminomethyl)-2-amino-7-bicyclo[2.2.1]heptane}dichloroplatinum(II) (2). An aquation step first occurs for both complexes, with the rate constants k ₁ = 1.12(0.02)×10^–4 s^–1 and 1.47(0.02)×10^–4 s^–1 respectively for 1 and 2 at 37  °C, values in agreement with those previously reported. It is followed by the actual platination step whose second-order rate constant has been determined for the first time by physicochemical techniques. The values for 1 and 2 respectively are: k ₂ = 2.08(0.07) M^–1 s^–1 and 3.9(0.4) M^–1 s^–1. These kinetic data are discussed in the context of a comparison of several biological properties of the two complexes. Received: 15 May 1998 / Accepted: 26 June 1998     

Chemical and physical-chemical characteristics of dietary fibres from Ulva lactuca (L.) Thuret and Enteromorpha compressa (L.) Grev.

Dietary fibres from Ulva lactuca (L.) Thuret (sea lettuce) and Enteromorpha compressa (L.) Grev. (A.O. nori) were measured according to a ‘standard’ method and a ‘physiological’ protocol simulating the gastric and intestinal environments. U. lactuca contained 15.8–8.0% soluble and 24.2–32.6% insoluble fibres according to the ‘standard’ and ‘physiological’ methods, respectively. For E. compressa, these values were 14.9–15.9 and 21.6–28.7%, respectively. For both algae, the composition suggests that the soluble fibres were xylorhamnoglycuronans sulphates and insoluble fibres were essentially composed of glucans. No marked chemical compositional variation was observed between soluble fractions extracted under the simulated gastric and intestinal conditions. Fibres in both algae are hydrophilic but the water holding capacities were higher after extraction of soluble fibres (5.5–9.5 g g⁻¹ for the dry algae; 14.0–16.0 g g⁻¹ for the standard insoluble fibres). Water soluble fibres demonstrated low intrinsic viscosities at 37 °C in buffers, particularly those from E. compressa (36.0–36.5 ml g⁻¹), and was affected by pH for those of U. lactuca (147.5 ml g⁻¹ at pH 3.0 and 175.0 ml g⁻¹ at pH 7.3).     

Flavodoxin from Wolinella succinogenes

A monomeric flavoprotein (18.8 kDa) was isolated from the soluble cell fraction of Wolinella succinogenes and was identified as a flavodoxin based on its N-terminal sequence, FMN content, and redox properties. The midpoint potentials of the flavodoxin (Fld) at pH 7.5 were measured as –95 mV (Fld_ox/Fld_s) and –450 mV (Fld_s/Fld_red) relative to the standard hydrogen electrode. The cellular flavodoxin content [0.3 μmol (g protein)^–1] was the same in bacteria grown with fumarate or with polysulfide as the terminal acceptor of electron transport. The flavodoxin did not accept electrons from hydrogenase or formate dehydrogenase, the donor enzymes of electron transport to fumarate or polysulfide. Pyruvate:flavodoxin oxidoreductase activity [180 U (g cellular protein)^–1] was detected in the soluble cell fraction of W. succinogenes grown with fumarate or polysulfide. The enzyme was equally active with Fld_ox or Fld_s at high concentrations. The K _m for Fld_s (80 μM) was larger than that for Fld_ox and for the ferredoxin isolated from W. succinogenes (15 μM). We conclude that flavodoxin serves anabolic rather than catabolic functions in W. succinogenes. Received: 15 May 1996 / Accepted: 21 June 1996     

Regulation of the synthesis of aryl metabolites by phospholipid sources in the white-rot fungus Bjerkandera adusta

The white-rot basidiomycete Bjerkandera adusta was cultivated in a liquid medium enriched with l-phenylalanine and various phospholipid sources (lecithin, egg yolk and asolectin). Three aromatic metabolites (benzaldehyde, benzyl alcohol and benzoic acid) were produced under these culture conditions. High concentrations of benzaldehyde (404 mg l^–1) were obtained when the cultures were supplemented with 10 g lecithin l^–1. Benzyl alcohol production was promoted when the strain was grown with 5 or 10 g lecithin l^–1. In the absence of or with a low concentration of lecithin (2.5 g l^–1), benzoic acid was the major aryl metabolite synthesized. The results presented here indicate that aryl alcohol oxidase, an extracellular enzyme catalyzing the oxidation of benzyl alcohol into benzaldehyde, was maximally detected when significant amounts of benzaldehyde were produced. Aryl alcohol oxidase activity was significantly enhanced in the presence of elevated concentrations of phospholipid sources. Together with lignin peroxidase, methoxylated and hydroxylated aryl metabolites were also synthesized under these culture conditions. The possible involvement of phospholipids in the synthesis of aryl metabolites is discussed. Received: 7 August 1998 / Accepted: 30 November 1998     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae

Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes α-isopropyl malate synthase I (α-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3′ end of the LEU4 ORF. Six different point mutations – four transitions and two transversions – were found in the remaining mutants. α-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn²⁺-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn²⁺-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity. Received: 30 September 1998 / Accepted: 20 October 1998     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro

 Efficacy of silver thiosulfate (STS) in reducing ethylene-induced culture abnormalities during minimal growth conservation of microplants was studied in seven potato (Solanum tuberosum L.) genotypes. Different concentrations of STS (0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 μg ml^–1) were tested in minimal growth medium based on MS medium supplemented with 20 g l^–1 mannitol and 40 g l^–1 sucrose. STS improved the microplant growth and reduced the culture abnormalities during prolonged maintenance of potato shoot cultures in vitro. The beneficial effect of STS was most prominent for number of green leaves per microplant and leaf senescence. After 16 months of storage, desirable microplant growth was observed in cultures conserved in medium containing 6.0–9.0 μg ml^–1 STS. The profile of the peroxidase isozymes of conserved cultures did not show any apparent genetic variation due to the presence of STS in the conservation medium. Received: 2 September 1998 / Revision received: 20 November 1998 / Accepted: 12 December 1998     

Ferric and cupric reductase activities in the green alga Chlamydomonas reinhardtii: experiments using iron-limited chemostats

Cells of the green alga Chlamydomonas reinhardtii Dangeard were grown in Fe-limited chemostat culture over a range of growth rates (0.15–1.5 d⁻¹). Greater cell densities and culture chlorophyll levels were achieved using an excess of chelator [ethylenediamine di-(o-hydroxyphenylacetic acid)] relative to FeCl₃ (80:1), compared to growth using a 1:1 chelator:FeCl₃ ratio. The C. reinhardtii cells reduced extracellular ferric chelates, and ferric chelate reductase activity increased with increasing Fe-limited growth rates. However Fe-sufficient cells exhibited a low rate of ferric chelate reductase activity, similar to severely Fe-limited cells. Iron-limited cells were capable of reducing a wide variety of ferric chelates, representing a wide range of stability constants, at similar rates, suggesting that the stability constants of ferric complexes are not important determinants of ferric reducing activity. Cupric reductase activity also increased with increasing Fe-limited growth rates, and Cu(II) was preferentially reduced compared to Fe(III). These results suggest that both reductase activities may represent the same plasma-membrane enzyme. The rate of cupric reduction was a function of the free [Cu²⁺], not the total [Cu(II)], suggesting that free Cu²⁺ is the actual substrate for cupric reductase activity. Received: 8 July 1998 / Accepted: 5 August 1998     

Kinetics of formation and stability of {Pt(dien)}2+ complexes with octamer and 14-mer DNA oligonucleotides containing a GG sequence

 Reaction of [Pt(dien)Cl]⁺ (1) with the 14-mer oligonucleotide 5′-d(ATACATGGTACATA) (I) gave rise to two major species which corresponded to the 5′-G and 3′-G platinated monofunctional adducts, and a minor amount of the bis-platinated adduct formed during the later stages of the reaction. The reaction of (1) with the related octamer 5′-d(ATACATGG) (II) was also investigated. Kinetic data obtained by HPLC showed that the 5′-G and 3′-G bases of the 14-mer oligonucleotide were platinated at similar rates: the second-order rate constant is 53×10^–2 M^–1 s^–1 at 298 K in 0.1 M NaClO₄. However, the platination rate of 5′-G of the octamer (II) (k=69×10^–2 M^–1 s^–1) was enhanced by a factor of three compared to the rate of platination at 3′-G (k=22×10^–2 M^–1 s^–1). All the adducts were separated by HPLC and characterized by NMR spectroscopy, enzymatic digestion and MALDI-TOF mass spectrometry. ¹H and ¹⁵N NMR shifts suggest that there are distinct conformational differences between 14-mer duplexes platinated at 5′-G (I5′ _ds) and 3′–G (I3′ _ds). Molecular mechanics modelling indicates that rotation around the Pt-N7 bond is more restricted in the case of the 5′-G adduct than in that of the 3′-G adduct. The binding of {Pt(dien)}²⁺ to 5′-GN7 and 3′-GN7 in the monofunctional adducts of (I) was shown to be reversible upon the addition of high concentrations of chloride ions. Received: 3 July 1998 / Accepted: 10 November 1998     

