The cellulase of Trichoderma viride. Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.     

Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from Trichoderma viride

Adsorption on crystalline cellulose of six endoglucanases (Endo I, II, III, IV, V and VI; 1, 4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) and two exoglucanases (Exo II and III; 1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.92), purified from a commercial cellulase preparation of Trichoderma viride origin, was studied. Endo I, III, and V adsorbed strongly on Avicel cellulose, while adsorption of Endo II, IV, and VI was much lower. Also, the two exoglucanases could be divided into one enzyme (Exo III) that had a high adsorption affinity and another enzyme (Exo II) that adsorbed only moderately. Adsorption data fitted the Langmuir-type adsorption isotherm. However, adsorption was only partially reversible with respect to dilution. No relation could be found between adsorption affinity and degree of randomness in cellulose hydrolysis, measured as the diversity of released hydrolytic products. Kinetic measurements indicated that only part of the adsorbed enzyme molecules are hydrolytically active.     

Purification and characterization of endoglucanase and exoglucanase components from Trichoderma viride

Major cellulase components—four endoglucanases (Endo I, II, III and IV) and one exoglucanase (Exo II)—were isolated from a commercial cellulase preparation derived from Trichoderma viride by a series of chromatographic procedures. The average molecular weights were determined by SDS-polyacrylamide gel electrophoresis. Endos I, III and IV, with M_rs of 52,000, 42,000 and 38,000, respectively, exhibited a more random hydrolytic mode on carboxymethylcellulose (CMC) than Endo II, which has an M_r of 60,000. Endo II showed low activity towards CMC, but out of the four purified endoglucanases this enzyme had the highest specific activity against Avicel. In the hydrolysis of H₃PO₄-swollen cellulose by Endos I, III and IV, cellobiose was the major product, but equimolar amounts of glucose and cellobiose were formed by Endo II. Exo II, with an M_r of 62,000, released cellobiose as the main product in the hydrolysis of H₃PO₄-swollen cellulose, but glucose was negligible. The combination of Endo I, II, III or IV with Exo II resulted in a synergistic effect in the degradation of Avicel at various combination ratios of these enzymes; the specific optimum ratio of endoglucanase to exoglucanase was largely dependent upon the random hydrolytic mode of the endoglucanase. On the other hand, adsorption of cellulase components was found apparently to obey the Langmuir isotherm, and the thermodynamic parameter (ΔH) was calculated from the adsorption equilibrium constant (K). The enthalpies of adsorption of the endoglucanases were in the range of −2.6–−7.2 KJmol⁻¹, much smaller than that of Exo II (−19.4 KJmol⁻¹). This suggest that Exo II shows stronger preferential adsorption than endoglucanases, and that the enthalpy of adsorption will be effective in distinguishing endoglucanase from exoglucanase.     

Purification, characterization, and synergistic action of endoglucanases from Fusarium lini

Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose.     

Escherichia coli apurinic-apyrimidinic endonucleases enhance the turnover of the adenine glycosylase MutY with G:A substrates

The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG). MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs. We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY. The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors. The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A- and OG:A-containing substrates was investigated. The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized. Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged. Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III. These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III. Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction.     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Transformation of arylamines into direct-acting mutagens by reaction with nitrite

Arylamines including aniline (I), 1-naphthylamine (II), 2-naphthylamine (III), 2-aminofluorene (IV), 1-aminoanthracene (V) and 1-aminopyrene (VI) were treated with 4 equivalent amounts of nitrite at pH3 and 37 degrees C for 4 h. The reaction mixtures of I, IV, V and VI showed mutagenicity to Salmonella typhimurium TA98 and TA100 strains without metabolic activation. The numbers of His+ revertant colonies to TA98 strain were 110/0.05 mumole I, 970/0.055 mumole IV, 620/0.10 mumole V and 870/0.02 mumole VI. These arylamines were converted into mutagens with diazoquinone, diazonium and nitro functions depending on their structures. The mutagen from I was p-diazoquinone (I2). The mutagen from IV was highly unstable fluorene-2-diazonium salt (IV1). The mutagens from V were N3O3-introduced anthracene (V1-1) and 1-nitroanthracene (V2), and those from VI were unidentified nitro-introduced compound (VI1) and 1-nitropyrene (VI2).     

