Modification of the Recalde method for the isolation of human monocytes

A modification of the method for monocyte isolation reported by Recalde (1984. J. Immunol. Methods. 69: 71-77) is described. Application of the modified method to 36 consecutive healthy adult donors gave a monocyte purity of 73 +/- 8% monocytes with less than 1% polymorphonuclear leukocytes and a yield of 3.44 +/- 0.93 x 10(5) monocytes/ml blood. While the monocyte purity of the modified Recalde method was lower than that obtained by elutriation (method BB in Fogelman et al. 1981. J. Lipid Res. 22: 1131-1141) in 15 donors (71 +/- 10% vs. 83 +/- 6%) the monocyte yield and the viability of the cells before and after culture were similar in both methods. The modified Recalde method does not require the expensive or complicated equipment required for elutriation and permits the isolation of human monocytes for culture in autologous serum from multiple donors in a single day.     

Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor

The inhibition of kallikrein and factor XIa by protein C inhibitor (PCI) was studied. The method of Suzuki et al. [Suzuki, K., Nishioka, J., & Hashimoto, S. (1983) J. Biol. Chem. 258, 163-168] for the purification of PCI was modified in order to avoid the generation of proteolytic activity and subsequent inactivation of PCI. With the use of soybean trypsin inhibitor, an efficient inhibitor of kallikrein and factor XIa, the generation of proteolytic activity was avoided. The kinetics for the inactivation of activated protein C (APC), kallikrein, and factor XIa by PCI were determined. In the absence of heparin, no inactivation of APC was observed, in contrast to kallikrein and factor XIa, which are inhibited with second-order rate constants of (11 +/- 4) X 10(4) and (0.94 +/- 0.07) X 10(4) M-1 s-1, respectively. Addition of heparin potentiated the inhibition of APC [(1.2 +/- 0.2) X 10(4) M-1 s-1] and factor XIa [(9.1 +/- 0.7) X 10(4) M-1 s-1] by PCI, whereas the inhibition of kallikrein by PCI was unchanged [(10 +/- 1) X 10(4) M-1 s-1]. The second-order rate constants for the inhibition of kallikrein or factor XIa by PCI were similar to the second-order rate constants for the inhibition of their isolated light chains by PCI, indicating a minor role for the heavy chains of both molecules in the inactivation reactions. With sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and immunoblotting, complex formation of APC, kallikrein, and factor XIa with PCI could be demonstrated. APC and kallikrein formed 1:1 molar complexes with PCI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium affinity of regulatory sites in skeletal troponin-C is attenuated by N-cap mutations of helix C

Site-directed mutagenesis was used to make amino acid substitutions at position 54 of skeletal troponin C, testing a relationship between the stability of helix C and calcium ion affinity at regulatory sites in the protein. Normally, threonine at position 54 is the first helical residue, or N-cap, of the C helix; where helices C and D, and the loop between, comprise binding site II. Mutations were made in the context of a previously described phenylalanine 29--> tryptophan (F29W) variant (Trigo-Gonzalez et al., Biochemistry 31, 7009-7015 (1992)), which allows binding events to be monitored through changes in the intrinsic fluorescence of the protein. N-Cap substitutions at position 54 were shown to attenuate the calcium affinity of regulatory sites in the N-terminal domain. Calcium affinities diminished according to the series T54 T54S > T54A > T54V > T54G with dissociation constants of 1.36 x 10(-6), 1.36 x 10(-6), 2.09 x 10(-6), 2.28 x 10(-6), and 4.24 x 10(-6) M, respectively. The steady state binding of calcium to proteins in the mutant series was seen to be monophasic and cooperative. Calcium off-rates were measured by stopped flow fluorescence and in every instance two transitions were observed. The rate constant of the first transition, corresponding to approximately 99% of the change in fluorescence, was between 900+/-20 and 1470+/-100 s(-1), whereas the rate constant of the second transitions was between 94+/-9 and 130+/-23 s(-1). The significance of two transitions remains unclear, though both rate constants occur on a time scale consistent with the regulation of contraction.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

