Isolation and properties of chloroplast particles of Scenedesmus obliquus D3 with high photochemical activity

An improved and standardized procedure for isolation of chloroplast particles from the unicellular green alga, Scenedesmus obliquus, D₃, is described. The method is generally applicable to heterotrophically- and autotrophically-grown cells of Scenedesmus as well as to Chlamydomonas reinhardti and Chlorella sorokiniana (7-11-05) cultures. Chloroplast particles with high NADP⁺ photoreducing capacity are obtained from heterotrophic cultures only when the cell types are random and the culture is in the logarithmic growth phase; maximal rates of 240–260 μmoles NADP⁺ reduced/h per mg chlorophyll are achieved. Optimal conditions for separation of such chloroplast particles require the use of Tricine buffer (20 mM, pH 7.5), 50 nM EDTA, 10 mM KCl and 0.5 mM dithiothreitol in the breaking medium; for the maintenance of high photochemical activity it is necessary to store particles in a solution consisting of 0.4 M sucrose, 30 mM KCl and 1% bovine serum albumin.

Optimum reaction conditions were developed and the properties of the isolated particles investigated. Maximal activities are obtained when the sucrose concentration is maintained below 0.4 M; the pH optimum with Tricine buffer is between 7.8–8.1; and at least 30 mM Cl⁻ is required. Red actinic light (wavelength >620 nm) with an intensity of 10⁶ ergs/cm² per s is required for saturation.

Ferrodoxin and ferredoxin-NADP⁺ oxidoreductase are lost from the particles during the preparatory procedures and maximum photochemical activity is attained only when they are added back in balanced amounts. Stimulatory effects of added plastocyanin and cytochrome c-553 are noted only with particles having an initially low photochemical activity.     

Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta)

In order to investigate the possible impacts of increased atmospheric CO₂ levels on algal growth and photosynthesis, the influence of CO₂ concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO₂ concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 M CO₂ in C. reinhardtii, C. pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO₂ was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N inC. pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p < 0.05) in high-CO₂-grown cells. Elevation of the CO₂ levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C. pyrenoidosa and S. obliquus when exposed to high photon flux density. The photoinhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO₂-grownC. pyrenoidosa and S. obliquus. Although pH and pCO₂ effects cannot be distinguished from this study, it can be concluded that increased CO₂ concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Cytochrome-c reductases from wild-type and mutant strains of Aspergillus nidulans

Summary The sedimentation coefficients of the NADPH: cytochrome-c oxidoreductase enzymes from wild-type and mutant strains of Aspergillus nidulans have been estimated by sucrose density gradient centrifugation. In the wild-type, two species of cytochrome-c reductase were found, with sedimentation coefficients of 13.7s and 7.6s respectively. The 13.7s species did not appear to be associated with the enzymes of nitrate reduction, whereas the 7.6s species was closely associated with NADPH: nitrate oxidoreductase. In mutant strains lacking nitrate reductase, a thir species of cytochrome-c reductase with a sedimentation coefficient of 4.5s was found. There is some evidence that this 4.5s cytochrome-c reductase is a subunit or breakdown product of nitrate reductase and a model is presented for the role of this 4.5s cytocnorome-c reductase in the assembly of the intact nitrate reductase molecule.     

Cardiovascular effects of prostaglandin D3 and D2 in anesthetized dogs

Prostaglandin (PG) D₃ has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 μg/kg) of PGD₃ and also PGD₂ were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD₃. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD₂, while PGD₃ was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 μg/kg/min) of PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD₃ infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD₂. In contrast, PGD₃ was less potent than PGD₂ as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.     

Formation of 1,24-24-trihydroxyvitamin D3 from 1,25-dihydroxyvitamin D3

Incubation of [26,27-³H₂]-25-hydroxyvitamin D₃ with kidney homogenates from rats fed a high (3%) calcium vitamin D-supplemented diet results in the production of a more polar metabolite which cochromatographs with 1,24,25-trihydroxyvitamin D₃. On the other hand, incubation with kidney homogenates from vitamin D-deficient or calcium-deficient rats did not produce the polar metabolite. Mitochondria but not microsomes carry out the reaction and evidence has been produced to demonstrate that the 1,24,25-trihydroxyvitamin D₃ can be produced in vivo from either 1,25-dihydroxyvitamin D₃ as previously reported.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.     