In vitro regeneration of a mature leguminous liana (Bauhinia vahlii Wight & Arnott)

An in vitro propagation protocol has been developed from mature lianas of Bauhinia valii. Browning was the major obstacle in the establishment of cultures. Explants collected during the growing season (April–June) showed maximum browning; however, browning was minimal during the dormant phase. This problem was circumvented by soaking the sterilized explants in a solution of antioxidant (50 mg l^–1 ascorbic acid+75 mg l^–1 citric acid). The explants were thereafter transferred to culture room conditions after an initial incubation in the dark at 4 °C for 48 h. Shoot proliferation (58%), shoot number (4.5) and shoot length (35 mm) was best in Murashige and Skoog (MS) medium supplemented with 2.5 μM kinetin + 100 mg l^–1 adenine sulfate. Seasonal fluctuations significantly affected the proliferation potential of the explants. March– April was found to be the best season for shoot initiation. Microshoots were rooted on a half-strength, growth regulator-free, agar-gelled Murashige and Skoog medium after a dip in half-strength MS liquid medium containing 1-naph-thaleneacetic acid + indole-3-butyric acid (10 μM). Rooted plantlets were potted and acclimatized under culture room conditions for 4 weeks before transfer to a polyhouse. Received: 9 March 1998 / Revision received: 14 August 1998 / Accepted: 23 September 1998     

Friable embryogenic callus and somatic embryo formation from cotyledon explants of African marigold (Tagetes erecta L.)

Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l^–1 2,4-D and 0.2 mg l^–1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l^–1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic callus was transferred to MS medium supplemented with 3 mg l^–1 ABA and 60 g l^–1 sucrose. The addition of 10–30 mM l-glutamine improved embryo development. Received: 13 May 1997 / Revision received: 24 February 1998 / Accepted: 28 March 1998     

Low expression of Neu2 sialidase in the thymus of SM/J mice—existence of neuraminidase positive cells “Neu-medullocyte” in the murine thymus

We have already reported that the homogenate of the A/J mouse thymus shows a high sialidase activity at the neutral pH region and that in both soluble and membrane fractions optimal pH was 6.5–7 (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). In the present study, we investigated the level of sialidase activities in the thymus of the SM/J mouse, a mouse strain that we know to have a Neu1^a allele that reveals a low level of sialidase activity in the liver. We found that while in the A/J thymus the soluble sialidase activity at pH 6.5 was high, the SM/J thymus lacked all such activity. A QTL analysis of SMXA recombinant inbred strains showed that soluble sialidase activity correlated well with the D1Mit8/9 marker on chromosome 1. The murine whole DNA-sequence data and the results of our FISH analysis (Kotani et al., Biochem. Biophys. Res. Comm., 286:250–258, 2001) showed that this location is consistent with the position of Neu2 gene. We confirmed that it is hard to detect the Neu2 enzyme of the SM/J mouse thymus by an anti-Neu2 antibody using a Western blot analysis. We also found that while the mRNA expression of Neu2 was quite normal in the SM/J mouse liver, it was very low in the SM/J mouse thymus. We therefore conclude that the lack of soluble sialidase activity in the SM/J mouse thymus is due to the thymus-specific low expression level of the Neu2 gene. We have previously shown that the sialidase positive cell which contains the Mac-1 and immunoglobulin, and which is located sparsely in the corticomedullar region or medullary region of the A/J mouse thymus (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). We showed now in this paper that the detection of this cell in the SM/J mouse thymus at pH 7.0 was difficult. We propose, therefore, to name the cell “Neu-medullocyte”.     

Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties

Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension cultures to a specific activity of 4.68 μmol (mg protein)⁻¹ min⁻¹. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of 5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843–854). There was 62% identity with the eucaryotic FDH consensus sequence. Michaelis constants of approx. 20 μm (formaldehyde), approx. 50 μm (glutathione) and approx. 31 μm (NAD⁺) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and ω-hydroxyfatty acids served as substrates for both FDH preparations. The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation products. Received: 1 April 1998 / Accepted: 18 November 1998