Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum L. cv. SR1) axillary buds establishes a role for cytokinins in tuberization and sink formation

The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink--source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.     

模拟打制实验及其在丁村角页岩石器研究中的应用

模拟打制是史前考古研究中的重要实验考古方法之一,对于正确认识考古标本的类型和技术、解读当时人类的认知水平和知识积累具有不可取代的作用。本文简要回溯了模拟打制实验的发展史,并评述其应用现状。模拟打制实验在欧美有较长的发展与应用历史,但最终趋向了不同的发展方向。传统的模拟打制实验研究在欧洲得到了更加深入的继承和发展,而美国在上世纪90年代后期逐渐发展起了以定量控制、数理统计为核心的实验方法。本文以近期在山西丁村开展的角页岩模拟打制实验为例,介绍传统的模拟打制实验的基本流程和内容,包括实验的内容设计、实施及记录和分析等。本次实验结果肯定了硬锤锤击技术在丁村角页岩石核剥片和修理中的广泛应用,并在一定程度上否定了以往对使用碰砧法的推测。文章最后对模拟打制实验存在的问题和应用前景作了讨论和展望。     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

Evolutionary diversification of structure and function in the family of intracellular calcium-binding proteins

Summary The maximum parsimony method was used to reconstruct the genealogical history of the family of intracellular calcium-binding proteins represented by six major present-day lineages, three of which - calcium dependent modulator protein, heart and skeletal muscle troponin Cs, and alkali light chains of myosin - were found to share a closer kinship with one another than with the other lineages. Similarly, parvalbumins and regulatory light chains of myosin were depicted as more closely related, whereas the branch of intestinal calcium-binding protein proved to have the most distant separation. The computer-generated amino acid sequence for the common ancestor of these six lineages described a four domain protein in which each domain of approximately 40 amino acid residues had a mid-region, 12 residue segment that bound calcium and had properties most resembling those of the calcium dependent modulator protein. It could then be deduced that parvalbumins evolved by deletion of domain I, inactivation of calcium-binding properties in domain II, and acquisition of increased affinity for Ca⁺⁺ and Mg⁺⁺ in domains III and IV. Regulatory light chains of myosin lost the cation binding property from three domains, retaining it in I, whereas alkali light chains of myosin lost this ability from each of the four domains. In skeletal muscle troponin C all domains retained their calcium-binding activity; however, like parvalbumins, domains III and IV acquired high affinity properties. Cardiac troponin C lost its binding activity from domain I but otherwise resembled the skeletal muscle form. Finally, intestinal calcium-binding protein evolved by deletion of domains III and IV.Positive selection could be implicated in these evolutionary changes in that the rate of fixation of mutations substantially increased in the mid portions of those domains which were loosing calcium-binding activity. Likewise, when the cation binding sites were changing from low to high affinity, an accelerated rate of fixed mutations was observed. Once this new functional parameter was selected these regions showed a remarkable conservatism, as did those binding sites which were maintaining the lower affinity. Moreover even in sequence regions not directly involved in cation binding, the lineage of troponin C became very conservative over the past 300 million years, perhaps because of the necessity for maintaining specific interfaces in order for the molecule to interact with troponin I and T in a functional thin myofilament. A similar phenomenon was observed in domain II of the regulatory light chains of the myosin lineage suggesting a possible binding site with the heavy chain of myosin.This paper is dedicated to the memory of Jean-Francois Pechère, deceased     

Dendritic cells and cytokines in human inflammatory and autoimmune diseases

Dendritic cells (DCs) produce cytokines and are susceptible to cytokine-mediated activation. Thus, interaction of resting immature DCs with TLR ligands, for example nucleic acids, or with microbes leads to a cascade of pro-inflammatory cytokines and skewing of T cell responses. Conversely, several cytokines are able to trigger DC activation (maturation) via autocrine, for example TNF and plasmacytoid DCs, and paracrine, for example type I IFN and myeloid DCs, pathways. By controlling DC activation, cytokines regulate immune homeostasis and the balance between tolerance and immunity. The increased production and/or bioavailability of cytokines and associated alterations in DC homeostasis have been implicated in various human inflammatory and autoimmune diseases. Targeting these cytokines with biological agents as already is the case with TNF and IL-1 represents a success of immunology and the coming years will expand the range of cytokines as therapeutic targets in autoinflammatory and autoimmune pathology.