Role of two forms of the D1 protein in the recovery from photoinhibition of photosystem II in the cyanobacterium Synechococcus PCC 7942

The study of turnover of two distinct forms of the photosystem II (PSII) D1 protein in cells of the cyanobacterium Synechococcus PCC 7942 showed that the 'high-light' form D1:2 is degraded significantly faster at 500 microE m(-2) s(-1) as compared with 50 microE m(-2) s(-1) while the degradation rates of the 'low-light' form D1:1 under low and high irradiance are not substantially different. Consequently, the D1:1 turnover does not match photoinactivation of PSII under increased irradiance and therefore the cells containing this D1 form exhibit a decrease in the PSII activity. Monitoring of the content of each D1 form during a recovery from growth-temperature photoinhibition showed a good correlation between the synthesis of D1:2 and restoration of the PSII activity. In contrast, when photoinhibitory treatment was conducted at low temperature, a fast recovery was not accompanied by the D1:2 accumulation. The data suggest that photoinactivation at growth temperature results in a modification of PSII that inhibits insertion of D1:1 and, therefore, for restoration of the photochemical activity in the photoinactivated PSII complexes the D1:2 synthesis is needed. This may represent the primary reason for the requirement of psbAII/psbAIII expression under increased irradiance.     

Effect of various photoperiods on testicular weight, weekly sperm output and plasma levels of LH and testosterone over the reproductive season in male turkeys

The effects of duration and variation in photoperiod on testis weight, testicular sperm production, semen output, and hormone status over the reproductive season in male turkeys were investigated. In Experiment 1, four groups of males raised from 17 to 23 wk of age under a constant short photoperiod were subjected to a constant short (Group 1: 7L:17D; Group 2: 10.5L:13.5D), constant long (Group 3: 14L:10D) or progressively increasing photoperiod (Group 4: 7L:17D to 14L:10D) up to 60 wk of age. In Experiment 2, four groups of males first raised as in Experiment 1 up to 23 wk of age were placed under a constant short (Group 5: 10.5L:13.5D), constant long (Group 6: 14L:10D), or night-interrupted photoperiod (Group 7: 6L:2.5D:1L:14.5D, referred to as subjective 9.5L:14.5D; Group 8: 6L:3.5D:1L:13.5D), referred to as subjective 10.5L:13.5D) up to 60 wk of age. Males in Groups 2-4 had similar reproductive characteristics, whereas sexual maturity was delayed from 29 to 49 wk in males from Group 1. In Experiment 2, males in Groups 5 and 8 had similar reproductive characteristics, whereas sexual maturity was delayed in males in Group 7 in a manner similar to that observed in Group 1. In both experiments, plasma LH and testosterone concentrations were poor indicators of testis development and semen production, irrespective of age and photoperiod. We conclude that a moderately short photoperiod such as 10.5L:13.5D or subjective 10.5L:13.5D may stimulate reproductive characteristics of male turkeys in a manner comparable to constant long or increasing photoperiods. We inferred the existence of a threshold of photosensitivity in male turkeys for photoperiods longer than 9.5L:14.5D, but shorter than or equal to 10.5L:13.5D.     

内含子中正筛选标记neo基因在转录中的剪切研究

目的:研究插入内含子中的正筛选标记基因neo在转录中的剪切情况。方法:克隆了猪血清白蛋白基因5'端调控序列,以猪基因组DNA为模板,P10/P11为引物,PCR扩增猪血清白蛋白基因翻译终止密码子后2.9kb的3'端调控序列;以pEGFP-1为模板,P400/P401为引物PCR扩增绿色荧光蛋白(EGFP)基因,插入猪血清白蛋白基因5'端调控区之后,在3'端调控序列的内含子中靠近N端的序列中插入正筛选标记基因neo,构建了表达EGFP的真核表达载体pEXp11。转染人肝癌细胞系HepG2,通过G418药物筛选获得稳定转染的抗药性细胞克隆。提取抗性细胞克隆基因组RNA并进行反转录,获得cDNA序列。结果:用引物D400/D401及分别位于neo基因和3'端调控序列上的一对引物D394/D357进行PCR检测,其中D394/D357并未扩增出目的条带。结论:插入内含子中的neo基因在转录过程中可随内含子一起被剪切。     