Grouping of Micromonospora based on their cultural-morphologic features, antibiotic-sensitivity and formation of antibiotics

500 Micromonospora cultures were subdivided into nine groups on the basis of their cultural-morphological properties, the ability to produce antibiotics of certain chemical classes, and the sensitivity to 18 different antibiotics: aurantiaca (I), cinnamomea (II), cinnamomea-vinacea (III), cinnamomea-olivacea (IV), nigra (V), nigra-violacea (VI), lilacinescens (VII), coerulea (VIII) and brunnea (IX). Cultures belonging to groups I, II, III, V and VI are moderately sensitive to most of the antibiotics and often occur in natural substrates. Black Micromonospora cultures (groups V and VI) mostly produce aminoglycoside antibiotics while brown cultures (groups II and III) form macrolide antibiotics. Cultures belonging to groups IV, VII, VIII and IX have a higher sensitivity to most of the antibiotics and are rarely isolated from natural substrates. These cultures have a weak ability to produce antibiotics.     

Mutagenicity of the anticancer drug, caracemide, and related compounds for salmonella

Caracemide, MeCON(CONHMe)(OCONHMe) (I), is a novel anticancer drug. Since it was derived from acetohydroxamic acid (II), a known mutagen, its potential metabolites and related compounds were synthesized and tested for mutagenicities in S. typhimurium TA98 and TA100. These compounds were: MeNHCONH(OCONHMe) (III), MeCONH(OCONHMe) (IV), MeCONOH(CONHMe) (V), MeNHCOONH2 X HCl (VI), MeNHCONHOH (VII), MeNHCOON(CONHMe)2 (VIII), and NOH(CONHMe)2 (IX). The mutagenicities in the absence of rat liver homogenate were: (VI) much greater than (IV) greater than (II), (III), (V). The other compounds were not mutagenic. (I) was mutagenic only in the presence of rat liver homogenate. The doses required to demonstrate mutagenicities of these compounds were from 0.05 to 5 mumoles/plate. The major hydrolytic products at 25 degrees C, pH 7, were (III), (IV), and (V) from (I); (II) and (III) from (IV); and (II), (III), (VII) and MeNHCONH(OCOMe) (X) from (V). (III) was stable at pH 7. Treatment of (IV) with HCl yielded (VI). Hydrolysis of (III) or (V) with ammonia yielded (VII). These results suggest that caracemide may be activated enzymatically or nonenzymatically by deacetylation or decarbamoylation, and its anticancer activity may be related to the reactivity of its metabolites with DNA. The synthetic procedures and characterizations of new compounds (IV), (V) and (X) are described.     

Subunit arrangement in beef heart complex III

Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Sch?gger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.     

Purification,properties and comparison of invertase,exoinulinases and endoinulinases of Aspergillus ficuum

Summary One invertase (Inv), five exoinulinases (Exo I; II; III; IV; V) and three endoinulinases (Endo I; II; III) were isolated from a commercial inulinase preparation derived from Aspergillus ficuum using ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sephacel and DEAE-Trisacryl, gel filtration on Ultrogel and Fast Protein Liquid Chromatography on a Mono Q column. The invertase (Inv) had a molecular weight of 84000 and was much more active on sucrose than on inulin: the ratio of activity on inulin and sucrose (I/S ratio) was 0.01. The five exoinulinases show the same molecular weight of 74000 and I/S ratios in the range 0.16–0.36. The three endoinulinases had molecular weight of 64000 and I/S ratios in the range 0.86–2.92. All the -fructofuranosidases were glycoproteins with a high sugar content (from 22 to 41% w/w). A. ficuum is the first described organism containing the three activities: invertase, exo and endoinulinase.     

Structures of withanosides I, II, III, IV, V, VI, and VII, new withanolide glycosides, from the roots of Indian Withania somnifera DUNAL. and inhibitory activity for tachyphylaxis to clonidine in isolated guinea-pig ileum.

