Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Selection of Arabidopsis cDNAs that partially correct phenotypes of Escherichia coli DNA-damage-sensitive mutants and analysis of two plant cDNAs that appear to express UV-specific dark repari activities

To resist terrestrial UV radiation, plants employ DNA-damage-repair/toleration (DRT) activities, as well as shielding mechanisms. Little is known about the structure and regulation of plant DRT genes. We isolated DRT cDNAs from Arabidopsis thaliana, by selecting for complementation of Escherichia coli mutants lacking all bacterial defenses against UV-light damage to DNA. These mutants are phenotypically deficient in recombinational and mutagenic toleration (RecA^–), excision repair (Uvr^–) and photoreactivation toreactivation (Phr^–). Among 840 survivors of heavily UV-irradiated (10^–7 survival) mutants harboring plasmids derived from an Arabidopsis cDNA library in the vector YES, we identified four unique plant cDNAs, designated DRT100, DRT101, DRT102, and DRT103. Drt101 and Drt102 activity were specific for UV-light damage, and complemented both UvrB^– and UvrC^– phenotypes in the dark. Apparent Uvr^– correction efficiencies were 1 to 40% for Drt101, and 0.2 to 15% for Drt102, depending on the UV fluence. Drt101 and Drt102 showed no extensive amino-acid homology with any known DNA-repair proteins. Drt100 appeared to correct RecA^–, rather than Uvr^–, phenotypes. Although the light dependence of Drt103 activity was consistent with its identification as a photoreactivating enzyme, its predicted amino-acid sequence did not resemble known photolyase sequences. The N-terminal coding sequence of Drt101 suggests that it is targeted to chloroplasts, as reported for Drt100. These cDNAs afforded only modest increases in survival during the original selection procedure. The fact that they were readily isolated nevertheless suggests that selections may be made powerful enough to overcome barriers to expression and function in bacteria, at least for cDNAs of reasonable abundance.     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

Tolerance of nitrifying sludge to p-cresol

p-Cresol at 17 mg l^–1 in a nitrifying culture inhibited by 70% nitrate formation whereas at 10 mg l^–1 there was no effect. p-Cresol at 220, 470, and 910 mg l^–1 was converted to intermediates after adaptation times of 8 h, 24 h, and 40 h, respectively. The sludge recovered 44% of its activity after transformation of p-cresol.     

Organochlorine and organophosphorus pesticide concentrations in water,sediment, and selected organisms in Lake Naivasha (Kenya)

Water, sediment, red swamp crayfish (Procambarus clarkii) and black bass (Micropterus salmoides) from Lake Naivasha were analyzed for selected organochlorine and organophosphorus pesticide residues. The mean p,p'-DDT, o,p'-DDT and p,p'-DDE residue levels recorded in black bass (28.3 (± 30.0), 34.2 (±54.0) and 16.1 (±16.1) g kg^–1, respectively) and crayfish (4.6 (±5.1), 3.2 (±2.8), and 1.4 (±1.1) g kg^–1, respectively), were higher than previously recorded. This indicated recent usage of technical DDT in the lake's catchment. Levels of p,p'-DDT, higher than those of p,p'-DDE further emphasized this. Mean lindane, dieldrin, -endosulfan and aldrin concentrations in black bass were 100.5, 34.6, 21.6 and 16.7 g kg^–1, respectively. The same residues were detected at lower concentrations in crayfish at 2.0, 2.0, 2.0 and 1.9 g kg^–1, respectively. The higher fat content (3.7 ± 2.7% SD) in black bass (compared to 0.6 ± 0.3% in crayfish) accounted for the significantly higher residue concentrations in black bass. Organophosphate pesticides were the most commonly used pesticides in the lake's catchment, but none was detected in any of the samples. The results indicate that there is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use throughout the catchment.     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Ecdysone 20-monooxygenase in a cricket,Gryllus bimaculatus (Ensifera,Gryllidae)—characterization of the microsomal midgut steroid hydroxylase in adult females