Subunit 3 of the COP9 signalosome is poised to facilitate communication between the extracellular matrix and the nucleus through the muscle-specific beta1D integrin

Yeast two-hybrid analysis (Fields and Song, 1989, Nature, 340:245-246) was used to screen a human heart library to isolate proteins interacting with the adult muscle-specific beta1D integrin but not with beta1A integrin. In addition to previously identified interactions (RACK 1(Liliental and Chang, 1998, Journal of Biological Chemistry, 273:2379-2383) and alpha-actinin (Otey et al., 1990, Journal of Cell Biology, 111:721-729), the authors isolated several novel candidates. These include subunit 3 (CSN3/Sgn3) of the COP9 signalosome complex, cyclins D1, D2, and D3, RanBPM, and a recently identified protein COG8/DOR1. These protein interactions were specific for beta1D integrin, as no binding to beta1A integrin cytoplasmic domain was measurable by two-hybrid analysis. This paper presents the initial characterization of the interaction of CSN3 with beta1D integrin, the localization of CSN3 and the other COP9 signalosome subunits in embryonic and adult cardiac myocytes and their response to muscle cell differentiation.     

A transient exchange of the photosystem II reaction center protein D1:1 with D1:2 during low temperature stress of Synechococcus sp. PCC 7942 in the light lowers the redox potential of QB

Upon exposure to low temperature under constant light conditions, the cyanobacterium Synechococcus sp. PCC 7942 exchanges the photosystem II reaction center D1 protein form 1 (D1:1) with D1 protein form 2 (D1:2). This exchange is only transient, and after acclimation to low temperature the cells revert back to D1:1, which is the preferred form in acclimated cells (Campbell, D., Zhou, G., Gustafsson, P., Oquist, G., and Clarke, A. K. (1995) EMBO J. 14, 5457-5466). In the present work we use thermoluminescence to study charge recombination events between the acceptor and donor sides of photosystem II in relation to D1 replacement. The data indicate that in cold-stressed cells exhibiting D1:2, the redox potential of Q(B) becomes lower approaching that of Q(A). This was confirmed by examining the Synechococcus sp. PCC 7942 inactivation mutants R2S2C3 and R2K1, which possess only D1:1 or D1:2, respectively. In contrast, the recombination of Q(A)(-) with the S(2) and S(3) states did not show any change in their redox characteristics upon the shift from D1:1 to D1:2. We suggest that the change in redox properties of Q(B) results in altered charge equilibrium in favor of Q(A). This would significantly increase the probability of Q(A)(-) and P680(+) recombination. The resulting non-radiative energy dissipation within the reaction center of PSII may serve as a highly effective protective mechanism against photodamage upon excessive excitation. The proposed reaction center quenching is an important protective mechanism because antenna and zeaxanthin cycle-dependent quenching are not present in cyanobacteria. We suggest that lowering the redox potential of Q(B) by exchanging D1:1 for D1:2 imparts the increased resistance to high excitation pressure induced by exposure to either low temperature or high light.     

Parallel expression of alternate forms of psbA2 gene provides evidence for the existence of a targeted D1 repair mechanism in Synechocystis sp. PCC 6803

The D1 protein of Photosystem II (PSII) is recognized as the main target of photoinhibitory damage and exhibits a high turnover rate due to its degradation and replacement during the PSII repair cycle. Damaged D1 is replaced by newly synthesized D1 and, although reasonable, there is no direct evidence for selective replacement of damaged D1. Instead, it remains possible that increased turnover of D1 subunits occurs in a non-selective manner due for example, to a general up-regulation of proteolytic activity triggered during damaging environmental conditions, such as high light. To determine if D1 degradation is targeted to damaged D1 or generalized to all D1, we developed a genetic system involving simultaneous dual expression of wild type and mutant versions of D1 protein. Dual D1 strains (nS345P:eWT and nD170A:eWT) expressed a wild type (WT) D1 from ectopic and a damage prone mutant (D1-S345P, D1-D170A) from native locus on the chromosome. Characterization of strains showed that all dual D1 strains restore WT like phenotype with high PSII activity. Higher PSII activity indicates increased population of PSII reaction centers with WT D1. Analysis of steady state levels of D1 in nS345P:eWT by immunoblot showed an accumulation of WT D1 only. But, in vivo pulse labeling confirmed the synthesis of both S345P (exists as iD1) and WT D1 in the dual strain. Expression of nS345P:eWT in FtsH2 knockout background showed accumulation of both iD1 and D1 proteins. This demonstrates that dual D1 strains express both forms of D1, yet only damage prone PSII complexes are selected for repair providing evidence that the D1 degradation process is targeted towards damaged PSII complexes. Since the N-terminus has been previously shown to be important for the degradation of damaged D1, the possibility that the highly conserved cysteine 18 residue situated in the N-terminal domain of D1 is involved in the targeted repair process was tested by examining site directed mutants of this and the other cysteines of the D1 protein. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Time Course of Sensitivity of the Photoinducible Phase to Light in the Redheaded Bunting, Emberiza bruniceps

