Differential expression of parvalbumin in neonatal phencyclidine‐treated rats and socially isolated rats

Decreased parvalbumin expression is a hallmark of the pathophysiology of schizophrenia and has been associated with abnormal cognitive processing and decreased network specificity. It is not known whether this decrease is due to reduced expression of the parvalbumin protein or degeneration of parvalbumin‐positive interneurons (PV⁺ interneurons). In this study, we examined PV⁺ expression in two rat models of cognitive dysfunction in schizophrenia: the environmental social isolation (SI) and pharmacological neonatal phencyclidine (neoPCP) models. Using a stereological method, the optical fractionator, we counted neurons, PV⁺ interneurons, and glial cells in the medial prefrontal cortex (mPFC) and hippocampus (HPC). In addition, we quantified the mRNA level of parvalbumin in the mPFC. There was a statistically significant reduction in the number of PV⁺ interneurons (p = 0.021) and glial cells (p = 0.024) in the mPFC of neonatal phencyclidine rats, but not in SI rats. We observed no alterations in the total number of neurons, hippocampal PV⁺ interneurons, parvalbumin mRNA expression or volume of the mPFC or HPC in the two models. Thus, as the total number of neurons remains unchanged following phencyclidine (PCP) treatment, we suggest that the decreased number of counted PV⁺ interneurons represents a reduced parvalbumin protein expression below immunohistochemical detection limit rather than a true cell loss. Furthermore, these results indicate that the effect of neonatal PCP treatment is not limited to neuronal populations.     

α-Parvalbumin reduces depolarizationminduced elevations of cytosolic free calcium in human neuroblastoma cells

We investigated whether the expression of human α-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca²⁺]_i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM.Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K⁺. During this period, [Ca²⁺]_i was monitored by video microfluorimetry using the Ca²⁺ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca²⁺]_i The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca²⁺]_i than non-parvalbumin-expressing cells. Resting [Ca 2+], did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca²⁺]_i in neuroblastoma cells can be suppressed by parvalbumin.     

Some properties of adenylate kinase from chemolithotrophically grown Thiobacillus novellus

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from Thiobacillus novellus was purified 54-fold. The preparation was, on the basis of densitometer scans of polyacrylamide gels, at least 85% pure. The optimum pH in the forward direction (2 ADP ATP+AMP) was about 8.7, and in the reverse 8.2 The enzyme was specific for AMP, ADP and ATP with apparent K _m values of 0.04, 0.34 and 0.09 mM respectively. A double reciprocal plot of specific activity vs. (ADP)² was linear. Both AMP and ATP inhibited the forward reaction with AMP the more inhibitory of the two. AMP inhibition was competitive with respect to ADP with a K _i of 0.125 mM. Although Mg²⁺ was necessary for maximal activity, about 20% of this was obtained in its absence. Co²⁺ and Mn²⁺ at similar concentrations gave 46% and 26% respectively of the activity found with Mg²⁺. Apparent K _m for Mg²⁺ was about 0.054 mM in the forward and 0.15 mM in the reverse direction. pHMB and HgCl₂ were potent inhibitors. Inhibition by pHMB but not HgCl₂ was reversible by GSH or cysteine. Arrhenius plots gave an E _a of 3.25 kcal/mole/degree C in the forward direction without discontinuity. In the reverse, there was a break at 26.7°C with an E _a of 3.62 kcal/mole/degree C for lower temperatures and 3.92 kcal/mole/degree C for higher temperatures.Molecular weights of the enzyme were 46,300±300 by SDS PAGE and 47,800±200 by SDS PAGE after treatment with 5 M urea and about 40,000 by sucrose density gradient centrifugation.Abbreviations APS adenosine-5-phosphosulfate - DEAE diethylaminoethyl - DTT dithiothreitol - E_a energy of activation - ECTEOLA epichlorohydrin triethanolamine - G6P glucose-6-phosphate - GSH glutathione (reduced) - PAGE Polyacrylamide gel electrophoresis - pHMB parahydroxymercuribenzoate - PEP phosphoenolpyruvate - PPi inorganic pyrophosphate - SDS sodium dodecyl sulfate - TEAE triethylaminoethyl Supported by an operating grant to A.M.C. from NSERCSummer student research trainee     