湖北秭归三叶虫Ushbaspis hubeiensis新材料及其研究意义

文中通过对Ushbaspis hubeiensis (Lin,1978)新材料的研究,对该种三叶虫躯体构造特征做了重要补充,使人们对它有更全面的认识,也将为今后进一步对乌什巴盾壳虫类(ushbaspid) 三叶虫的分类、系统演化的研究以及对寒武纪第二世生物地层划分,提供一些重要依据。文中还就张文堂等（1980）将采自贵州湄潭的Kepingaspis sp.标本鉴定为Metaredlichioides constrictus Qian and Yao,1974提出质疑。     

峡东地区早寒武世水井沱组古盘虫类三叶虫的再研究

对峡东和邻近地区早寒武世古盘虫类三叶虫的再研究,支持将Emeidiscus Li,1980;Mianxrindiscus S．Zhang et Zhu in Zhang et al,1980;Mianxiandiscus(Liangshandiscus)S．Zhang in Zhang et al．,1980;Hupeidiscus Chang in Lu et al．,1974;Guizhoudiscus S．Zhang in Zhang et al．,1980;Shizhudiscus S．Zhang et Zhu in Zhang et al．,1980等属作为Tsunyidiscus Chang,1966的同义名的意见。并对前人所建立的有关属的种,进行了重新研究和整理,作了大量的归并和修订。就目前所知道,峡东地区早寒武世水井沱组的古盘虫类三叶虫仅有Tsunyidiscus Chang,1966和Sinodiscus Chang in Lu et al,1974两属。Hupeidiscus经修订归入Tsunyidiscus Chang,1966后,原Hebediscus orientalis Chang,1953(即原Hupeidiscus的模式种)一种的种名与Tsunyidiscus orientalis(Walcott,1905)重名,故将前者重新命名为Tsunyidiscus pertenus nom．nov．。文中还记述了湖北宜昌、秭归等地的早寒武世古盘虫类三叶虫2属3种,包括Tsunyidiscus yanjiazhaiensis S．Zhang,Zhou et Yuan in Yin and Li,1978;Tsunyidisacutus(Sun,1983);Sinodiscus changyangensis S．Zhang in Zhou et al．,1977。     

Characterization of the mechanical properties of skin by inverse analysis combined with the indentation test

This study proposes a new method to determine the mechanical properties of human skin by the use of the indentation test [Pailler-Mattei, 2004. Caractérisation mécanique et tribologique de la peau humaine in vivo, Ph.D. Thesis, ECL-no. 2004-31; Pailler-Mattei, Zahouani, 2004. Journal of Adhesion Science and Technology 18, 1739-1758]. The principle of the measurements consists in applying an in vivo compressive stress [Zhang et al., 1994. Proceedings of the Institution of Mechanical Engineers 208, 217-222; Bosboom et al., 2001. Journal of Biomechanics 34, 1365-1368; Oomens et al., 1984. Selected Proceedings of Meetings of European Society of Biomechanics, pp. 227-232; Oomens et al., 1987. Journal of Biomechanics 20(9), 877-885] on the skin tissue of an individual's forearm. These measurements show an increase in the normal contact force as a function of the indentation depth. The interpretation of such results usually requires a long and tedious phenomenological study. We propose a new method to determine the mechanical parameters which control the response of skin tissue. This method is threefold: experimental, numerical, and comparative. It consists combining experimental results with a numerical finite elements model in order to find out the required parameters. This process uses a scheme of extended Kalman filters (EKF) [Gu et al., 2003. Materials Science and Engineering A345, 223-233; Nakamura et al., 2000. Acta Mater 48, 4293-4306; Leustean and Rosu, 2003. Certifying Kalman filters. RIACS Technical Report 03.02, 27pp. http://gureni.cs.uiuc.edu/~grosu/download/luta + leo.pdf; Welch and Bishop, An introduction to Kalman filter, University of North Carolina at Chapel Hill, 16p. http://www.cs.unc.edu/~welch/kalman/]. The first results presented in this study correspond to a simplified numerical modeling of the global system. The skin is assumed to be a semi-infinite layer with an isotropic linear elastic mechanical behavior [Zhang et al., 1994. Proceedings of the Institution of Mechanical Engineers 208, 217-222] This analysis will be extended to more realistic models in further works.     