This study investigated the differential sensitivity of the photoinducible phase (Φi) to light in the redheaded bunting (Emberiza bruniceps). Using a skeleton paradigm, we assessed the rate and magnitude of testicular response as a function of the duration of an inducing light pulse and of the time of its introduction in Φi. For a period of 7 weeks, birds received at an intensity of ~100 lux, the same (6 h) entraining light stimulus with a varied inducing light pulse: 1, 2, 4 or 6 h beginning at zeitgeber time (zt) 11 (6L:5D:1L:12D, 6L:5D:2L:11D, 6L:5D:4L:9D or 6L:5D:6L:7D), or 1h pulses at zt 12 (6L:6D:1L:11D) or zt 16 (6L:10D:1L:7D). The testes were stimulated in all LD alternations, but duration- and time-dependent effects of the light pulse on the rate and magnitude of the testicular response were clearly evident. Illumination of a larger portion of Φi seems to result in higher rates of gonadal growth but there is a duration limit above which there will be no further increase of testicular response.     

Time Course of Sensitivity of the Photoinducible Phase to Light in the Redheaded Bunting,Emberiza bruniceps

This study investigated the differential sensitivity of the photoinducible phase (Φi) to light in the redheaded bunting (Emberiza bruniceps). Using a skeleton paradigm, we assessed the rate and magnitude of testicular response as a function of the duration of an inducing light pulse and of the time of its introduction in Φi. For a period of 7 weeks, birds received at an intensity of ~100 lux, the same (6 h) entraining light stimulus with a varied inducing light pulse: 1, 2, 4 or 6 h beginning at zeitgeber time (zt) 11 (6L:5D:1L:12D, 6L:5D:2L:11D, 6L:5D:4L:9D or 6L:5D:6L:7D), or 1h pulses at zt 12 (6L:6D:1L:11D) or zt 16 (6L:10D:1L:7D). The testes were stimulated in all LD alternations, but duration- and time-dependent effects of the light pulse on the rate and magnitude of the testicular response were clearly evident. Illumination of a larger portion of Φi seems to result in higher rates of gonadal growth but there is a duration limit above which there will be no further increase of testicular response.     

Effect of photoperiod length on body mass and testicular growth in the house sparrow (Passer domesticus) and brahminy myna (Sturnus pagodarum)

Two experiments studied the relative effects on body mass and testicular growth of stimulatory photoperiods applied simultaneously to two photosensitive species, the house sparrow (Passer domesticus) and brahminy myna (Sturnus pagodarum). Experiment 1 on the house sparrow consisted of two parts. In experiment 1A, beginning on 24 March 2002, short day pretreated sparrows were exposed for 12 weeks to 13L: 11D (13 h light: 11 h darkness), 20L: 4D and NDL (control). Experiment 1B was similar to 1A except that it used sparrows that were not treated with short days. This experiment was repeated at three different times in the year. Beginning on 29 December 2002 (for 24 weeks), 26 March 2003 (for 12 weeks) and 16 August 2003 (for 8 weeks), sparrows captured from the wild and acclimated to captive condition for 1 week were exposed to 13L: 11D and 20L: 4D. Each time, a group was maintained in NDL and served as the control. Experiment 2 was performed on myna and used an identical protocol. Beginning on 24 March 2002, myna that were captured from the wild and acclimated to captivity conditions were exposed for 16 weeks to 13L: 11D and 20L: 4D; a group was maintained in NDL and served as the control. There was photostimulation and subsequent regression of the testes on all day lengths except in the August group of experiment 1B. The effect on body mass was variable. Interestingly, however, the response to 20L:4D was relatively smaller as compared to 13L:11D. Taken together, these results confirm that the two species use photoperiods in control of their reproductive cycle, and tend to indicate that exposure to unnatural long photoperiods may in fact be unfavorable and could compromise gonadal growth and development.     