斜生栅藻对不同形态锑胁迫的响应及抗性研究

该研究以斜生栅藻(Scenedesmus obliquus)为研究对象,研究不同浓度的Sb(Ⅲ)、Sb(Ⅴ)胁迫下对斜生栅藻生长速率、叶绿素a、抗氧化酶活性和细胞表面结构的影响,以探讨重金属胁迫下斜生栅藻的抗性机理。结果表明:(1)在高浓度Sb(Ⅲ)、Sb(Ⅴ)胁迫处理中,斜生栅藻生长对Sb(Ⅲ)胁迫的响应更加迅速;随着Sb(Ⅲ)、Sb(Ⅴ)胁迫浓度增加,斜生栅藻体内叶绿素a含量呈下降趋势,且对Sb(Ⅲ)胁迫浓度的变化反应更为敏感。(2)在800μmol/L Sb(Ⅲ)和Sb(Ⅴ)胁迫下,斜生栅藻体内丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性分别在胁迫第7~8天和第6~7天时变化更为明显;过氧化氢酶(CAT)活性在胁迫处理的第4~5天就迅速增加,对Sb胁迫响应比SOD更为迅速。(3)在高浓度Sb(Ⅲ)、Sb(Ⅴ)胁迫下,斜生栅藻细胞结构受到严重损伤,而且Sb(Ⅲ)对斜生栅藻细胞的损伤更加明显。研究发现,Sb(Ⅲ)、Sb(Ⅴ)对斜生栅藻生长、叶绿素a及细胞结构表现出不同程度的抑制和破坏作用,其细胞受到的影响程度与Sb(Ⅲ)、Sb(Ⅴ)的处理浓度及处理时间均密切相关,但斜生栅藻依靠自身抗氧化酶系统在一定程度上能够缓解Sb胁迫的不利影响。     

不同藻密度下布洛芬浓度对萼花臂尾轮虫生命表统计学参数的影响

在不同斜生栅藻(Scenedesmus obliquus)密度(1.0×10~6、2.0×10~6个细胞/m L和4.0×10~6个细胞/m L)下,研究了不同浓度的(0、1、10、100、1000μg/L和5000μg/L)布洛芬对萼花臂尾轮虫(Brachionus calyciflorus)生命表统计学参数的影响。结果表明,与各藻密度下的对照组相比,当藻密度为1.0×106个细胞/m L时,100—5000μg/L布洛芬处理组中轮虫的生命期望和平均寿命显著缩短,100μg/L布洛芬处理组中轮虫的世代时间显著缩短,1.0μg/L布洛芬处理组中轮虫的净生殖率显著提高,10—5000μg/L布洛芬处理组中轮虫的后代混交率显著提高。当藻密度为2.0×10~6个细胞/mL时,10—5000μg/L布洛芬处理组中轮虫的生命期望和平均寿命显著缩短,1000μg/L和5000μg/L布洛芬处理组中轮虫的世代时间显著缩短,1、10、1000μg/L和5000μg/L布洛芬处理组中轮虫的种群内禀增长率显著提高,1μg/L和1000μg/L布洛芬处理组中轮虫的后代混交率显著提高。当藻密度为4.0×10~6个细胞/mL时,10—5000μg/L布洛芬处理组中轮虫的生命期望、平均寿命和世代时间显著缩短。藻密度对轮虫的世代时间、净生殖率和种群内禀增长率有显著性影响(P0.05),布洛芬浓度对轮虫的生命期望、平均寿命、世代时间、净生殖率和种群内禀增长率有显著性影响(P0.05),藻密度和布洛芬浓度的交互作用对轮虫的生命期望、平均寿命和后代混交率有显著性影响(P0.05)。在实验设置的布洛芬浓度范围内,2.0×10~6个细胞/m L藻密度下,轮虫的生命期望、平均寿命和世代时间与布洛芬浓度之间均具有显著的剂量—效应关系(P0.05); 4.0×10~6个细胞/m L藻密度下,轮虫的生命期望、平均寿命和净生殖率与布洛芬浓度之间均具有显著的剂量—效应关系(P0.05)。     