NADH-dependent Fe3+EDTA and oxygen reduction by plasma membrane vesicles from barley roots

Brüggemann, W. and Moog, P. R. 1989. NADH-dependent Fe³⁺EDTA and oxygen reduction by plasma membrane vesicles from barley roots. Biochemical properties of pyridine-dinucleotide-dependent Fe³⁺-EDTA reductase were analysed in purified plasma membranes (PM) from barley (Hordeum vulgare L. cv. Marinka) roots. The enzymatic activity preferred NADH over NADPH as electron donor and it was 3-fold increased in the presence of detergent. The reductase showed a pH optimum of 6.8 and saturable kinetics for NADH with K_m (NADH) of 125 μM and V_max of 143 nmol Fe (mg protein)^-1 min^-1 in the presence of 500 μM Fe³⁺EDTA. For the dependence of the reaction rate on the iron compound, K_m(Fe³⁺EDTA) of 120 μM and V_max of 184 nmol (mg protein)^-1 min^-1 were obtained. The activity was insensitive to superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and antimycin A, but stimulated by an oxygen-free reaction medium. It could be solubilized by 0.25% (w/v) Triton X-100. The solubilized enzyme revealed one band in native polyacrylamide gel electrophoresis (PAGE) and in isoelectric focussing (IEF) at pl 7.4 by enzyme staining. Major polypeptides with molecular weights of 94, 106, 120 and 205 kDa corresponded to the enzyme-stained band from native PAGE. Analysis of oxygen consumption by the membranes revealed the existence of NADH:CK oxidoreductase activity, which was stimulated by salicylhydroxamic acid (SHAM), chinhydron, Fe³⁺EDTA and Fe³⁺EDTA but not by K₃ [Fe(CN)₆] or K₄[Fe (CN)₆). The stimulating effect of the iron chelates on oxygen consumption was due to Fe²⁺ and could be suppressed by bathophenanthroline disulfonate (BPDS), SOD and p-chloromercurophenylsulfonic acid (PCMS). The results are discussed with respect to the nature of the stimulation.     

Reproductive biology of the lessepsian Reticulated leatherjacket Stephanolepis diaspros (Fraser - Brünner, 1940) in the Gulf of Gabes (Eastern Mediterranean Sea)

Fundamental information on the reproductive biology of the lessepsian fish Reticulated leatherjacket Stephanolepis diaspros from the Gulf of Gabes (Eastern Mediterranean Sea) was considered in this study. The study was carried out on a sample of 1,053 specimens of the Reticulated leatherjacket caught by trawlers and were monthly collected from May 2003 to December 2005. The length frequency distribution indicated that males are taller than females. The sex-ratio was 0.87:1 which significantly deviated from the hypothetical distribution of 1:1. The size and the season have an effect on this sex-ratio. Gonadosomatic Index (GSI%) values were highest in July for both sexes and this species accumulates energy reserves in the liver to be spent during the maturity of gonads and the spawning. First maturation occurred at 7.96 cm for females and 10.58 cm (L₅₀) for males. The fecundity of S. diaspros ranged from 28,250 to 218,000 with a mean of 100,778.76 ± 17,702.1. The relationships between absolute fecundity and total length, eviscerated weight and weight of gonads were will described and the correlation coefficients calculated between fecundity and each of these independent variables, are judged to be either significant (R² > 0.8) indication of a very good model for the fecundity study.     