Effect of a light pulse during the dark on photoperiodic regulation of the rate of thyroxine-induced, spontaneous, and prolactin-inhibited metamorphosis in Rana pipiens tadpoles

Since Rana pipiens tadpoles injected with thyroxine (T4) early in the dark develop more slowly than those injected in the light, we studied the effect of giving a light pulse of 1 hr early in the dark. Tadpoles injected under a 7.5-W red light bulb in a darkened room with 0.2 microgram T4 daily at 2200 hr went through metamorphosis faster on a 12L:3D:1L:8D cycle with a light pulse after injection than on a 12L:12D cycle without a light pulse, and even faster on a 12L:1.5D:1L:9.5D cycle with a light pulse before the injection. Thus a 1-hr light pulse counteracted the metamorphic delay resulting from administration of T4 in the dark, and set in motion the conditions that resulted in a more rapid response to an injection of T4. However, a 1-hr light pulse in the early dark had no effect on growth and development of older or younger untreated tadpoles or those constantly immersed in 30 micrograms/liter T4. Larvae on 21L:3D with T4 injection in the dark and on 12L:3D:1L:8D with T4 injection at 0700 hr just before the start of the main light phase progressed faster than 12L:3D:1L:8D with injection at 2200 hr in the dark before only a 1-hr light pulse. Thus the length of the light phase immediately after T4 injection was significant. There was no difference on 12L:12D and 12L:3D:1L:8D cycles in the effectiveness of daily injections of 10 micrograms prolactin (PRL) in the early dark at 2200 hr in promoting tail growth or antagonizing tail resorption induced by T4 immersion. Under these conditions, PRL utilization did not appear to be inhibited by the light pulse.     

Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. RESULTS: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM. CONCLUSIONS: The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.     

The relationships among the species of the Drosophila virilis group inferred from the gene Ras1 sequences

Comparative analysis of a group of closely related Drosophila species (D. virilis, D. lummei, D. novamexicana, D. americana texana, D. flavomontana, D. montana, D. borealis, D. lacicola, D. littoralis, D. kanekoi, and D. ezoana) was conducted based on an incomplete sequence of gene Ras1. The pattern of the relationships among the species corresponded to that expected from analysis of morphological and cytogenetic characters. Statistical data favoring neutrality of the substitutions examined in the Ras1 gene are presented. This character of the gene Ras1 evolution confers more reliability to reconstruction of phylogenetic relationships among closely related species. The resultant tree for main phylads of the group is as follows: (D. virilis, D. lummei, D. montana, D. ezoana).     

Presumptive Identification of Group D Streptococci: the Bile-Esculin Test

Six tests commonly used for the presumptive identification of group D streptococci were evaluated. Strains tested included 282 group D streptococci and 366 non-group D. Ratios of percentages of group D to non-group D strains which gave positive reactions for each test are as follows: bile-esculin, 100:2; salt tolerance, 88:24; heat tolerance, 100:80; SF broth, 86:1; KF broth, 99:40; and methylene blue milk reduction, 90:17. These data indicate that the bile-esculin test provided a reliable means of identifying group D streptococci and differentiating them from non-group D streptococci. Methodology for reading and interpreting positive reactions and time of incubation of the bile-esculin medium was defined. Evidence of the need for standardization of salt and heat-tolerance tests was obtained.     

Homozygosity mapping of the Achromatopsia locus in the Pingelapese.