Seven new withanolide glycosides called withanosides I, II, III, IV, V, VI, and VII were isolated from an Indian natural medicine, Ashwagandha, the roots of Indian Withania somnifera DUNAL. (Solanaceae), together with four known compounds, withaferin A, 5 alpha,20 alpha(F)(R)-dihydroxy-6 alpha,7 alpha-epoxy-1-oxowitha-2,24-dienolide, physagulin D, and coagulin Q. The structures of withanosides I, II, III, IV, V, VI, and VII were determined based on chemical and physicochemical evidence. Principal constituents, withanoside VI (10 and 30 microM) and withaferin A (10 microM), attenuated the tachyphylaxis to clonidine on electrically stimulated guinea-pig ileum in vitro.     

Purification and characterisation of two endoglucanases from Arthrobotrys oligospora

Two endoglucanases, designated Endo I and Endo II, were purified from the culture filtrates of a nematode trapping fungus, Arthrobotrys oligospora. The purification procedure entailed ammonium sulphate precipitation, gel filtration and preparative PAGE. Both the preparations (Endo I and Endo II) were homogeneous by PAGE, had molecular weights of 24,300 and 44,500 respectively as determined by non-denaturing PAGE, and yielded only cellobiose as the main product of CM-cellulose hydrolysis. The optimum pH and temperature for Endo I were 6.0 and 50 degrees C, and for Endo II, pH 5.6-6.4 and 50 degrees C. The two enzymes differed with respect to their Km (Endo I, 5.04 mg/ml; Endo II, 3.2 mg/ml) and energy of activation values (Endo I, 10.7 kCal; Endo II, 9.5 k Cal). Both enzymes were completely inhibited by 1.25 mH Hg2+ and partially by Pb2+, DTNB and p-HMB while DTT and GSH enhanced their activities.     

Photobiological behaviour of bacteria and phages supplemented with aza-analogoues of nucleic acid bases.

The photochemical stability of anomalous nucleic acid bases of the azatype, 5-azacytosine (I), 5-azacytidine (II), 6-azacytosine (III), 6-azacytidine (IV), 6-azathymine (V), 6-azauracil (VI), and 8-aza-adenine (VII) to U. V. light of the wavelength 254 nm differs from the U. V. stability of the normal constituents. Changes of the U.V. inactivation of Escherichia coli K12 C600, E. coli B, Bacillus cereus, as well as E. coli phages gamma cb2 and gamma b2b5 supplemented with azaderivatives prior to irradiation were investigated. It was found that I, II, III, IV, and VII are more, V and VI less sensitive to U. V. light compared with corresponding natural nucleic acid bases. Their changed U. V. sensitivities are reflected in the survival curves after U. V. -irradiation in as far as azabases are incorporated into the nucleic acids in vivo. This explains the increase in U.V. sensitivity of E. coli K12 C600, E. coli B, and B. cereus supplemented with I, II, III, IV, and VII and the decrease in U.V. sensitivity of Streptococcus faecalis supplemented with V (the latter information was taken from Gunther and Prusoff 1967). The lack of any significant influence on inactivation curves of E. coli K12 C600 by V and VI, and on E. coli phages gamma cb2 and gamma c2b5 by II, is discussed in terms of too small incorporation rates. No discrimination was put forward with respect to DNA and RNA incorporation.     

Synthesis and biological activity of position 1 analogs of LH-RH.

(Formyl-sarcosine)1-LH-RH (I), (acetyl-sarcosine)1-LH-RH (II), (2-pyrrolidone-4-carboxylic acid)1-LH-RH (III), (N-methyl-2-pyrrolidone-4-carboxylic acid)1-LH-RH (IV, hydroxyproline1-LH-RH (V) and (cylcopentane-carboxylic acid)1-LH-RH (VI) were synthesized by solid phase methods on a benzhydrylamine resin support. Peptides I-IV were assayed for LH- and FSH-releasing activity over a 4-h period after subcutaneous infection into immature male rats in order to detect any prolongation ofactivity. The peptides were found to have the following integrated LH-releasing activities compared with LH-RH: I, 64%; II, 72%; III, 19%; IV, 58%. None of the peptides were found to be longer acting than LH-RH. Peptides V and VI were far less active, 0.001% and 1.4%, respectively.     