Adaptation of the Photosynthetic Apparatus of Anacystis nidulans to Irradiance and CO2-Concentration

Homocontinuous cultures of the cyanobacterium Anacystis nidulans (syn. Synechococcus sp. PCC 6301) were grown at white light intensities of 2 and 20 W/m², and supplied with 0.03 and 3 % CO₂ enriched air. The mutual influence of these growth factors on the development of the photosynthetic apparatus was studied by analyses of the pigment content, by low temperature absorbance and fluorescence spectroscopy, by analyses of oxygen evolution light-saturation curves, and by SDS PAGE of isolated phycobilisomes. The two growth factors, light and CO₂, distinctly affect the absorption cross section of the photosynthetic apparatus, which is expressed by its pigment pattern, excitation energy distribution and capacity. In response to low CO₂ concentrations, the phycocyanin / allophycocyanin ratios were lower and one linker polypeptide L³⁰_R, of the phycobilisomes was no longer detectable in SDS PAGE. Apparently, low CO₂ adaptation results in shorter phycobilisome rods. Specifically, upon adaptation to low light intensities, the chlorophyll and the phycocyanin content on a per cell basis increase by about 50% suggesting a parallel increase in the amount of phycobilisomes and photosystem core-complexes. Low light adaptation and low CO₂ adaptation both cause a shift of the excitation energy distribution in favor of photosystem I. Variations in the content of the “anchor” polypeptides L⁶⁰_CM and L⁷⁵_CM are possibly related to changes in the excitation energy transfer from phycobilisomes to the photosystem II and photosystem I core-complexes.     

Purification and Characterization of β-N-Acetylhexosaminidase from the Liver Prawn,Penaeus japonicus

β-N-Acetvlhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS–PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS–PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The aminoterminal amino acid sequence was NH₂-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-VaI-?-Val-Lys-Gly-Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50°C, respectively. The enzyme was stable from pH 4 to 11, and below 55°C. It was 39% inhibited by 10mM HgCl₂.

Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAc_n, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-β-GlcNAc_n, n= 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of K_m and k_cat at 25°C and pH 5.5 were 0.137 mM and 598s^–1 for pNp-β-GlcNAc, 0.117 mM and 298s^–1 for GlcNAc₂, 0.055 mM and 96.4s^–1 for GlcNAc₃, 0.044 mM and 30.1 s^–1 for GlcNAc_4, 0.045 mM and 14.7 s^–1 for GlcNAc₅, and 0.047 mM and 8.3 s^–1 for GlcNAc₆, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.     

PURIFICATION AND CHARACTERIZATION OF PHOSPHODIESTERASE I FROM Walterinnesia aegyptia VENOM

A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5′-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60°C, respectively. The energy of activation (Ea) was 96.4, the V_max and K_m were 1.14 µM/min/mg and 1.9 × 10^?3 M, respectively, and the K_cat and K_sp were 7 s^?1 and 60 M ^?1 min^?1 respectively. Cysteine was a noncompetitive inhibitor, with K_i = 6.2 × 10^?3 M and an IC₅₀ of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with K_i = 0.8 × 10^?3 M and an IC₅₀ of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg²⁺ slightly potentiated the activity. PDE-I hydrolyzed thymidine-5′-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3′-5′-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0 mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.     

Isolation and characterization of parvalbumins from skeletal muscles of a tropical amphibian,Leptodactylus insularis

Summary Parvalbumins were isolated from skeletal muscles of a tropical amphibian, Leptodactylus insularis, and three new isotypes were identified. The total concentration of parvalbumins in L. insularis was the same as the total amounts found in an amphibian from the temperate or variable zone (Rana temporaria). Muscles of the thigh and foreleg had the maximum parvalbumin concentration (0.35 mmol · kg wet weight^-1). Samples from pectoralis and rectus abdominis muscles had significantly less (0.29 mmol · kg^-1). Three previously unknown parvalbumin isotypes (IV, IIIa, and IIIb) were isolated from the tropical amphibian. They were different from the isotypes (IVa and IVb) predominant in R. temporaria skeletal muscle. Parvalbumins are thought to have a role in the short-term removal of myoplasmic Ca²⁺ during muscle relaxation. Hence, the unique isotypes in L. insularis may reflect optimal molecular adaptations retained during the animal's evolution in a constantly warm environment.Abbreviations DEAE diethylaminoethyl - ELISA enzyme linked immuno sorbent assay - SPDP N-succininydyl-3-(2-pyridyldithio) propionate - SR sarcoplasmic reticulum     

