Native Mr Research Articles

Influence of proton T1 on oxymetry using Overhauser enhanced magnetic resonance imaging

In Overhauser enhanced magnetic resonance imaging (OMRI) for in vivo measurement of oxygen partial pressure (pO2), a paramagnetic contrast agent is introduced to enhance the proton signal through dynamic nuclear polarization. A uniform proton T1 is generally assumed for the entire region of interest for the computation of pO2 using OMRI. It is demonstrated here, by both phantom and in vivo (mice) imaging, that such an assumption may cause erroneous estimate of pO2. A direct estimate of pixel-wise T1 is hampered by the poor native MR intensities, owing to the very low Zeeman field (15-20 mT) in OMRI. To circumvent this problem, a simple method for the pixel-wise mapping of proton T1 using the OMRI scanner is described. A proton T1 image of a slice through the center of an SCC tumor in a mouse clearly shows a range of T1 distribution (0.2 approximately 1.6 s). Computation of pO2 images using pixel-wise T1 values promises oximetry with minimal artifacts by OMRI.

Purification, characterization and kinetic properties of the Taenia solium glutathione S-transferase isoform 26.5 kDa.

Glutathione S-transferases are major phase II detoxification enzymes. Taenia solium, a parasite of humans and pigs, is exposed to toxic products. The aim of this work was to purify and characterize a T. solium glutathione S-transferase isoform of 26.5 kDa (SGST26.5) in order to obtain its kinetic parameters. Homogeneous SGST26.5 was obtained by a simple purification procedure. SGST26.5 showed a p I of 7.07, and a native Mr of 60 kDa with 26.5 kDa subunits. The optimum activity for SGST26.5 was found at pH 6.5-7.0 in the range 10-42 degrees C. SGST26.5 had a specific enzyme activity of 78, 7.1, 6.6, and 0.7 microM min(-1) mg(-1) with CDNB, 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal and trans-2-nonenal as substrates, respectively. It also had a kcat/ K(mCDNB)=2.15 x 10(3) M(-1 )s(-1), kcat/ KmGSH)=4.5 x 10(3) M(-1 )s(-1) and Vmax for GSH and CDNB=74 and 77 microM min(-1) mg(-1), respectively. SGST26.5 was inhibited in a noncompetitive form by cibacron blue, bromosulfophthalein and triphenyltin chloride. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer. Bireactant system analysis show that it follows an ordered sequential mechanism.

Evidence for the Involvement of a Tsetse Midgut Lectin-Trypsin Complex in Differentiation of Bloodstream-Form Trypanosomes

We have previously described a bloodmeal-induced molecule (lectin-trypsin complex) from the midgut of the tsetse fly, Glossina longipennis, with both lectin and trypsin activities (Osir et al., 1995). In this paper, we report on the isolation of a similar molecule from the midguts of Glossina fuscipes fnscipes and provide direct evidence for its involvement in the development of African trypanosomes. The molecule (native Mr ∼65,700) has two non-covalently linked subunits, Mr ∼28,800 and Mr ∼35,700. The native molecule was found to be capable of inducing differentiation of bloodstream-form trypanosomes into procyclic (midgut forms) in vitro. In the assays, specific antibodies against procyclin were used to monitor the transformation of the bloodstream-form trypanosomes into procyclic forms. This induction was specifically inhibited by D-glucosamine. Cis-aconitate was also capable of inducing the transformation process with the same efficiency as that of the lectin-trypsin complex. While increasing the concentrations of the lectin-trypsin complex (≥100 μg protein/ml) in the incubation assays resulted into higher transformation rates, it also led to high parasite mortality. These results provide evidence for the involvement of the midgut lectin-trypsin complex in the differentiation of bloodstream-form trypanosomes within tsetse midgut.

Combining two single-shot imaging techniques with slice-selective and non-slice-selective inversion recovery pulses: new strategy for native MR angiography based on the long T1 relaxation time and inflow properties of blood.

Combining two single-shot imaging techniques with slice-selective and non-slice-selective inversion recovery pulses: new strategy for native MR angiography based on the long T1 relaxation time and inflow properties of blood.

Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.

Purification and characterization of a microbial dehydrogenase: a vanillin:NAD(P)+ oxidoreductase.

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native Mr value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.

Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine

The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank® AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native Mr of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with Km values of 562μM and 1592μM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (Ki 1.15μM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys95) and cysteine (Cys96, Cys338 and Cys377) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys96, each mutation caused a substantial loss in enzyme activity. Most notably, Lys95 increased the Km for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the kcat for ODC and lysine decarboxylase (LDC) activity respectively. The Cys377 → Ala mutant possessed a kcat that was lowered by 23-fold, and the Km value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.