不同藻密度下Zn2+ 浓度对萼花臂尾轮虫实验种群增长参数的影响

为了比较不同食物密度下污染物浓度对受试生物的慢性毒性,筛选出以轮虫为受试生物对水环境中Zn2+污染进行监测的敏感指标,在不同斜生栅藻(Scenedesmus obliquus)密度(1.0×106、2.0×106和4.0×106个/m L)下不同浓度(0、0.1、0.3、0.5、0.7、0.9 mg/L)的Zn2+对萼花臂尾轮虫(Brachionus calyciflorus)实验种群增长参数的影响。结果表明,25℃以及1.0×106、2.0×106和4.0×106个/m L藻密度下Zn2+对萼花臂尾轮虫的24 h LC50值分别是6.647、8.102和5.873 mg/L。与各食物密度下的对照组相比,当食物密度为1.0×106个/m L时,各浓度的Zn2+对萼花臂尾轮虫的各种群增长参数均无显著性影响(P0.05)。当食物密度为2.0×106个/m L时,各浓度的Zn2+均显著延长了轮虫的生命期望、世代时间和平均寿命,提高了轮虫的净生殖率;除0.3 mg/L外,其他浓度的Zn2+显著提高了轮虫的种群内禀增长率。当食物密度为4.0×106个/m L时,0.1、0.3和0.7mg/L的Zn2+显著提高了轮虫的种群内禀增长率,0.7和0.9 mg/L的Zn2+显著提高了轮虫的后代混交率。藻密度对轮虫的生命期望、世代时间、净生殖率、种群内禀增长率、平均寿命和后代混交率均有极显著性影响(P0.01),Zn2+浓度对轮虫的生命期望、世代时间、净生殖率、种群内禀增长率和后代混交率均有极显著性影响(P0.01),藻密度和Zn2+浓度之间的交互作用对轮虫的生命期望、种群内禀增长率和后代混交率均有显著性影响(P0.05)。2.0×106个/m L食物密度下,Zn2+浓度与轮虫的生命期望、世代时间、净生殖率和平均寿命之间具有显著的剂量-效应关系;4.0×106个/m L食物密度下,Zn2+浓度与轮虫的后代混交率间也具有显著的剂量-效应关系。     

Direct modulation of 25-hydroxyvitamin D3 hydroxylation in kidney tubules by 1,25-dihydroxyvitamin D3

Kidney tubules obtained from chicks fed a high-calcium low-phosphorus diet retained 25-hydroxyvitamin D₃-1-hydroxylase activity after a 10 h incubation in serum-free minimum essential medium. Inclusion of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) in the medium prompted a suppression of 25-hydroxyvitamin D₃-1-hydroxylase and the induction of 25-hydroxyvitamin D₃-24-hydroxylase activities. The enzyme switch-over response could be prompted by 1.6 × 10^?7 M 1,25-dihydroxyvitamin D₃ and occurred within 6 h following treatment. Medium calcium appeared to augment the metabolite's switch-over action.     

Growth and photosynthetic activities of wild-type and antenna-deficient mutant strains of Rhodobacter capsulatus

Cells of Rhodobacter capsulatus wild-type strains (37b4, B 10) and mutant strains, lacking lightharvesting (LH) complex II (B800–850) and defective in formation of LH I (B870) complex [U 43 (pTXB 87), U43 (pTXA6-10)] were grown photosynthetically at high and low light intensities in a turbidostate. The mutant strain U43 (pTXA6-10), lacking any LH system, was able to grow at high and low light intensities with doubling times of 4.6 and 9.8 h, respectively. In this mutant the concentration of photochemical reaction centers (RC) per cell and per membrane protein was several times higher than in wild type cells, but the bacteriochlorophyll content, the size of the photosynthetic unit and the rate of photophosphorylation were lower than in wild type cells. Reversible bleaching of reaction center and photophosphorylation were measured under different excitation light intensities. The charge recombination in the RC between the primary donor and Q_B was very slow in the mutant strains. Two membrane fractions differing in absorption spectra and light saturation behaviour of reversible bleaching and photophosphorylation were isolated from the mutant strains. The experimental data indicate that photosynthetic units of different composition and/or organization are present in the mutant cells.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Braunschweig     

Permeabilization induced by lipid II-targeting lantibiotic nisin and its effect on the bioconversion of vitamin D3 to 25-hydroxyvitamin D3 by Rhodococcus erythropolis