Purification and Properties of Mycodextranase from Streptomyces sp. J-13-3

An enzyme hydrolyzing nigeran (alternating α-l,3-and α-l,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-I00. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS–PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50°C. The enzyme activity was inhibited significantly by Hg⁺, Hg²⁺, and p-chloromercuribenzoic acid. The K_m (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the α-l,4-glucosidic linkages in nigeran.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly²²-Asn³⁴, in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C₁₅, C₁₆ and C₁₇ chain lengths and C₁₅ chain length, respectively, and it was considered that these fatty acids are bound to glycerol–Cys¹⁸. Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys¹⁸ → Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.     

Reproductive biology of female striped marlin Kajikia audax in the western Pacific Ocean

Length and mass data for 1260 (536 females, 683 males, 41 sex unknown) striped marlin Kajikia audax were collected at the fish markets of Tungkang, Singkang and Nanfangao from July 2004 to September 2010. Of these samples, 534 gonads (236 females and 298 males) ranging from 95 to 206 cm in eye‐to‐fork length (L _EF ) and 8 to 88 kg in round mass (M _R), were collected. Chi‐square tests indicated sex ratios were homogeneous among months in 2004 and 2006–2008, but not in 2005, 2009 and 2010; and there were significant differences in sex ratio by size. The overall sex ratio (R _S ) differed significantly from the expected 0·5. Kajikia audax are sexually dimorphic and the proportions of females increased with size between 140 and 210 cm L _EF . Reproductive activity was assessed using a gonado‐somatic index (I _G ), external appearance of the gonads and histological examination and results indicated that the spawning season occurred from April to August with a peak in June to July. Based on histological observations and the distribution of oocyte diameters, K. audax are multiple spawners and their oocytes develop asynchronously. The estimated length‐at‐50% maturity (L _EF50 ) was c . 181 cm (c . 4·8 years of age) for females. The proportion of reproductively active females in the spawning season with ovaries containing postovulatory follicles (0·27) indicated that they spawned every 3·7 days on average. The hydrated oocyte method estimated mean ± S.D. batch fecundity (F _B ) to be 4·4 ± 2·02 million eggs; average relative fecundity was 53·6 ± 13·9 oocytes g^?1 M _R; and the average annual fecundity was 181·3 ± 48·3 million eggs. The parameters estimated in this study are key information for stock assessments of K. audax in the north‐western and central Pacific and will contribute to the conservation, management and sustainable yield of this species.     

Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well.In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors.SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents.UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose.De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (¹²⁵I)-labelled lectin is bound to Mannan-Sepharose affinity matrix. The sugar inhibition pattern of this binding shows -methyl mannopyranoside and M-6-P to be equally effective as inhibitors. Scatchard analysis of the binding of UA to (¹⁴C)-mannose shows a Ka of 6.43×10⁵ (M^–1) and that 1 mole of UA can bind to 8 moles of mannose. The possible role of the protein in implantation has also been discussed.Abbreviations b.w. body weight - BSA Bovine Serum Albumin - Con A Concanavalin A - cpm counts per minute - Endo H endoglycosidase H - GlcNAc N-acetyl glucosamine - Man mannose - M-6-P mannose-6-phosphate - MEM-deficient Minimum Essential Medium Eagle-deficient modification - NaBH₄ sodium borohydride - NaN₃ sodium azide - (NH₄)₂SO₄ ammonium sulphate - p.c. post coitum - PMSF phenyl methyl sulphonyl fluoride - PTA phosphotungstic acid - RCA Ricinus communis Agglutinin - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - UA Uterine Agglutinin - WGA Wheat-germ Agglutinin     

Three Types of Acidic Polysaccharides Associated with Coccolith of Pleurochrysis haptonemofera: Comparison with Pleurochrysis carterae and Analysis Using Fluorescein-Isothiocyanate-Labeled Lectins

