Characterization of mammalian synemin,an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells: co-localization and interaction with type III intermediate filament proteins and keratins

Using a monoclonal antibody, we have detected a high molecular weight muscle protein, co-localized and co-isolating with desmin. Searching a human cDNA database with partial amino acid sequences of the protein, we found a cDNA clone encoding a 1565-amino-acid polypeptide, identified as a mammalian (human) synemin, a member of the intermediate filament (IF) protein family. Immunoblotting showed the presence of a 180-kDa polypeptide in skeletal muscle and 180- and 200-kDa polypeptides in cardiac and smooth muscles. Interestingly, synemin was also found in myoepithelial cells, which have keratin filaments instead of desmin. Moreover, synemin was also found in astrocytes of optic nerves and non-myelin-forming Schwann cells, together with glial fibrillary acidic protein (GFAP) and vimentin. Blot overlays pointed to molecular interactions of synemin with desmin, vimentin, GFAP and keratin 5 and 6, but not with keratin 14. The experimental data also suggested a possible link with nebulin, a skeletal muscle protein. Purified synemin was coassembled with desmin in different molar ratios, and at 1:25, as typically found in vivo, IFs were formed which were comparable in length to desmin filaments. However, at molar ratios of 3:25 and 6:25, much shorter and irregular shaped filamentous polymers were generated. The fact that synemin is present in all four classes of muscle cells and a specific type of glial cells is indicative of important functions. Its incorporation may give structural and functional versatility to the IF cytoskeleton.This work was supported by grants from the Ministry of Education, Science, and Culture of Japan.     

Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10⁻⁵ M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm²). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24–72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.     

A functional unit consisting of an eversible gland with neurosecretory innervation and a proprioreceptor derived from a complex sensillum in an insect

Summary The eversible sac of the antennal tip in Hypogastrura socialis (Collembola) has been reconstructed from serial ultrathin sections. The organ contains 3 specialized epidermal glandular cells and the dendrites of 2 sensory cells encapsulated by an enveloping cell. The following features of the system are particularly remarkable and have been analyzed in detail: (1) a neurosecretory innervation of the glandular cells, (2) the structure of the dendritic outer segments within the sac, and (3) the structure of the complex sensillum, from which these dendrites may be derived. The system may be thought of as providing an example of phylogenetic transformation of an exteroceptor into a mechanoreceptive proprioceptor. A functional model is proposed which involves control of the mechanism of evagination as well as of the secretory discharge.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

A structural investigation of complex I and I + III2 supercomplex from Zea mays at 11-13 Å resolution: Assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I + III₂ supercomplex from the C₄ plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III₂ supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.     

Variability and stability of tea weevil‐induced volatile emissions from tea plants with different weevil densities,photoperiod and infestation duration

Abstract After herbivore attack, many plants emit herbivore‐induced plant volatiles (HIPVs). HIPVs can attract carnivores and/or repel herbivores, thereby mediating tritrophic plant–herbivore–carnivore interactions. HIPVs act as chemical information between organisms; hence, their variability and stability are vital. In the present study, variations in the volatile emissions, from the tea plant Camellia sinensis (O. Ktze) damaged by the tea weevil Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae), with weevil densities, photoperiod and infestation duration, were investigated. The volatiles induced by high‐density weevils were more abundant in composition and amount than those induced by low‐density weevils, whether at noon, night or after weevil removal. The induced volatile emissions were similar on the second and third day after infestation, and the emissions of the major induced compounds displayed diurnal cycles. Linalool, (E,E)‐α‐farnesene, and benzyl nitrile were emitted mainly at noon, whereas 1,3,8‐p‐menthatriene and (E)‐β‐ocimene were maximally emitted at night. Given the different emission dynamics, significant differences were found between noon‐ and night‐induced volatiles. In summary, tea plants damaged by different weevil densities emitted a relatively stable signal at a particular time. This stability could be attributed to the similarities under the two densities of the main induced volatile compounds, their relative ratios and the emission dynamics of the induced volatiles.     

不同产地黄芩中无机元素含量及其与根际土壤无机元素的关系

药用植物中各无机元素含量的不仅影响药用植物的生长发育,也是药材有效成分的构成因子。通过对全国范围内16个不同产地(即居群)的92个野生黄芩(Scutellaria baicalensis Georgi)样本及其相应的根际土壤中10种无机元素含量的分析,发现不同产地黄芩及其根际土壤无机元素都有很大变异,且不同产地黄芩根际土壤中无机元素的变异远大于黄芩药材中无机元素的变异。总体来看,黄芩中Mg(9级)含量较其他植物含量高;P(1级)、K(2级)、Mn(3级)含量与其他植物相比处处较低水平;黄芩对Sr(富集系数达到3.52)有较强富集。并且通过无机元素分布曲线分析建立了无机元素指纹谱,主成分分析筛选出黄芩主要特征无机元素为Mg、K、Ca、Fe、Zn。本研究还表明,黄芩对各元素的吸收能力受产地的影响较大,提示黄芩对无机元素的吸收与各产地根际土壤无机元素有一定关联性。     

PspGI,a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.     

Variability of haemocyte and haemolymph parameters in European flat oyster Ostrea edulis families obtained from brood stocks of different geographical origins and relation with infection by the protozoan Bonamia ostreae

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM^®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.