Summary Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml^–1. K⁺ and Na⁺ (10^–3–10^–1 M), Ca²⁺ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg²⁺ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent K_m for ecdysone of 3.7·10^–7 M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10^–6 M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I₅₀ of 8·10^–4 M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein^–1 was calculated for midgut microsomes of 4-day-old females.Abbreviations GO carbon monoxide - EDTA ethylenediamine tetraacetic acid - HPLC high performance liquid chromatography - I ₅₀ concentration for 50% inhibition - KCN potassium cyanide - K ₁ inhibition constant - K _m Michaelis-Menten constant - MOPS 3-morpholinopropanesulfonic acid - NADH/NAD ⁺ nicotinamide adenine dinucleotide reduced/oxidized - NADPH/NADP ⁺ nicotinamide adenine dinucleotide phosphate reduced/oxidized - Na ₂ S ₂ O ₄ sodium dithionite - SEM Standard error of mean - TLC thin-layer chromatography - TRIS 2-amino 2-hydroxymethyl-1,3-propanediol (trishydroxymethyl aminomethane) - V _max maximal reaction velocity     

Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences

Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a K_m value of 0.6 mM for arginine and a V_max value of 13 μmole Pi min⁻¹ mg protein⁻¹ for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1608 and 1239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66–73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63–71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55–57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.     

Quantitative assessment of the interaction between the antagonistic fungus Talaromyces flavus and the wilt pathogen Verticillium dahliae on eggplant roots

Quantitative aspects of the interaction between the antagonist Talaromyces flavus, the pathogen Verticillium dahliae and eggplant roots, were studied. When eggplant roots were inoculated with T. flavus, prior to the infection with the pathogen, the population density of T. flavus on V. dahliae-infected roots was at least 3 times higher than on healthy uninfected roots, and the proliferation of T. flavus on diseased eggplant roots was related to the severity of wilt symptoms, in the two levels of application of T. flavus studied. However, in all classes of disease severity tested (disease index, 0–3), the population density of T. Flavus on eggplant roots treated with 10⁶ ascospores g^–1 rooting mixture was significantly (p=0.05) higher than with 10⁵ ascospores g^–1. In roots treated with 10⁵ and 10⁶ T. flavus ascospores g^–1 rooting mixture, the population density of V. dahliae was reduced by 51% and 69%, respectively. When testing the relationships between the population density of V. dahliae in the roots and disease severity, no significant (p=0.05) difference was found between disease indexes 2 and 3. However, the density of V. dahliae on roots of plants with disease index 1 was significantly (p=0.05) lower than disease indexes 2 and 3. The positive relationship between the inoculum concentration of V. dahliae and the population density of T. flavus developed on eggplant roots was significant (p=0.001), linear, and highly correlated (r=0.945) on a logarithmic scale. In addition, the analysis of these data revealed a significant (p=0.05), high, negative and linear correlation (r=–0.985) between the log concentration of V. dahliae inoculum and the disease reduction achieved by T. flavus.     

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K_m and V_max values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min^–1 mg^–1 protein and 1,149 mol min^–1 mg^–1 protein, respectively. The turnover rate (k_cat) and catalytic efficiency (k_cat/ K_m) for Leu-p-NA and LeuGlyGly were 10,179 s^–1 and 49,543 s^–1 and 15,470 mM^–1 s^–1 and 1981.7 mM^–1 s^–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co²⁺ .     

Kunitz-type trypsin inhibitor with high stability from <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. seeds

The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI-trypsin complex (K _i = 2.3·10⁻⁶ M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 131–140.     

Production of cellulases in batch culture using a mutant strain of Trichoderma reesei growing on soluble carbon source

Trichoderma reesei Rut-C30 is a highly derepressed mutant which synthesised active cellulases in culture media containing glucose and lactose as the only carbon sources. The maximum biomass, filter paper and specific filter paper activities for cell growth on 20 g glucose l^–1 were 20 g dry cell wt l^–1, 1.9 FPU ml^–1 and 4.8 FPU mg^–1 protein respectively, while on 40 g glucose l^–1 were 25 g dry cell wt l^–1, 4.5 FPU ml^–1 and 6.2 FPU mg^–1 protein, respectively. This strain had a higher specific filter paper activity (6.2 FPU mg^–1 protein) than was produced by other T. reesei mutants (3.6 FPU mg^–1 protein).     