In the coccolithophorid microalgae acidic polysaccharides are considered to be involved in the formation of the calcified scale, coccolith. Characteristics of the acidic polysaccharides extracted from the cell surface of the coccolithophorid Pleurochrysis haptonemofera were analyzed. The acidic polysaccharides on the cell surface can be detected by measuring fluorescence of cells after fluorescein-isothiocyanate-labeled lectin staining by flow cytometry. Flow cytometric analyses revealed that the acidic polysaccharides remained on the cell surface even after CaCO₃ in the coccolith was dissolved by lowering pH, but they were extracted by subsequent EDTA or EGTA treatment, suggesting that they are bound not into the CaCO₃ crystals of the coccolith, but onto the surface via Ca²⁺. Analyses of the acidic polysaccharides by anion exchange chromatography, colloidal precipitation with divalent cations, and polyacrylamide gel electrophoresis (PAGE) revealed that P. haptonemofera has 3 types of acidic polysaccharides (Ph-PS-l, -2, and -3). The PAGE patterns suggested that Ph-PS-2 has a repeated structure with a broad range of molecular weight, as in Pleurochrysis carterae, while Ph-PS-1 and -3 contain several minor components in addition to a major component, respectively. The minor components in Ph-PS-1 and -3 that have not been found in P. carterae might be characteristic of P. haptonemofera. Analyses of both the cell surface treated by various concentrations of EDTA and EGTA and the extracts suggested that Ph-PS-2, which is distinguishable by a higher affinity to concanavalin A, is bound onto the coccolith surface more intensely than the other two types of acidic polysaccharides.     

Purification and characterization ofFus sI3596, a 65 kd allergen ofFusarium solani

A component ofFusarium solani (F. solani), identified as the major allergen,Fus sI₃₅₉₆ ^* was purified to homogeneity from culture filtrate (CF) by means of anion-exchange column chromatography, gel filtration and FPLC. The homogeneity ofFus sI₃₅₉₆ ^* was assessed by IEF, PAGE, SDS-PAGE (non-reducing), immunoblot and HPLC.Fus sI₃₅₉₆ ^* was isolated as a glycoprotein of MW 65 kd and pI 3.6. The IgE ELISA-inhibition assay after periodate treatment of the fraction showed a lower IgE binding capacity suggesting involvement of carbohydrate moiety in IgE binding reactions of the allergen. Peptide fragments ofFus sI₃₅₉₆ ^* obtained after CNBr and trypsin treatment were analysed by immunoblotting for their allergenicity. This study indicated that there could be at least 3 allergenic determinants in the major allergen,Fus sI₃₅₉₆ ^* ofF. solani CF.Abbreviations DEAE diethyl aminoethyl - HPLC high performance liquid chromatography - SDS sodium dodecyl sulphate - PBS-Tween phosphate buffer saline containing 0.1% tween - DTT dithiothreitol - TFA triflouroacetate - FPLC fast protein liquid chromatography     

DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a ³²P-postlabeling/thin layer chromatography (TLC) method and a ³²P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36–36.4 μM) for 3 h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2′-deoxyadenosin-N⁶-yl)-3-aminobenzanthrone-3′-phosphate (dA3′p-N⁶-C2-ABA) and 2-(2′-deoxyguanosin-N²-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-N²-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3′p-N⁶-C2-ABA and its relative adduct labeling (RAL) value at 36.4 μM of 3-NBA was 200.8 ± 86.1/10⁸ nucleotide.     

