Thymidine kinase enzyme variants in Physarum polycephalum. Kinetics and properties of the enzyme variants

Thymidine kinase variants of Physarum polycephalum separated by repeated DEAE-cellulose chromatography have been characterized. The enzyme variants show similar catalytic properties (e.g., substrate specificity, pH optimum) and molecular weights, as judges by their sedimentation in sucrose gradients. However, they differ significantly with respect to pI, inhibition by dTTP and thermostability, and they have slightly different Km values for deoxythymidine as a substrate.     

Thymidine kinase enzyme variants in Physarum polycephalum. In vitro interconversion of the enzyme variants.

Isoelectric focusing of plasmodial extracts of Physarum polycephalum demonstrated the presence of several multiple enzyme variants of thymidine kinase, which appear sequentially during the nuclear division cycle. Variants (A) + (A1) are the only enzyme variants found in the late G2-phase, whereas the variants (C) + (C1) are only present at the time of mitosis and S-phase (1, 2). Evidence is presented that multiple forms of thymidine kinase (A) + (A1) with high pI arise by dephosphorylation of a primary translation product with low pI (C and/or C1). The thymidine kinase fractions (A) + (A1) and (C) + (C1) + (c1) were separated and partially purified by DEAE-cellulose chromatography. The enzyme variants (C) + (C1) are converted in vitro by an endogenous enzymatic factor as well as by bacterial alkaline phosphatase into the variants (A) + (A1).     

Fluctuations in S-adenosyl-L-methionine (AdoMet) during mitotic cycle of the myxomycete Physarum polycephalum

A sensitive radioisotope dilution method was used to measure the S-adenosyl-L-methionine (AdoMet) content in macroplasmodia of the slime mold Physarum polycephalum during the mitotic cycle. The AdoMet pool had two maxima, one during mitosis, the other in the middle of G2 phase.     

Fluctuations in proline and other free amino acids during the mitotic cycle of the myxomycete Physarum polycephalum.

1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis. 2. Proline formed 20--25% of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55% of the pool. 3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.     

Rapid and efficient method for the N-formylation of C-blocked peptides

A simple and rapid method for the formylation of carboxyl-blocked peptides from their corresponding N-t-Boc precursor is described. Several examples illustrate the methodology's usefulness and compatibility with sensitive functionalities.     

A kinetic and binding study of the reactivity of Escherichia coli ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

Escherichia coli H+-ATPase (ECF1) was inactivated in a time- and concentration-dependent manner by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a selective carboxyl group reagent. Among the subunits of ECF1, only the beta subunit was modified by EEDQ. The reaction of 1 mol of EEDQ per mol of ECF1 resulted in total inactivation, in spite of the fact that the enzyme possesses three beta subunits.     

Fluctuations in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate during the mitotic cycle of the acellular slime mould Physarum polycephalum.

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic guanosine 3′:5′-monophosphate (cyclic GMP) have been determined at half-hourly intervals throughout the mitotic cycle of Physarum polycephalum. Cyclic AMP was constant at 1pmole/mg protein throughout except for a transient peak of 17pmoles/mg protein in the last quarter of G2. Cyclic GMP was more variable (2–4pmole/mg protein) rising to 9.5pmole/mg protein during the 3 hour S period and to 7pmole/mg protein during the last hour of G2. The significance of these changes is discussed.     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

Profiles of gonadotropins and steroid hormone-like substances in the hemolymph of mud crab Scylla paramamosain during the reproduction cycle

To elucidate the role of gonadotropins-like substances in mud crab Scylla paramamosain, hemolymph samples were measured for concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH) and steroid hormones by enzyme-linked immunosorbent assay (ELISA). Hormonal concentration data were analyzed in association with the stages of gonadal development. ELISA has shown that in the female crab, the level of FSH reaches its peak in the early stage of ovary development, while estradiol and LH peaked during the late maturing stage of the ovary. In the male crab, testosterone and FSH culminated during the spermatid stage, and the level of LH peaked during the sperm stage. These results indicated that substances resembling the vertebrate FSH and LH are present in the hemolymph of S. paramamosain, and they may be involved in the development of the gonad.     

Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig: cDNA cloning, expression in tissues and expression pattern in the reproductive axis during the estrous cycle

Since its discovery, gonadotropin-inhibitory hormone (GnIH) has appeared to act as a key neuropeptide in the control of vertebrate reproduction. GnIH acts via the novel G protein-coupled receptor 147 (GPR147) to inhibit gonadotropin release and synthesis. To determine the physiological functions of GnIH in the pig, a study was conducted to clone and sequence the cDNA of the GnIH precursor and GPR147. Our results demonstrated that the cloned pig GnIH precursor cDNA encoded three LPXRF and that its receptor possessed typical transmembrane features. Subsequently, tissue expression studies revealed that GnIH was mainly expressed in the brain, corresponding largely with the tissue expression patterns of GPR147 in the pig. The expression patterns in the reproductive axis of the female pig across the estrous cycle were also systemically investigated. The hypothalamic levels of both GnIH and its receptor mRNA were lowest in estrus and peaked in the proestrus and diestrus phases. The highest pituitary GnIH mRNA level was detected in the metestrus, and its receptor displayed a somewhat similar pattern of expression to that of the ligand. However, the expression patterns of GnIH and GPR147 were negatively correlated in the ovary. Immunolocalization in the ovary during the estrous cycle revealed that the immunoreactivities of GnIH and GPR147 were mainly localized in the granulosa and theca cells of the antral follicles during proestrus and estrus and in the luteal cells during metestrus and diestrus. Taken together, this research provided molecular and morphological data for further study of GnIH in the pig.     

