Expression and characterization of the C345C/NTR domains of complement components C3 and C5

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.     

Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.     

Drosophila lacks C20 and C22 PUFAs

Drosophila melanogaster has been considered a model organism for investigating human diseases and genetic pathways. Whether Drosophila is an ideal model for nutrigenomics, especially for FA metabolism, however, remains to be illustrated. The aim of this study was to examine the metabolism of C20 and C22 PUFAs in Drosophila. Analysis of FA composition revealed a complete lack of C20 and C22 PUFAs in the body tissue of larvae, pupae, and adult flies fed either a base or supplemented diet abundant in the PUFA precursors linoleic acid and α-linolenic acid. PUFA with >C20 could only be found in flies supplemented with specific FAs. Interestingly, the supplemented C22 PUFAs docosahexaenoic acid (22:6n-3) and docosatetraenoic acid (22:4n-6) were largely converted to the shorter chain C20 PUFAs eicosapentaenoic acid (20:5n-3) and arachidonic acid (20:4n-6), respectively. Furthermore, a genome sequence scan indicated that no gene encoding Δ-6/ Δ-5 desaturases, the key enzymes for the synthesis of C20/C22 PUFA, was present in Drosophila. These findings demonstrate that Drosophila lacks the capability to synthesize the biologically important C20 and C22 PUFAs, and thereby argue that Drosophila is not a valid model for the study of lipid metabolism and related diseases.     

Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

贺谈老百岁华诞

谈家桢教授是国际著名遗传学家,我国现代遗传学的奠基人之一,他也是一位卓越的教育家和社会活动家。 1909年9月15日,谈家桢先生出生于浙江宁波。他就读于苏州东吴大学,1930年获理学学士学位。随后赴北京燕京大学攻读硕士学位,导师是我国现代遗传学奠基人之一的李汝祺教授,1932年获硕士学位。经导师推荐,谈家桢先生赴美国深造,师从当代遗传学宗师摩尔根,在遗传学家杜布赞斯基的指导下完成博士研究生学业,于1936年获美国加州理工学院哲学博士学位。     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

C3 and C6 complement types in schizophrenia

C3 and C6 complement types were studied in schizophrenic patients and controls. The distributions of the three common C3 types (F, FS and S) among the patients was significantly different from that in the controls (p less than 0.005) and the frequency of the C3F gene was significantly increased (p less than 0.0005) among the patients. There were no significant differences in C6 gene or phenotype frequencies between patient and controls.     

Phosphorus responses of C3 and C4 species

An hypothesis was formulated that phosphorus (P) partitioningin tissues of C₄ leaves would permit C₄ plants to resist P deficiencybetter than C₃ plants. To test this hypothesis, 12 C₃, C₄, andC₃–C₄ intermediate species were grown at adequate, deficient,and severely deficient P supply in a solid-phase-buffered sandculture system to characterize photosynthetic and growth responses.Species differed considerably in response to P stress. The growthof C₃ species was more sensitive to P supply than C₄ species,but C₃ and C₄ species had similar photosynthetic P use efficiency,and C₄ species did not have low leaf P content, contrary toour hypothesis. In fact, leaf photosynthetic rates were notcorrelated with growth responses. Moncots had lower leaf P contentand better maintenance of leaf production under P stress thandicots, because of greater inhibition of branching (dicots)than of tillering (monocots). The most P efficient species inthis survey was Brachiaria, a C₄ monocot that increased rootbiomass allocation under stress while maintaining P allocationto the shoot. It is concluded that C₄ species are not inherentlymore P efficient than C₃ species, but that monocots are moreP efficient than dicots, because of contrasting P and biomassallocation under stress. Key words: Phosphorus deficiency, C₃ plants, C₄ plants, growth response     

Insect herbivory on C3 and C4 grasses

Summary This study tested the hypothesis that grasses with the C₄ photosynthetic pathway are avoided as a food source by insect herbivores in natural communities. Insects were sampled from ten pairs of C₃–C₄ grasses and their distributions analyzed by paired comparisons tests. Results showed no statistically significant differences in herbivore utilization of C₃–C₄ species. However, there was a trend towards heavier utilization of C₃ species when means for both plant groups were compared. In particular, Homoptera and Diptera showed heavier usage of C₃ plants. Significant correlations between insect abundances and plant protein levels suggest that herbivores respond to the higher protein content of C₃ grasses. ¹³C values for six of the most common grasshopper species in the study area indicated that three species fed on C₃ plants, two species fed on C₄ plants, and one species consumed a mixture of C₃ and C₄ tissue.Welder Wildlife Refuge Contribution Number 213     

Structure and activity of C1r and C1s

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.     

C3、C4和C3-C4中间型植物的进化

介绍了有关C3、C4和C3-C4中间型植物进化的形态学、生理学、分子生物学、遗传学等方面的证据;推断地球上首先出现C3植物,然后是C3-C4中间类型植物,最后出现C4植物.     

Genetics of complement C4. Two homoduplication haplotypes C4S C4S and C4F C4F in a family

Summary A family in which two homoduplicated C4 haplotypes (or supergenes) segregate is described. One haplotype C4F ^* 3 C4F ^*2.2 is composed of two C4F alleles and the other C4S ^* 5.1 C4S ^*1 of two C4S alleles. The C4F duplication haplotype is a partial inhibitor of the Rodgers antigen, and judged from our family and population material, it seems to be rather frequent and associated with HLAB ^*35, Bf ^* F, and HLAD/DR ^*1. The C4S duplication haplotype is Rg(a-) and is not identified in individuals without another S, Ch(a+) variant.This work was supported by grant No 12-1727 from the Danish Medical Research Council     

Dehydroxylation of cholic acid at C12 and epimerization at C5 and C7 by Bacteroides species

Freshly isolated cultures (2060) of human intestinal bacteria of the predominant flora, among them 1029 strains of saccharolytic Bacteroides species, were tested for cholic acid transformation. Eight Bacteroides strains reduced cholate to chenodeoxycholate, while 73 strains dehydroxylated at C7, producing deoxycholate. Concurrent oxidation of hydroxyl groups, mainly at C7, was seen with many strains. No strain was able to dehydroxylate simultaneously at C7 and C12. One isolate, identified as a mixed culture of Bacteroides fragilis and B. uniformis, epimerized cholic acid at C5 and simultaneously epimerized, oxidized and dehydroxylated at C7. The following transformation products were identified: 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic acid (ursocholic acid), 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic acid and a 3 alpha,12 alpha-dihydroxy-5 alpha-cholenoic acid. Dehydroxylating and epimerizing abilities were detected when fresh isolates were tested first for cholate transformation. They were no longer recognizable after some serial transfers. Dehydroxylation at C12 of cholate could not be demonstrated with mixed fecal cultures. The possible intermediate, however, 3 alpha,7 alpha-dihydroxy-5 beta-chol-11-enoate, was abundantly hydrogenated by stool suspensions.