Phylogenetic evidence against evolutionary stasis and natural abiotic reservoirs of influenza A virus

Zhang et al. (G. Zhang, D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S. O. Rogers, J. Virol. 80:12229-12235, 2006) have claimed to have recovered influenza A virus RNA from Siberian lake ice, postulating that ice might represent an important abiotic reservoir for the persistence and reemergence of this medically important pathogen. A rigorous phylogenetic analysis of these influenza A virus hemagglutinin gene sequences, however, indicates that they originated from a laboratory reference strain derived from the earliest human influenza A virus isolate, WS/33. Contrary to Zhang et al.'s assertions that the Siberian "ice viruses" are most closely related either to avian influenza virus or to human influenza virus strains from Asia from the 1960s (Zhang et al., J. Virol. 81:2538 [erratum], 2007), they are clearly contaminants from the WS/33 positive control used in their laboratory. There is thus no credible evidence that environmental ice acts as a biologically relevant reservoir for influenza viruses. Several additional cases with findings that seem at odds with the biology of influenza virus, including modern-looking avian influenza virus RNA sequences from an archival goose specimen collected in 1917 (T. G. Fanning, R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger, J. Virol. 76:7860-7862, 2002), can also be explained by laboratory contamination or other experimental errors. Many putative examples of evolutionary stasis in influenza A virus appear to be due to laboratory artifacts.     

1H, 13C, and 15N NMR assignments and global folding pattern of the RNA-binding domain of the human hnRNP C proteins.

The hnRNP C1 and C2 proteins are abundant nuclear proteins that bind avidly to heterogeneous nuclear RNAs (hnRNAs) and appear to be involved with pre-mRNA processing. The RNA-binding activity of the hnRNP C proteins is contained in the amino-terminal 94 amino acid RNA-binding domain (RBD) that is identical for these two proteins. We have obtained the 1H, 13C, and 15N NMR assignments for the RBD of the human hnRNP C proteins. The assignment process was facilitated by extensive utilization of three- and four-dimensional heteronuclear-edited spectra. Sequential assignments of the backbone resonances were made using a combination of 15N-edited 3D NOESY-HMQC, 3D TOCSY-HMQC, and 3D TOCSY-NOESY-HSQC as well as 3D HNCA, HNCO, and HCACO spectra. Side-chain resonances were assigned using 3D HCCH-COSY and 3D HCH-TOCSY spectra. Four-dimensional 13C/13C-edited NOESY and 13C/15N-edited NOESY experiments were used to unambigously resolve NOEs. The overall global folding pattern was established by calculating a set of preliminary structures using constraints derived from the sequential NOEs and a small number of long-range NOEs. The beta alpha beta-beta alpha beta domain structure exhibits an antiparallel beta-sheet with the conserved RNP 1 and RNP 2 sequences [Dreyfuss et al. (1988) Trends Biochem. Sci. 13, 86-91] located adjacent to one another as the two inner strands of the beta-sheet.     

Relationship of DNA methylation level to the presence of heterochromatin in mealybugs.

Purified nuclear DNA from two mealybug species was analyzed for its 5-methylcytosine (m5C) content by reversed-phase high-pressure liquid chromatography. We observed that the percent m5C (percentage of cytosines which are methylated) varied between the two species, between males and females of the same species, and between lines with and without supernumerary B chromosomes. This is the first case of a sex-specific difference in overall DNA methylation level. In contrast to a recent report (Deobagkar et al., J. Biosci. [India] 4:513-526, 1982), we found no other modified bases in the DNA. Overall, the percent m5C in Pseudococcus obscurus was two to three times higher than in Pseudococcus calceolariae. In both species, the percent m5C in males was higher than in females, although only in P. calceolariae was the difference statistically significant (0.68 +/- 0.02 versus 0.44 +/- 0.04). The high m5C content in males was correlated with the presence of a paternally derived, genetically inactive set of chromosomes which is facultatively heterochromatic. The presence of constitutive heterochromatin, however, was associated with a lower m5C content. Thus, for example, the percent m5C in females of a P. obscurus line with heterochromatic B chromosomes (1.09 +/- 0.04) was significantly lower than that of a related line lacking such chromosomes (1.26 +/- 0.06). Our findings are discussed with respect to the possible relationship between DNA methylation and heterochromatization.     