The endo-(1→4)-β- -glucanase system of Penicillium pinophilum cellulase: Isolation, purification, and characterization of five major endoglucanase components

Five major endo-(1→4)-β- -glucanases (I–V) have been isolated from a cellulase preparation of P. pinophilum. The pI values for I–V were 7.4, 4.8, 4.1, 3.7, and 4.0, respectively, and the respective molecular weights were 25,000, 39,000, 62,500, 54,000, and 44,500, when determined by SDS-gel electrophoresis. Endoglucanase V was optimally active at 65–70° and I–IV were most active at 50–60°. The pH optima of I and III–V were in the range 4.0–5.0. Antiserum prepared to I reacted only with I; II antiserum reacted only with II. Endoglucanases I and V were more random in their attack on CM-cellulose and H₃PO₄-swollen cotton cellulose, and showed no activity against cello-oligosaccharides containing less than five -glucose residues, whereas III and IV were active against all the cello-oligosaccharides tested and acted in a less random manner, and II was intermediate in its catalytic action. III was adsorbed completely on both Avicel PH101 and H₃PO₄-swollen cellulose, whereas IV was not adsorbed. The endoglucanases I–V have distinct roles in the digestion of cellulose.     

Effects of aqueous cinnamon extract on chemically-induced carcinoma of hamster cheek pouch mucosa

This study aimed to investigate the effects of aqueous cinnamon extract (ACE) on 7, 12-Dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch (HCP) mucosa. Sixty male Syrian hamsters were randomly divided into six equal groups. The hamsters of groups I, II and III received no treatment, DMBA and ACE respectively, for 16 weeks. Groups IV and V were handled as group II and concomitantly treated with ACE for the same period and additionally group V received ACE for other 16 weeks after the stoppage of DMBA application. Group VI hamsters were handled as group III and additionally received DMBA for other 16 weeks after the stoppage of ACE supplementation. Hamsters of each group were euthanized according to the experimental schedule. The buccal pouches were and prepared for H&E stain, PAS reagent, CD3 and PDGF immunohistochemical reactivity. All groups showed dysplastic changes with varying degrees except groups I and III. Deep invasive carcinomas were recorded in 90% of the samples of group II, 60% of group IV, 50% of group V and 40% of group VI. From the previous results, it can be concluded that ACE has the potentiality preventing oral cancer initiation better than inhibiting oral cancer progression.     

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).     

Onset of cryptic vicariance in the Japanese dormouse Glirulus japonicus (Mammalia, Rodentia) in the Late Tertiary, inferred from mitochondrial and nuclear DNA analysis

The sequences of the mitochondrial cytochrome b gene and restriction site variation in the spacer region of the nuclear ribosomal RNA gene [rDNA-restriction fragment length polymorphism (RFLP)] were analysed to determine the phylogeographic structure of the Japanese dormouse ( Glirulus japonicus ), which is threatened by deforestation and has been designated an endangered species in Japan. The phylogenetic tree of cytochrome b grouped G. japonicus into six geographical populations: north-eastern Honshu (I), central Honshu (II), west-central Honshu/Kii Peninsula (III), western Honshu (IV), Shikoku (V), and westernmost Honshu/Kyushu (VI); the genetic distances among these groups suggest divergence in the Late Tertiary. The lineage of group VI was located at the basal position in the phylogenetic tree, followed by the radiation of the other lineages. An rDNA-RFLP analysis of 15 restriction sites roughly supported such genetic isolation; groups I, II, III, IV, V and VI have five, two, one, one, one and four unique restriction sites, respectively, revealing four geographic groups as cryptic species: I, II, III + IV + V and VI. Our results reveal the ancient divergences of the local population, which has a complicated evolutionary history, and should be useful in developing a framework for the conservation of this species.