Purification and Molecular Analysis of a Monoamine Oxidase Isolated from Narcissus tazetta

Semicarbazide-sensitive amine oxidase activity was detected in Narcissus tazetta. The enzyme was purified to homogeneity by the criterion of native polyacrylamide gel electrophoresis (PAGE) with DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose columns. The molecular mass of the enzyme, determined using a GS-520 HQ column, was estimated to be 135 kDa. SDS–PAGE yielded two bands of, 75 kDa and 65 kDa. The enzyme, which had catalytic activity for some aliphatic and aromatic monoamines, belongs to a class of monoamine oxidases (MAOs). The K _m value for n-propylamine was 5.9 × 10^?5 M. A substrate analog, 2-bromoethylamine, inhibited enzyme activity. Redox-cycling staining detected a quinone in the MAO protein. By inductively coupled plasma mass analysis, it was determined that there were 2.44 moles of copper atoms per mole of the enzyme. Protein sequence analysis revealed that there was no identity between two N-terminal residues of the 75 kDa and 65 kDa proteins of narcissus MAO.     

Purification and characterization of a novel extracellular β-1,3-glucanase complex (Glu<Emphasis Type="Italic">Ggt</Emphasis>) secreted by <Emphasis Type="Italic">Gaeumannomyces graminis</Emphasis> var. <Emphasis Type="Italic">tritici</Emphasis>

β-1,3-Glucanases produced by the take-all disease pathogen Gaeumannomyces graminis var. tritici (Ggt) have been suggested to be implicated in infection and colonization process of the pathogen in wheat. For further studying the role of these enzymes in pathogenesis by cytochemical technique, an extracellular β-1,3-glucanase complex (GluGgt), was purified from culture filtrate of Ggt by (NH₄)₂SO₄ precipitation, hydrophobic-interaction, anion-exchange and size-exclusion chromatography. The complex consisted of two interconvertible isoforms (Ia and Ib), both active proteins Ia and Ib appeared to be glycosylated in native polyacrylamide gel electrophoresis (PAGE). The pI of the active proteins Ia and Ib determined by IEF-PAGE were 6.3 and 6.4, respectively. GluGgt yielded two subunits with molecular masses of 66.2 and 56.0 kDa, respectively, in 15% SDS–PAGE. The complex had a pH optimum of 5.0 and the optimal temperature was 50°C. The enzyme complex proved to be stable at a temperature up to 60°C and retained an optimal high activity in the pH range from 4.0 to 7.0. Enzyme activity of GluGgt was obviously stimulated by Mn²⁺ ions and moderately inhibited in the presence of Hg²⁺ and Co²⁺ ions and KMnO₄. The K _m of GluGgt was estimated to be 1.08 mg ml⁻¹ for laminarin as substrate and the V _max 0.20 μmol min⁻¹.     

A Kinetic Analysis of 6-[18F]Fluoro-l-Dihydroxyphenylalanine Metabolism in the Rat

Abstract: A previous study of the metabolism of 6-[¹⁸F]-fluoro-l -3,4-dihydroxyphenylalanine (FDOPA) in rats pretreated with carbidopa contained information amenable to kinetic analysis. Using these data, tracer transfer coefficients and metabolic rate constants were estimated. After intravenous injection, FDOPA in circulation was O-methylated (k^D₀ = 0.055 min^?1), and the metabolite (O-methyl-FDOPA) escaped from plasma with a rate constant (k^M_?1) of 0.01 min^?1. The initial clearance of FDOPA to striatum (K^D₁) was 0.07 ml g^?1 min^?1, and the equilibrium distribution volume (V^D_e) was 0.67 ml g^?1. The initial clearance of O-methyl-FDOPA to striatum (K^M₁) was 0.08 ml g^?1 min^?1, and the equilibrium distribution volume (V^M_e) was 0.75 ml g^?1. The rate constant of FDOPA decarboxylation (k^D₃) was 0.17 min^?1 in striatum. The elimination of 6-[¹⁸F]fluorodopamine (FDA) from striatum suggested an apparent rate constant for monoamine oxidase activity (k′₇) of 0.055 min^?1. 6-[¹⁸F]Fluorohomovanillic acid (FHVA) was formed from 6-[¹⁸F]fluoro-l -3,4-dihydroxyphenylacetic acid with a rate constant (k₁₁) of 0.083 min^?1, and FHVA was eliminated from striatum (k₉) with a rate constant of 0.12 min^?1. The steady-state concentration ratios of FDA and its metabolites were shown to be functions of these rate constants.