Purification, solubility, and pKa of veratridine

The alkaloid neurotoxin veratridine is widely used by cell physiologists to increase membrane sodium permeability. The compound is only sporadically available from commercial sources, but can be purified (Kupchan et al., 1953, J. Amer. Chem. Soc. 75, 5519-5524) from veratrine, a mixture of several alkaloids. We describe here a purification procedure only slightly modified from that of Kupchan et al., and include important details not mentioned in the original paper. Ultraviolet and infrared spectra are presented. We have also determined the pKa and solubility of veratridine in 150 mM NaCl at 25 degrees C. The solubility is steeply pH dependent, ranging from 0.61 +/- 0.02 mM above pH 12 to 18.5 mM at pH 8.07. The pKa, determined from the solubility versus pH curve, was found to be 9.54 +/- 0.02.     

Revision on Cephalostachyum Munro (Poaceae: Bambusoideae) in China

Species of Cephalostachyum Munro (Poaceae: Bambusoideae) from China are distributed in Yunnan and Tibet, mainly in Yunnan. In this paper, we discussed species of Cephalostachyum and compiled a key to species from China, based on recent studies on micromorphological characters of leaf epidermis and molecular phylogenetics of paleotropical woody bamboos. There is a total of seven species of Cephalostachyum distributed in China, all in Yunnan: Cchinense (Rendle) DZ. Li et HQ. Yang, Cfuchsianum Gamble et Hook. f., Cmannii (Gamble) Stapleton et DZ. Li, Cpallidum Munro, Cpingbianense (Hsueh et YM. Yang ex Yi et al.) DZ. Li et HQ. Yang, Csanguineum (WP. Zhang) DZ. Li et HQ. Yang and Cscandens Bor. Leptocanna Chia et HL. Fung and Cvirulentum YM Yang et F. Du are synonyms of Cephalostachyum Munro and Cfuchsianum Gamble et Hook. f. respectively. On the other hand, Cpergracile Munro and Cvirgatum (Munro) Kurz are morphologically closer to Schizostachyum Nees than to Cephalostachyum, and they should be treated as members of Schizostachyum. This paper is of significance to a worldwide revision of Cephalostachyum.     

Accumulation of ester- and ether-linked phosphatidates by HeLa cells in response to ionophore A23187 through activation of phospholipase D.

Phosphatidates seem to play an important role in the control of cell proliferation modified by ligands (M. Kaszkin et al. 1991, Cancer Res. 51, 4328-4335). In this study the potency of calcium ionophore A23187 to alter phosphatidate levels in HeLa cells as a model was studied in detail. HeLa cells prelabeled with [14C]arachidonic acid responded to calcium ionophore A23187 with a rapid accumulation of labeled 1,2-diacylglycerophosphate (acyl-PA) and 2-acyl-1-O-alkylglycerophosphate (alkyl-PA) with a first peak at 5 min and a second increase starting at 20-30 min. In cells prelabeled with [14C]oleic acid the ionophore mobilized relatively more of labeled acyl-PA. The total amount of phosphatidates mobilized was in the order of 0.2 micrograms/10(6) cells, i.e. an almost 10(-4)M concentration. The transphosphatidylation of labeled acyl- and alkyl-PA to 1-butanol in all cases showed that activation of phospholipase D had occurred. The reaction became detectable at 10(-6)M ionophore and was fully expressed at 10(-5)M. Butyl phosphatidate generated during 1 h treatment with ionophore amounted to approx. 0.5 nmol per 10(6) cells (i.e. 10(-4)M conc. within cells) as shown by the use of [14C]butanol. The 3-5-fold rise of the overall phosphatidate level is probably sufficient to alter physically cellular membranes, particularly if the new phosphatidate is restricted to certain compartment(s).     