The impact of insulin-like growth factor-1 on the pattern of cardiac elongation factor-2 variants in a model of overload

Because of its key role in proteosynthesis, the total content of elongation factor-2 (EF-2) and the distribution of six main EF-2 variants were investigated after Pseudomonas Exotoxin A catalyzed [³²P]ADP-ribosylation using 1D-PAGE and isoelectric focusing (IEF) in a rat model of hemodynamic overload with variable degrees of cardiac hypertrophy: Chronic NO-synthase inhibition by L-NAME (N-omega-nitro-L-arginine-methyl-esther; 0.75 mg/ml drinking water) induced arterial hypertension without hypertrophy but myocardial apoptosis; additional treatment with IGF-1 (osmotic micropumps) did not modify hypertension but reduced apoptosis allowing moderate hypertrophy of the left ventricles. Total EF-2 did not significantly increase in rats with hemodynamic overload with or without IGF-1 supplementation. A positive correlation was found between an acidic EF-2 variant and apoptosis (p = 0.01). Hypertrophy under additional IGF-1 was combined with a shift of the EF-2 variants to basic subtypes (p < 0.01). This finding may be indicative of the trophic potency of IGF-1.     

Amino acid deletion products resulting from incomplete deprotection of the Boc group from Npi-benzyloxymethylhistidine residues during solid-phase peptide synthesis.

In peptide synthesis, the use of N(alpha)-tert-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(pi-Bom)] raises the problem of the Bom group generating formaldehyde during the hydrogen fluoride (HF) cleavage reaction. This can lead to modification of the functional groups on amino acids in the peptide chain. Besides this side reaction, the failure of N(alpha)-Boc deprotection from the His(pi-Bom) residue occurs during TFA treatment for the standard solid-phase peptide synthesis (SPPS) even in the case of a non 'difficult sequence'. This gives amino acid deletion products generated at the N-terminus of the His(pi-Bom) residues. Reviewing the removability of the Boc group on amino acid derivatives showed that the group on the His(pi-Bom) residue was much more resistant under the deprotecting conditions than expected. To circumvent this problem, special precautions, i.e. prolonged deprotection steps and/or increased concentrations of TFA, should be taken for a successful SPPS.     

Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells

BACKGROUND INFORMATION: The actin cytoskeleton forms distinct actin arrays which fulfil their functions during cell cycle progression. Reorganization of the actin cytoskeleton occurs during transition from one actin array to another. Although actin arrays have been well described during cell cycle progression, the dynamic organization of the actin cytoskeleton during actin array transition remains to be dissected. RESULTS: In the present study, a GFP (green fluorescent protein)-mTalin (mouse talin) fusion gene was introduced into suspension-cultured tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow) cells by a calli-cocultivation transformation method to visualize the reorganization of the actin cytoskeleton in vivo during the progression of the cell cycle. Typical actin structures were indicated by GFP-mTalin, such as the pre-prophase actin band, mitotic spindle actin filament cage and phragmoplast actin arrays. In addition, dynamic organization of actin filaments was observed during the progression of the cell from metaphase to anaphase. In late metaphase, spindle actin filaments gradually shrank to the equatorial plane along both the long and short axes. Soon after the separation of sister chromosomes, actin filaments aligned in parallel at the cell division plane, forming a cylinder-like structure. During the formation of the cell plate, one cylinder-like structure changed into two cylinder-like structures: the typical actin arrays of the phragmoplast. However, the two actin arrays remained overlapping at the margin of the centrally growing cell plate, forming an actin wreath. When the cell plate matured further, an actin filament network attached to the cell plate was formed. CONCLUSIONS: Our results clearly describe the dynamic organization of the actin cytoskeleton during mitosis and cytokinesis of a plant cell. This demonstrates that GFP-mTalin-transformed tobacco BY-2 cells are a valuable tool to study actin cytoskeleton functions in the plant cell cycle.     