Achromatopsia, or total color blindness (also referred to as "rod monochromacy"), is a severe retinal disorder characterized clinically by an inability to distinguish colors, impaired visual acuity in daylight, photophobia, and nystagmus. Inherited as an autosomal recessive trait, achromatopsia is rare in the general population (1:20,000-1:50,000). Among the Pingelapese people of the Eastern Caroline Islands, however, the disorder occurs at an extremely high frequency, as recounted in Oliver Sacks's popular book The Island of the Colorblind: 4%-10% of this island population have the disorder and approximately 30% carry the gene. This extraordinary enrichment of the disease allele most likely resulted from a sharp reduction in population in the late 18th century, in the aftermath of a typhoon and subsequent geographic and cultural isolation. To obtain insights into the genetic basis of achromatopsia, as well as into the genetic history of this region of Micronesia, a genomewide search for linkage was performed in three Pingelapese kindreds with achromatopsia. A two-step search was used with a DNA pooling strategy, followed by genotyping of individual family members. Genetic markers that displayed a shift toward homozygosity in the affected DNA pool were used to genotype individual members of the kindreds, and an achromatopsia locus was identified on 8q21-q22. A maximal multipoint LOD score of 9.5 was observed with marker D8S1707. Homozygosity was seen for three adjacent markers (D8S275, D8S1119, and D8S1707), whereas recombination was observed with the flanking markers D8S1757 and D8S270, defining the outer boundaries of the disease-gene locus that spans a distance of <6.5cM.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1546

An acidic O-polysaccharide isolated by mild acid hydrolysis from the lipopolysaccharide of Hafnia alvei PCM 1546 is composed of D-Gal, D-Glc, D-GlcA, D-GalNAc and O-acetyl groups in the ratios 1:1:1:2:1.6. On the basis of sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: [see equation in text].     

Structural analysis of the alpha-D-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme

The neutral exopolysaccharide EPS180 produced from sucrose by the glucansucrase GTF180 enzyme from Lactobacillus reuteri 180 was found to be a (1-->3,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis, periodate oxidation, and 1D/2D 1H and 13C NMR spectroscopy of the intact EPS180, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of EPS180, a composite model, that includes all identified structural features, was formulated as follows: [Formula: see text].     

Photoperiodic stimulation of pubertal development in male deer mice: involvement of the circadian system

Pubertal development in prairie deer mice (Peromyscus maniculatus bairdii) is accelerated by exposure of juveniles to a long-day photoperiod, and, conversely, retarded by exposure to short days. The purpose of the present study was to evaluate the possible involvement of the circadian system in the photoperiodic regulation of puberty. Weanling males, previously housed on a short-day light cycle of 6L:18D, were subjected to a "resonance" protocol in which they received one of the following light cycles: 6L:18D, 6L:30D, 6L:42D, 6L:54D, or 16L:8D. Post-weaning exposure to cycles of 16L:8D, 6L:30D, and 6L:54D stimulated reproductive organ growth as measured at 6 weeks of age. Exposure to cycles of 6L:18D and 6L:42D failed to stimulate reproductive development. These data support the hypothesis that young male deer mice use a circadian rhythm of responsiveness to light to measure photoperiodic time and, consequently, regulate pubertal development.     

Terminal plasmin degradation products D and E of duck fibrinogen and their effect on polymerization of fibrin

Duck fibrinogen (Mr 320 000) treated with streptokinase-activated human plasminogen in the presence of calcium ions was hydrolysed to terminal core fragments D and E. They were isolated from the digest by: (1) ion-exchange chromatography on DEAE-cellulose, (2) gel filtration on Sephadex G-100, and (3) affinity chromatography with the use of fibrin monomers coupled to CNBr-activated Sepharose. When the native D fragment, D1 was additionally digested by plasmin in the presence of EDTA, more degraded forms D2 and D3 appeared. Molecular weight of D1, D2, D3 and E estimated on SDS-polyacrylamide gel electrophoresis is 100 000, 89 000, 80 000 and 50 000, respectively. It was found that after reduction with 2-mercaptoethanol the fragments D1 and D3 consisted each of three polypeptide chains: alpha, beta, gamma: the gamma-chain of D3 remnant was more degraded (Mr 24 000) as compared with the gamma-chain of D1 remnant (Mr 42 000). Polymerization of both duck and pig fibrin monomers was inhibited by fragments D1 but not by D3.     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.