Molecular weight of poly(C9). 12 to 18 C9 molecules form the transmembrane channel of complement

Poly(C9), the tubular 27 S complex forming the transmembrane channel of the membrane attack complex of complement, was purified to homogeneity by gel filtration and sucrose density gradient ultracentrifugation. The molecular weight of poly(C9) was determined by two independent methods in addition to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. First, sedimentation equilibrium analysis using 0.2% SDS or 1% deoxycholate containing buffer as solvents yielded a point weight average molecular weight exclusive of bound detergent of 0.9 to 1.3 X 10(6) and a weight average molecular weight of all poly(C9) complexes of 1,050,000 +/- 40,000 (S.D.). SDS and deoxycholate binding to poly(C9) was measured in an air-driven ultracentrifuge and was determined to be 0.53 +/- 0.065 (S.D.) g of SDS and 0.26 +/- 0.015 (S.D.) g of deoxycholate/g of poly(C9), respectively. Second, the mass of 27 S poly(C9) devoid of bound detergent was determined by electron scattering of unstained specimens in the scanning transmission electron microscope. The molecular weight obtained by this method was 1,078,000 +/- 194,000. The inner diameter of poly(C9) tubules imaged in top view projections by negative staining electron microscopy varied between 9 and 12 mm. The accumulated data suggest a true heterogeneity of the molecular weight of poly(C9) due to polymers with varying protomer numbers. Using a mean value of 73,500 for the molecular weight of monomeric C9, the protomer number of poly(C9) tubules appear to vary between 12 and 18 C9 subunits. Approximately 50-75% of the tubules have 14 to 16 subunits as deduced from the mass distribution determined by electron scattering and from ring size measurements. It is suggested that poly(C9) tubules with various protomer numbers may arise due to limited flexibility in the C9-C9 interaction.     

Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C

Binding of Ca(2+) to the regulatory domain of troponin C (TnC) in cardiac muscle initiates a series of protein conformational changes and modified protein-protein interactions that initiate contraction. Cardiac TnC contains two Ca(2+) binding sites, with one site being naturally defunct. Previously, binding of Ca(2+) to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TDeltaS) of 2 and 0.5 kcal mol(-1) for the functional and nonfunctional sites, respectively, using (15)N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dynamics of the Ca(2+)-free regulatory domain are investigated by backbone amide (15)N relaxation measurements at eight temperatures from 5 to 45 degrees C. Analysis of the relaxation measurements yields an order parameter (S(2)) indicating the degree of spatial restriction for a backbone amide H-N vector. The temperature dependence of S(2) allows estimation of the contribution to protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (C(p,j)) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cal mol(-1) K(-1). The average heat capacity for Ca(2+) binding site 1 is larger than that for site 2 by 1.3 +/- 0.8 cal mol(-1) K(-1), and likely represents a mechanism where differences in affinity between Ca(2+) binding sites for EF hand proteins can be modulated.     

Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F- actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin''s ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.     

Chemiluminescent simultaneous determination of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the liver and brain of the rat.

The quantification of phospholipid hydroperoxides in biological tissues is important in order to know the degree of peroxidative damage of membrane lipids. For this purpose, optimal conditions for the chemiluminescent simultaneous assay of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in rat liver and brain were determined. A chemiluminescence detection-high performance liquid chromatography (CL-HPLC) method that incorporates cytochrome c and luminol as a post-column hydroperoxide-specific luminescent reagent was used (Miyazawa et al. 1987. Anal. Lett. 20: 915-925; Miyazawa. 1989. Free Radical Biol. Med. 7: 209-217). An n-propylamine-bound silica column with hexane-2-propanol-methanol-water 5:7:2:1 (v/v/v/v) (flow rate 1.0 ml/min) as eluant was used to determine both PCOOH and PEOOH, which were separated from each other and from other lipids and lipid-soluble antioxidants. High reproducibility and sensitivity as low as 10 pmol hydroperoxide-O2 were observed with a mixture of 10 micrograms/ml cytochrome c and 2 micrograms/ml luminol in 50 mM borate buffer (pH 10.0, flow rate 1.1 ml/min) as luminescent reagent and a post-column mixing joint temperature of 40 degrees C. Using the established analytical conditions, it was confirmed that both PCOOH (1324 +/- 122 pmol/g liver, 114 +/- 18 pmol/g brain, mean +/- SD) and PEOOH (728 +/- 89 pmol/g liver, 349 +/- 60 pmol/g brain, mean +/- SD) are present in the liver and brain of Sprague-Dawley rats bred on a slightly modified AIN-76A semisynthetic diet for 3 months. The phospholipid hydroperoxide content in the rat liver was shown to be affected by dietary oils, but not significantly affected in the brain.     