云南大中山黑颈长尾雉栖息地选择周年变化

采用比较利用和可利用栖息地的方法,分别对云南大中山黑颈长尾雉秋冬季、春季和夏季栖息地展开调查。检验和Bonferroni置信区间分析结果表明,黑颈长尾雉不同季节均偏向选择常绿阔叶林,偏向选择或回避选择的乔木、灌木和草本的种类不尽相同。利用和可利用样方差异性检验显示,不同季节对地形因素的选择性不明显,仅夏季距水源距离一个因子差异显著;植被因子中,春季差异显著的仅有乔木层盖度因子,夏季有乔木密度、乔木层盖度、落叶层盖度和草本层盖度等4个因子,秋冬季有乔木层盖度、草本层盖度和藤本密度等3个因子。主成分分析表明,不同季节利用样方负荷绝对值较大的因子在各主成分中的序位不尽相同,即栖息地选择的主要生态因子随季节发生变化。单因素方差分析和逐步判别分析表明,秋冬季与春季栖息地特征较接近,而与夏季差别较大。χ2     

3,4-二羟基苯乙酮胁迫对天山云杉种子萌发过程中内源植物激素含量变化的影响

3,4-二羟基苯乙酮(DHAP)是天山云杉(Picea schrenkiana ssp. tianschanica)叶和凋落物中存在的主要自毒物质, 是导致天山云杉林天然更新障碍的原因之一。为了解释自毒物质发生作用的生理机制, 该文设计多个浓度梯度的DHAP溶液处理天山云杉种子, 以发芽率和发芽势为种子萌发参数, 运用反相超高效液相色谱(UPLC)分析技术, 检测了种子萌发过程中内源植物激素玉米素(ZT)、赤霉素(GA₃)、吲哚乙酸(IAA)和脱落酸(ABA)含量水平的变化。研究结果表明: DHAP处理对天山云杉种子萌发影响具有浓度效应, 表现为5.0 mmol·L^-1 > 0.1 mmol·L^-1 > 1.0 mmol·L^-1 >对照, 即5.0 mmol·L^-1 DHAP处理组对种子萌发的抑制作用最强、0.1 mmol·L^-1 DHAP处理组次之、1.0 mmol·L^-1 DHAP处理组最弱; DHAP处理组的种子内源ZT、GA₃浓度水平降低, ABA含量升高, GA₃浓度峰值出现时间延迟, IAA浓度在高浓度(5.0 mmol·L^-1 DHAP)处理组短时间内(3-6天)过量积累, 1.0和5.0 mmol·L^-1 DHAP处理组的种子内源ABA浓度峰值出现时间延迟; DHAP处理种子萌发1-6天时, ZT/(GA₃+IAA)比值降低, IAA/ZT、ABA/ZT比值增大; ABA/(ZT + GA₃ + IAA)比值在0.1 mmol·L^-1 DHAP处理组增大, 在5.0 mmol·L^-1 DHAP处理组降低。DHAP处理引发种子内源激素含量水平及激素含量间比值变化, 可能是抑制、延迟天山云杉种子萌发的主要原因。     

Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex of Pisum sativum L.

Calmodulin and NAD kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5–5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 mm. In-vitro NAD-kinase activity was found to increase 17-fold per cell over the apical 30 mm, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and NAD kinase were found to be highly dependent on extraction procedures.Abbreviation EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Metabolism of phosphoenolpyruvate in the C4 cycle during photosynthesis in the phosphoenolpyruvate-carboxykinase C4 grass Spartina anglica Hubb.

The aim of this work was to investigate the fate of phosphoenolpyruvate (PEP) produced by decarboxylation of oxaloacetate during photosynthesis in the bundle sheaths of leaves of the PEP-carboxykinase C₄ grass Spartina anglica Hubb. Mesophyll protoplasts and bundle sheath cells were separated enzymically and used to investigate activities and distributions of putative enzymes of the C₄ cycle and the photosynthetic carbon metabolism of bundle sheath cells. The results indicate that neither conversion of PEP to pyruvate nor its conversion to 3-phosphoglycerate can account for all of the carbon flux through the C₄ cycle during photosynthesis. It is likely, therefore, either that PEP moves directly from bundle sheath to mesophyll or that more than one pathway of regeneration of PEP is involved in the C₄ cycle in this plant.Abbreviations Chl chlorophyll - PEP phosphoenolpyruvate - Pi phosphate - RuBP ribulose-1,5-bisphosphate     

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Immunoreactive-beta-endorphin, -adrenocorticotropin and -calcitonin in extracts of anaplastic or differentiated (rat) medullary thyroid carcinoma.

Fifteen analogs of luliberin (2, LRH) were synthesized by the solid phase method and examined for their ability to block ovulation in the rat. Two analogs, [Ac-DAla¹,DPhe²,DTrp^3,6]-LRH and [Ac-DPhe¹,DPhe²,DTrp^3,6]-LRH, each blocked ovulation at a single injection dose of 250 μg administered at noon on the day of proestrus; three peptides, [Ac-DPro¹,DPhe²,DTrp^3,6]-LRH, [Ac-DThi¹,DPhe²,DTrp^3,6]-LRH and [Ac-DTrp¹,DPhe²,DTrp^3,6]-LRH, were effective at doses of 500 μg each; and four others, [Ac-DTrp¹,DPhe²,DTrp³,DTrp(Nps)⁶]-LRH, [Chlorambucil-DPhe¹, DPhe², DTrp^3,6]-LRH, [1,DThi²,DTrp^3,6]-LRH and [(2-DLys⁶]-LRH, gave partial inhibition at doses tested.