Purification, characterization, and substrate and inhibitor structure-activity studies of rat liver FAD-AMP lyase (cyclizing): preference for FAD and specificity for splitting ribonucleoside diphosphate-X into ribonucleotide and a five-atom cyclic phosphodiester of X, either a monocyclic compound or a cis-bicyclic phosphodiester-pyranose fusion.

An enzyme with FAD-AMP lyase (cyclizing) activity, splitting FAD to AMP and riboflavin 4',5'-phosphate (cFMN), was recently identified [Fraiz, F., et al. (1998) Biochem. J. 330, 881-888]. Now, it has been purified to apparent homogeneity from a rat liver supernatant, by a procedure that includes affinity for ADP-agarose (adsorption required the activating cation Mn2+ and desorption required its removal), to a final activity of 2.2 units/mg after a 240-fold purification with a 15% yield. By SDS-PAGE, only one protein band was observed (Mr = 59 000). The correspondence between protein and enzyme activity was demonstrated by renaturation after SDS-PAGE, by gradient ultracentrifugation followed by analytical SDS-PAGE, and by native PAGE with visualization of enzyme activity by fluorescence. A native Mr of 100 000 (ultracentrifugation) or 140 000 (gel filtration) indicated that FAD-AMP lyase could be a dimer. The enzyme required millimolar concentrations of Mn2+ or Co2+, exhibited different optimum pH values with these cations (pH 8.5 or 7.3, respectively), and was strongly inhibited by ADP or ATP, but not by dADP, dATP, or the reaction products AMP and cFMN. A specificity study was conducted with 35 compounds related to FAD, mostly nucleoside diphosphate-X (NDP-X) derivatives. Besides FAD, the enzyme split 11 of these compounds with the pattern NDP-X --> NMP + P=X. Structure-activity correlations of substrates, nonsubstrates, and inhibitors, and the comparison of the enzymic reactivities of NDP-X compounds with their susceptibilities to metal-dependent chemical degradation, pinpointed the following specificity pattern. FAD-AMP lyase splits ribonucleoside diphosphate-X compounds in which X is an acyclic or cyclic monosaccharide or derivative bearing an X-OH group that is able to attack internally the proximal phosphorus with the geometry necessary to form a P=X product, either a five-atom monocyclic phosphodiester or a cis-bicyclic phosphodiester-pyranose fusion. For instance, NDP-glucose and GDP-alpha-L-fucose were substrates, but dTDP-glucose, NDP-mannose, and GDP-beta-L-fucose were not. Judging from kcat/Km ratios, we found the best substrate to be FAD, followed closely by ADP-glucose (kcat/Km only 2-fold lower, but not a physiological compound in mammals), whereas other substrates exhibited 50-500-fold lower kcat/Km values. However, there was no evidence for specific flavin recognition. Instead, what seems to be recognized is the NDP moiety of NDP-X, with a strong preference for ADP-X. Splitting would then depend on the presence of an adequate X-OH group. The possibility that, besides FAD, there could be in mammals other ADP-X substrates of FAD-AMP lyase is discussed, with emphasis placed on some ADP-ribose derivatives.

Cryotherapy of malignant tumors: studies with MRI in an animal experiment and comparison with morphological changes

Aim of our study was to investigate the efficacy of 7 F cryoprobes for percutaneous use morpho- and histologically, to examine the role of apoptosis after cryotherapy, and to compare contrast-enhanced MRI with histopathological findings at different time intervals in a tumor-mouse model. Percutaneous cryotherapy was performed in 15 immunocompromised nude mice with subcutaneously implanted tumors using the non-small-cell lung cancer cell line Lu 1. In group a) 7 mice were sacrificed after definite time intervals and histological examinations were done for evaluation of necrosis and apoptosis (HE; TUNEL assay); 2 mice are in long-term follow-up. In group b) in 6 mice tumor destruction and perfusion before and after freezing were investigated with native and contrast-enhanced MR imaging (T1- and T2-weighted spin-echo) and compared with histopathological findings. Histological control were done in 2 untreated mice. We observed fast tumor-reduction within two weeks (ca. 50%). On long-term follow-up (> 6 months) no recurrence has been noticed so far. Tumors were well vascularized prior to treatment and did not-show contrast enhancement an any time after cryotherapy. A narrow contrast-enhanced zone was seen on the tumor border subcutaneously as a sign of peripheral hyperemia and central vascular stasis after cryotherapy. On histology there was evidence of both apoptosis and necrosis. We have established a tumor-mouse model for further investigations. Two minutes freezing of a 2-cm tumor in the mouse model is sufficient for tumor ablation with scarred healing. Apoptosis may play a role in cryotherapy of experimental tumors. Contrast-enhanced MRI is suitable for the estimation of the cryolasion.