Interaction of RecBCD enzyme with DNA damaged by gamma radiation

Summary The DNA of a gene 2 mutant (T4 2 ^–) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm. Under normal conditions, recBCD ⁺ cells are therefore incapable of supporting the growth of phage T4 2 ^–. Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2 ^–-infected bacteria able to form plaques. We found that recBCD ⁺ cells can form plaques if, before infection with T4 2 ^–, they have been exposed to gamma radiation. It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme. This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes. Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure. The reappearance of the nucleolytic action of RecBCD enzyme on T4 2 ^– DNA accompanies the cessation of degradation of bacterial DNA. Both, this cessation and the reappearance of the nucleolytic action of RecBCD enzyme on T4 2 ^– DNA depend on a functional recA gene product. These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome. By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e. during postirradiation DNA degradation. It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.     

DSP toxin contents inDinophysis fortii and scallops collected at Mutsu Bay,Japan

Cell densities of toxic phytoplankton species responsible for diarrhetic shellfish poisoning (DSP) were monitored at a sampling site in Mutsu Bay, Japan, in 1995.Dinophysis fortii almost completely dominated the toxic phytoplankton community. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) contents in bothD. fortii cells and midgut glands of scallops collected at the same sampling site were determined by HPLC — fluorometry. DTX1 was detected fromD. fortii and scallops. The contents of DTX1 inD. fortii changed markedly during the experimental periods (5–252 pg cell^–1). The highest concentration of DTX1 in the midgut glands of scallops coincided with the period of relatively high cell densities ofD. fortii with the highest content of DTX1 (252 pg cell^–1). The results demonstrate that toxin content in the cells is an important factor affecting the toxicity of shellfish.     

Nature of the water channels in the internodal cells ofNitellopsis

Summary The hydraulic resistance was measured on internodal cells ofNitellopsis obtusa using the method of transcellular osmosis. The hydraulic resistance was approximately 2.65 pm^–1 sec Pa, which corresponds to an osmotic permeability of 101.75 m sec^–1 (at 20°C).p-Chloromercuriphenyl sulfonic acid (pCMPS) (0.1–1mm, 60 min) reversibly increases the hydraulic resistance in a concentration-dependent manner.pCMPS does not have any effect on the cellular osmotic pressure.pCMPS increases the activation energy of water movement from 16.84 to 32.64 kJ mol^–1, indicating that it inhibits water movement by modifying a low resistance pathway.pCMPS specifically increases the hydraulic resistance to exosmosis, but does not influence endosmosis. By contrast, nonyltriethylammonium (C₉), a blocking agent of K⁺ channels, increases the hydraulic resistance to endosmosis, but does not affect that to exosmosis. These data support the hypothesis that water moves through membrane proteins in characean internodal cells and further that the polarity of water movement may be a consequence of the differential gating of membrane proteins on the endo- and exoosmotic ends.     

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3 ^+/+ and Mgat3 ^–/– mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3 ^–/– mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3 ^–/– mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3 ^–/– mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc. Published in 2003.     

Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning,Expression in Escherichia coli,and Biochemical Characterization of the Recombinant Protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60–65°C, pH 11.0, K _M = 0.055 mM with p-nitrophenyl phosphate. The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60°C and being completely inactivated in 2 h at 80°C. In substrate specificity assays, the highest activity was observed with p-nitrophenyl phosphate and dATP. Vanadate, inorganic phosphate, and SDS were inhibitory, while thiol-reducing agents had virtually no effect. The enzyme activity strongly depended on exogenous Mg²⁺ and declined in the presence of EDTA.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan