Action Spectra for Conversions of Phycochrome c from Nostoc muscorum

The reversibly photochromic pigment, phycochrome c, was extracted from the blue-green alga Nostoc muscorum strain A. Action spectra were determined for in vitro conversions of the pigment from the short wavelength to the long wavelength form and vice versa. The action peak for the absorbance decrease at 650 nm is at 630 nm. During this decrease there is only a slight increase of the absorbance in the green region. Green and yellow light (maximum efficiency at 580 nm) completely restores absorbance at 650 nm. The observations are explained by the existence of three spectrally different forms of phycochrome c: P^c₆₃₀ and P^c₆₅₀ which equilibrate in darkness and P^c₅₈₀ which is reversibly photoconvertible to P^c₆₃₀. We have also measured the absorbance changes brought about by saturating irradiations with light of various wavelengths (“photostationary state spectrum”). Extreme photostationary states were obtained with about 650 nm and 500 nm light.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Developmental stability in brown trout: are there any effects of heterozygosity or environmental stress?

We studied the developmental stability of brown trout, Salmo trutta L., in 10 populations (five acidified, five control) in Norway, measured as fluctuating asymmetry (FA) and departure from the morphological norm. We measured four meristic and four morphometric characters, and scored the level of biochemical heterozygosity at 49 loci (20 polymorphic). We reared eggs of a single population in a hatchery using four different water qualities (three replicates of each treatment) to test the effect of acidification stress on developmental instability. There were no significant differences in the level of FA, in departure from the morphological norm between brown trout sampled from lakes with acidified or control water qualities, or in brown trout hatched at different water qualities. There was no correlation between level of heterozygosity and FA or departure from the morphological norm, either when tested within populations or among populations. There were no single-locus effects on developmental stability tested for 11 loci. We conclude that measures of developmental stability or morphological variability are not useful for detecting acidification stress in brown trout. Furthermore, we conclude that developmental stability in our material varies independently of heterozygosity.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

de novo转录组学分析华南地区入侵植物五爪金龙代谢特征

为探究华南地区严重入侵植物五爪金龙(Ipomoea cairica)生物入侵的分子机制,对五爪金龙及其近缘种七爪金龙(I. digitata)和裂叶牵牛(I. nil)进行de novo转录组测序和组装,得到56551条unigenes,其中56522条得到注释,7815条GO注释,15615条COG注释,180201条KEGG数据库注释。转录组分析结果表明,五爪金龙氮代谢通路关键酶基因的表达高于对照。次生代谢关键酶(PAL、4CL、CAD、查耳酮合酶、苯基丙乙烯酮异构酶、槲皮黄3-O-甲基转移酶等)基因在五爪金龙与七爪金龙及裂叶牵牛中均得到协同性的差异表达,而这些代谢通路指导的产物合成对五爪金龙的抗逆境能力、生长、化感作用等均起关键作用。关键基因的RT-qPCR验证结果与转录组结果具有一致性。因此,这从分子生物学层面上对解释五爪金龙在华南地区的入侵机制提供了新的证据。     

The Ultrastructure of the Fungus Trichoderma viride and Investigation of its Growth on Cellulose

Changes in the ultrastructure of Trichoderma viride during growth in shake cultures on cellobiose and cellulose fibres were examined. Electron micrographs of thin sections of germinating conidia, septate hyphae with ascomycete pores and other cell organelles are presented. Extensive autolysis of hyphae was observed after growth for 20 h on cellobiose. The fungus grew in the lumina and within the walls of cellulose fibres. The hyphae followed the directions of the laminar structure but did not grow across them. The observations indicated that the hyphae penetrated the fibres by causing cracks and by dissolving enzymatically the cellulose.     

Phylogeographic analyses reveal distinct lineages of the liverworts Metzgeria furcata (L.) Dumort. and Metzgeria conjugata Lindb. (Metzgeriaceae) in Europe and North America

Seed plant genera often exhibit intercontinental disjunctions where different species are found on different continents. Many morphologically circumscribed bryophyte species exhibit similar disjunctions. We used nucleotide sequences from the plastid and nuclear genomes to test hypotheses of phylogeography within representatives of the genus Metzgeria: Metzgeria furcata, Metzgeria conjugata, and Metzgeria myriopoda. The first two species have sexual and asexual populations, exhibit disjunctions between North America and Europe, and have been split into separate species, numerous subspecies or varieties. The third species occurs in eastern North America but is not reported from Europe. Phylogenetic analyses resolved three distinct lineages within the morphologically defined species, M. furcata: one in North America, and two in Europe. Similarly, three morphologically cryptic clades of M. conjugata were resolved by the molecular data: northern North America, Europe, and south‐eastern North America. For both species, molecular divergence among taxa occurred in the absence of morphological change. In the case of M. myriopoda, all plants from eastern North America were both morphologically uniform and genetically homogeneous (although not identical). The present study provides significant insight into a plant group with complex taxonomy, and indicates that these liverwort taxa with wide distributions, extreme sex ratios, and continental disjunctions harbor cryptic lineages. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 745–756.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity

p18(INK4c) is a member of the INK4 family of proteins that regulate the G(1) to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16(INK4a) member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16(INK4a) mutations reduce protein thermodynamic stability. Based on this observation, we used p18(INK4c) as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18(INK4c) mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18(INK4C) proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16(INK4a)-mediated cancers.     

Rapid and unexpected effects of piscivore introduction on trophic position and diet of perch (Perca flavescens) in lakes recovering from acidification and metal contamination

1. Yellow perch (Perca flavescens) are often the only surviving fish species in acidified lakes. We studied four lakes along a gradient of recovery from acidification and that had different food web complexities. All had abundant yellow perch, two had low piscivore abundance, one had a well‐established piscivore population and one was manipulated by introducing piscivorous smallmouth bass (Micropterus dolomieu). We hypothesised that there would be strong effects on perch abundance, behaviour and diet induced by the presence of piscivores. 2. In the manipulated lake, the bass reduced yellow perch abundance by 75% over a 2‐year period. Concomitantly, perch use of the pelagic habitat fell from 48 to 40%. 3. In contrast to findings from less disturbed systems, yellow perch in the littoral zone of the manipulated lake did not strongly shift from zooplankton to benthic food sources after the arrival of piscivores. Diet analysis using stable carbon isotopes revealed a strong continued reliance on zooplankton in all lakes, independent of the degree of piscivory. The failure to switch to benthos in the refuge area of the littoral zone is most likely related to the depauperate benthos communities in these formerly acidified lakes. 4. Yellow perch in lakes recovering from acidification face a considerable ecological challenge as the necessary switch to benthic diet is hindered by a low abundance of benthos. The arrival of piscivores in these recovering lakes imposes further restrictions on perch access to food items. We infer that future recovery of perch populations (and higher trophic levels) will have to be preceded by the re‐establishment of diverse benthic macroinvertebrate communities in these lakes.