Vitamin D₃ (VD₃) is a fat-soluble prohormone in mammals. VD₃ is inert and must be activated by hydroxylation at the C-25 and C-1α positions to exert its biological activity. We recently accomplished the bioconversion of VD₃ to 25(OH)VD₃ with a recombinant strain of Rhodococcus erythropolis and found that the permeability of VD₃ into the cytoplasm may be the rate-limiting step of 25(OH)VD₃ production (Sallam et al., 2010). When the cells were treated with the lipid II-targeting lantibiotic nisin, the permeability of green chemiluminescent cyclodextrin (GCCD), which is used as a model substrate instead of VD₃-partially methylated-β-cyclodextrin (PMCD) complex, was drastically induced. Nisin also induced VD₃ hydroxylation, and the rate was correlated with the expression levels of Vdh and its redox partner proteins. In the bioconversion reaction, the stability of the redox partner proteins and the additional NADH-regenerating system are crucial for VD₃ hydroxylation. The degradation rate of the [2Fe–2S] cluster of ferredoxin ThcC from R. erythropolis NI86/21 is faster than that of AciB from Acinetobacter sp. OC4. Therefore, the nisin-treated R. erythropolis cells coexpressing Vdh and AciBC (1176.5 μg) exhibited much greater 25(OH)VD₃ production than the cells coexpressing Vdh and ThcCD (431.7 μg) after four consecutive 16 h reactions. These results suggest that nisin forms nisin-lipid II pore complexes in the Rhodococcus membrane that increase the accessibility of VD₃–PMCD complexes to the inside of the cells. Furthermore, nisin-treated Rhodococcus cells can be utilized for the bioconversion of other fat-soluble chemicals.     

Immunomodulatory actions of 1,25-Dihydroxyvitamin D3

The sterol, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), has immunosuppressive activity. The hormone inhibits the production of lymphokines (IL-2, IFN-γ) and monocyte-derived cytokine (IL-12) leading to inhibition of helper T cell subset type 1 (Th₁). When given in vivo, the hormone prevents the development of spontaneous and induced models of autoimmunity. Analogs of 1,25(OH)₂D₃, with reduced hypercalcemic effects, display an enhanced activity in autoimmunity compared to the sterol and prolong graft survival in experimental transplantation. This paper reviews our understanding of the cellular actions of the hormone and the therapeutic application of 1,25(OH)₂D₃ and analogs in autoimmunity and transplantation.     

Metabolic pathway to 25-hydroxyvitamin D3-26,23-lactone from 25-hydroxyvitamin D3

mRNA was prepared from autopsy liver samples from a homozygote for α₁-antitrypsin deficiency (PiZZ) and from a normal (PiMM) subject. Both preparation gave equivalent synthesis of α₁-antitrypsin in a wheat germ cell-free system. This suggests that the deficiency of plasma α₁-antitrypsin associated with the Z variant is due to a failure of processing and secretion of the protein rather than of its synthesis. It is likely that it is the resultant intracellular accumulation of the Z protein rather than a deficiency of protease inhibitor that is the primary cause of the liver pathology associated with this variant.     

Influence of phosphorus concentration on the growth kinetics and stoichiometry of the microalga Scenedesmus obliquus

The growth of the freshwater microalga Scenedesmus obliquus was studied at 30°C in a mineral culture medium with phosphorus concentrations of between 0 and 372 μ . The values for the specific growth rates, between and , fitted a semistructured substrate-limitation model with μ_m1 = 0·0466 h⁻¹, μ_m2 = 0·0256 h⁻¹ and . The specific uptake rate of phosphorus reached a maximum value of q_Sm1 = 658·01 × 10⁻⁴ μmol P mg⁻¹ biomass h⁻¹.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL.Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size.The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1xpab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned.These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.     

Biphasic action of prostaglandin E2 on conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in chick renal tubules

The effect of PGE₂ on the conversion of 25-hydroxyvitamin D₃ (25 OH D₃) to 1,25-dihydroxyvitamin D₃ (1,25- (OH) ₂D₃) by isolated renal tubules from vitamin D deficient chicks was studied under a variety of experimental conditions. In the absence of added vitamin D metabolites, PGE₂ (2 × 10⁻⁶M) caused an immediate inhibition of formation of 1,25-(OH) ₂D₃, followed by a delayed stimulation, apparent after 15 h exposure to PGE₂. Pretreatment of the tubules with 1,25-(OH) ₂D₃ prevented the immediate inhibitory action of PGE₂, and allowed the stimulation to be apparent after 4 h exposure to PGE₂. The cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX) significantly stimulated the formation of 1,25-(OH) ₂D₃. PGE₂ significantly inhibited 1,25-(OH) ₂D₃ formation in tubules which had been stimulated by IBMX. PGE₂ stimulated the adenylate cyclase activity in a crude particulate fraction from the chick kidney, and raised cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels in the renal tubules.It is concluded that PGE₂ can either stimulate or inhibit 1,25-(OH) ₂D₃ formation in chick renal tubules. The stimulatory effect may be partly due to elevation of cyclic AMP. The mechanism of the inhibitory effect requires further investigation.