Production and purification of Clostridium botulinum type C and D neurotoxin

Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.     

Hydrogen and carbon isotopic fractionations of lipid biosynthesis among terrestrial (C3, C4 and CAM) and aquatic plants

Compound-specific hydrogen and carbon isotopic compositions in n-alkanoic acids, phytol and sterols were determined for various plant classes (terrestrial C3-angiosperm; C3-gymnosperm; C4; crassulacean acid metabolism (CAM); and aquatic C3 plants) in order to investigate isotopic fractionations among various plant classes. In all plants, lipid biomolecules are depleted in both D (up to 324 per thousand ) and 13C (up to 14.7 per thousand ) relative to ambient water and bulk tissue, respectively. In addition, the magnitude of D- and 13C-depletion of lipid biomolecules is distinctive depending on plant classes. For example, C3 angiosperm n-alkanoic acids are less depleted in D (95+/-23 per thousand ) and 13C (4.3 +/- 2.5 per thousand ) relative to ambient water and bulk tissue, respectively, while C4 plant n-alkanoic acids are more depleted in D (119 +/- 15 per thousand ) and 13C (10.2 +/- 2.0 per thousand ). On the other hand, C3 angiosperm phytol and sterols are much more depleted in D (306 +/-12 per thousand for phytol, 211+/-15 per thousand for sterol) with less depletion in 13C (4.1 +/- 1.1 per thousand for phytol, 1.3 +/- 0.9 per thousand for sterol) relative to ambient water and bulk tissue, respectively, while C4 plant phytol and sterols are less depleted in D (254 +/- 7 per thousand for phytol, 186 +/- 13 per thousand for sterols) with much more depletion in 13C (9.0 +/- 1.2 per thousand for phytol, 5.0 +/- 1.1 per thousand for sterols). Among various plant classes, there is a positive correlation between the D- and 13C-depletion for n-alkanoic acids, while a negative correlation was found for phytol and sterols from the same plants.     

Correction of glucose carbon recycling for the determination of 'true' hepatic glucose production rates by (1-13C1)glucose

Hepatic glucose production (HGP) and glucose carbon recycling are traditionally estimated by the combined use of hydrogen and carbon-labeled glucose tracers. A single-isotope method such as that of Reichard et al. for the determination of HGP and glucose carbon recycling requires the determination of activities in different glucose carbons by chemical degradation. Since the 13C content in the glucose carbon skeleton can be determined from mass fragmentography, the use of 13C-labeled glucose and mass fragmentography can provide a single-isotope method for the quantification of the recycled carbons. Correction for the recycling makes it possible to determine the true HGP. In this study, (1-13C1)glucose and mass fragmentography were used for the determination of HGP and glucose carbon recycling in six colon cancer patients. Molar enrichment of the molecular ion (m/z 328 cluster of glucose aldonitrile pentaacetate) was used to determine 'uncorrected' HGP, which was 1.93 +/- 0.11 mg kg-1 min-1 (mean +/- s.e.m.). The difference in molar enrichment of the molecular ion C1-C6 (m/z 328) and the ion corresponding to C1-C4 fragment (m/z 242) was used to determine the contribution of recycled label carbon. After this correction, the 'corrected' HGP was 2.04 +/- 0.12 mg kg-1 min-1, which is not significantly different from the 'true' HGP rate of 2.05 +/- 0.15 mg kg-1 min-1 determined by using (6-3H)glucose. HGP determined from the enrichment of the molecular ion C1-C6 underestimates true HGP, as expected. The corrected HGPs correlate well with those from 6-3H method (r = 0.86, y = 1.06x - 0.12; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)