Evidence for two distinct classes of high affinity growth hormone binding proteins in pregnant rat serum.

These studies have established the presence of two major classes of high affinity growth hormone binding proteins in pregnant rat serum, designated GHBPa and GHBPb, with apparent native Mr of 257 K and 98 K respectively. GHBPa, which has not been identified previously, exhibits a binding affinity (2-5 nM(-1)) that is up to 20-fold higher than GHBPb (0.2-0.8 nM(-1)) and is the least abundant form, being approximately 15-20% of total serum GH-binding capacity. Western immunoblot analysis revealed that each GHBP is composed of several immunoreactive proteins which were reactive with carboxy-terminal (RB1615) and/or N-terminal (MAb263) domain antibodies, suggesting the presence of GHBPs with and without the hydrophilic tail. Of importance is that GHBPa exhibited significantly higher Mr (78-182 K, +DTT) than that predicted by GHBP cloning, suggesting that they may be covalently bound to other non-GH-binding proteins or may be distinct entities. GHBPb, on the other hand, was composed of smaller Mr (43/48 K, +DTT) "hydrophilic" tail-containing proteins, some of which were disulphide linked to a larger complex of approximately 110 K. These novel findings challenge the current view of the mechanism for generation of the rat serum GHBP and raise the intriguing possibility that the two classes of GHBP may play distinct and important roles in GH physiology.

Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native Mr of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native Mr 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.

3-Hydroxybenzoate:coenzyme A ligase from cell cultures of Centaurium erythraea: isolation and characterization.

In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a seven-step-procedure. The enzyme was an AMP-forming CoA ligase with a Km = 14.7 microM for 3-hydroxybenzoic acid, 8.5 microM for coenzyme A and 229 microM for ATP. The pH and temperature optima were 7.5 and 35 degrees C, respectively. In SDS-PAGE, two polypeptides of Mr 41,500 and 40,500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their N-termini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation. 3HBL had a native Mr of approx. 50,000 and is thus active as a monomer.

Two Forms of Vitellogenin, Yielding Two Distinct Lipovitellins, Play Different Roles during Oocyte Maturation and Early Development of Barfin Flounder, Verasper moseri, a Marine Teleost that Spawns Pelagic Eggs

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, Mr 107,000, and vLvH B, Mr 94,000) and light chains (vLvL A, Mr 30,000, and vLvL B, Mr 28,000) when analyzed by SDS–PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (Mr 168,000) and Vg B (Mr 175,000), which give rise to vLvH A–vLvL A and vLvH B–vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and β′-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native Mr 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development.

Evidence that platelet and tumour heparanases are similar enzymes

In order to enter tissues, blood-borne metastatic tumour cells and leucocytes need to extravasate through the vascular basal lamina (BL), a process which involves a battery of degradative enzymes. A key degradative enzyme is the endoglycosidase heparanase, which cleaves heparan sulphate (HS), an important structural component of the vascular BL. Previously, tumour-derived heparanase activity (which has been shown to be related to the metastatic potential of murine and human melanoma cell lines) was reported to cleave HS and be inhibited by heparin, as distinct from human platelet heparanase, which cleaved both substrates [Nakajima, Irimura and Nicolson (1988) J. Cell Biochem. 36, 157-167]. We recently reported the purification of human platelet heparanase and showed that the enzyme is a 50-kDa endoglucuronidase [Freeman and Parish (1998) Biochem. J. 330, 1341-1350]. We now report the purification and characterization of heparanase activity from highly metastatic rat 13762 MAT mammary adenocarcinoma and human HCT 116 colonic carcinoma cells and from rat liver using essentially the same procedure that was reported for purification of the human platelet enzyme. The rat 13762 MAT tumour enzyme, which has a native M(r) of 45 kDa when analysed by gel-filtration chromatography and by SDS/PAGE, was observed to be an endoglucuronidase that degraded heparin and HS to fragments of the same sizes as the human platelet enzyme does. N-deglycosylation of both the human platelet and rat 13762 MAT tumour enzymes gave, in each case, a 41-kDa band by SDS/PAGE analysis, demonstrating that the observed difference in M(r) between the platelet and tumour enzymes may have been due largely to differences in the relative amounts of N-glycosylation. Two peptides were isolated following Endoproteinase Lys-C digestion of both the human platelet and rat 13762 MAT tumour heparanases and were shown to be highly similar. Both the rat liver and human colonic carcinoma heparanases also degraded both heparin and HS to fragments of the same sizes as the human platelet enzyme does. Western-blot analysis of an SDS/PAGE gel using antibodies raised against human platelet heparanase demonstrated that human platelet, human tumour and rat tumour heparanases were immunochemically cross-reactive. In conclusion, because of the similarities in their sizes, substrate specificities, peptide sequences and immunoreactivities, we propose that heparanase activities present in human platelets, rat liver and in rat and human tumour cells are, in fact, mediated by a similar enzyme.

Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus.

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.

Characterization of pedicel β‐glucuronidase activity in developing maize (Zea mays) kernels

A conspicuous endogenous maize (Zea mays L.) β‐glucuronidase (GUS) activity was observed in histochemical assays of non‐transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue‐specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p‐nitrophenyl‐β‐D‐glucuronide or 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG‐based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with Km values for MUG of 2.2 and 2.7 µM for the early‐ and late‐eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native Mr of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid‐1,4‐lactone. Only the early‐eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5‐bromo‐4‐chloro‐3‐indoyl‐β‐D‐glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme. In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid‐1,4‐lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli‐derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.

Characterization of beta-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role in initiation of fatty acid biosynthesis.

The Streptomyces glaucescens fabH gene, encoding beta-ketoacyl-acyl carrier protein (beta-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an Mr of 37,000, while gel filtration analysis determined a native Mr of 72,000 +/- 3,000 (mean +/- standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a Km value of 3.66 microM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with Km values of 2.4 microM for acetyl-CoA, 0.71 microM for butyryl-CoA, and 0.41 microM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.

Clinical and nuclear magnetic resonance tomography diagnosis of glenoid labrum injuries

Clinical and radiological evaluation of labral tears remains challenging. It has been shown that intravenous administration of contrast agents produces an MR arthrographic effect without the need for intraarticular injection. This is the first study evaluating this new technique of indirect MR arthrography in diagnosis of glenoid labrum tears. 28 patients with clinically suspected labral injuries were prospectively investigated (1.5 Tesla, flexible surface coil). A native MR exam of the shoulder (transverse and oblique-coronar orientation, T1-weighted spin-, proton density- and T2*-weighted gradient echo sequences) and indirect MR arthrography (transverse and oblique-coronar orientation, fat-suppressed T1-weighted spin-echo sequences, intravenous injection of gadopentetate dimeglumine [0.1 mmol/kg], followed by 10-15 min of joint movement) were performed. Results were confirmed by arthroscopy and/or open surgery. Additionally sensitivity evaluation of clinical tests for investigation of labral tears were performed. Indirect MR-Arthrography improved delineation of the glenoid labrum and hyaline cartilage significantly (p < 0.05). Sensitivity and specificity of indirect MR arthrography in diagnosis of labral injuries were 90% and 89% respectively, compared to 79% and 67% of the native MR exam. The reliability of the checked clinical tests is not sufficient enough to determine labral lesions (predictive value between 50 and 70%). Indirect MR-arthrography is a promising non-invasive technique in the evaluation of the glenoid labrum.

Physiological regulation, purification and properties of urease fromMethylophilus methylotrophus

The methylotrophic bacterium Methylophilus methylotrophus hydrolyses urea to ammonia using a cytoplasmic urease (EC 3.5.1.5). During growth in continuous culture under various nutrient limitations, urease was induced by urea and short-chain amides (formamide and urea≫acetamide), and repressed by excess ammonia. The enzyme was purified using ammonium sulfate fractionation and fast protein liquid chromatography (FPLC), and exhibited a narrow substrate specificity (urea≫formamide and acetamide; no activity with other amides) with a Km for urea of 3.8 mM. Gel filtration FPLC and SDS-PAGE showed that the enzyme had a native Mr of approximately 190 000 and was composed of α (Mr 64 000), β (Mr 15 500) and γ (Mr 15 000) subunits in the probable ratio α2β2γ2. The physiological regulation and biochemical properties of the M. methylotrophus urease are compared with those of other bacterial ureases.

Degradation of chloroaromatics: purification and characterization of a novel type of chlorocatechol 2,3-dioxygenase of Pseudomonas putida GJ31.

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native Mr value under nondenaturating conditions by gel filtration gave a molecular mass of 135 +/- 10 kDa, indicating a homotetrameric enzyme structure (4 x 33.4 kDa). The pI of the enzyme was estimated to be 7.1 +/- 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40 degrees C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.