PhaP is involved in the formation of a network on the surface of polyhydroxyalkanoate inclusions in Cupriavidus necator H16

Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2- to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of ~20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, ~50-nm globular structures that are raised ~1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is ~15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from Cupriavidus necator H16 ΔphaP were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of phaP on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.     

Fibrous and tubular inclusions in four xylariaceous fungi

Summary Intranuclear and cytoplasmic fibrous inclusions and cytoplasmic tubular inclusions have been studied using electron microscope techniques. The fibrous inclusions are composed of closely packed, rod-like structures. Each rod has an outside diameter of ± 24 nm, a hollow centre and lateral projections along its length. The tubular inclusions occur as densely packed bundles or loose arrays of 10–13 nm diameter tubules which are composed of subunits arranged in a double helical structure. The distribution, origin and possible function of these and similar inclusions is discussed.     

Involvement of macroautophagy in the dissolution of neuronal inclusions

Ubiquitinated inclusions are a common feature of many neurodegenerative conditions. We have proposed that, at least in part, such inclusions may be formed due to dysfunction of the proteasome. We have modeled such proteasomal dysfunction by applying pharmacological inhibitors to cultured embryonic rat cortical neurons. This treatment leads to neuronal death and formation of ubiquitin/-synuclein-positive cytoplasmic inclusions. At late time points following proteasomal inhibition such inclusions are no longer discerned. Instead, many neurons accumulate small ubiquitinated aggregates, which may represent remnants of the inclusions. In this work we have examined a potential mechanism for inclusion dissolution. Electron microscopy images showed activation of macroautophagy at late time points after proteasomal inhibition. Labeling with LysoTracker Red, a dye that accumulates in acidic compartments, or immunostaining for the lysosomal enzyme Cathepsin D, showed an increase in globular staining. Cathepsin D co-localized partially with small ubiquitinated aggregates, but not inclusions. Application of an inhibitor of macroautophagy or of the vacuolar ATPase led to an increase in the number of inclusions and a decrease in small aggregates, whereas an activator of autophagy had the opposite effects. There was no significant change in apoptotic death following these manipulations. We conclude that, following proteasomal inhibition of cultured cortical neurons, there is activation of macroautophagy and of the lysosomal pathway. This activation results in dissolution of ubiquitinated inclusions into small aggregates, without directly impacting neuronal cell death. These data further support the idea that in this model inclusions and neuronal cell death are independent processes.     

聚羟基脂肪酸酯纳米微球：结构特征、生物合成及其在生物技术和生物医药领域的应用

聚羟基脂肪酸酯（polyhydroxyalkanoate）PHA 纳米微球是很多微生物在营养失衡的情况下,在体内合成的一种可生物降解的细胞内聚酯,主要作为微生物的碳源及能量储备。天然 PHA 微球的内部是由疏水的聚酯链构成的疏水核心,其外层是由磷脂界膜及膜上嵌入或附着的包括 PHA合酶 PhaC 和 PHA 颗粒相关蛋白 PhaP 等蛋白构成的边界层。PhaC 通过共价键连接在PHA微球表面,而 PhaP 通过疏水相互作用吸附在 PHA 微球表面。通过将外源性功能蛋白与 PhaC 或 PhaP 进行融合表达,在重组微生物体内就能直接合成表面带有功能蛋白的纳米微球复合体。由于该纳米微球在微生物细胞内是以独立的包涵体形式存在,因此通过细胞破碎及离心等方法就能简便、有效地使其从细胞中分离并得以纯化。鉴于 PHA 微球这种表面易被修饰改造的特性,越来越多的功能蛋白通过与 PHA 微球表面蛋白（PhaC 或 PhaP）的融合表达,呈递在了 PHA 微球表面,使其成为一种廉价、高效的蛋白固定化及呈递的新技术。本文在介绍了 PHA 微球的结构特性及生物合成的基础上,着重综述了目前关于功能化 PHA 微球在蛋白纯化、固定化酶、生物分离、靶向递药、疾病诊断、成像技术及新型疫苗开发方面的研究现状及其未来在生物医药等领域的广泛应用前景。     

alpha-synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.     

Protist-like inclusions in amber, as evidenced by Charentes amber

The mid-Cretaceous amber of France contains thousands of protist-like inclusions similar in shape to some ciliates, flagellates and amoebae. The sheer abundance of these inclusions and their size variation within a single amber piece are not concordant with true fossil protists. French amber is coniferous in origin, which generally does not preserve well protists without cell walls. Thus, it would be surprising if French Cretaceous amber had preserved millions of protists. Here, we present a survey of the protist-like inclusions from French amber and attempt to elucidate their origins.Diverse Cretaceous ambers (from Spain, Germany and Lebanon), also derived from conifer resins, contain thousands of protist-like inclusions. In contrast, Tertiary ambers and modern resins are poor in protist-like fossils. This suggests these inclusions originated from early Cretaceous plant resins, probably secreted with the resin by trees that did not survive after the Cretaceous (such as the Cheirolepidiaceae). A review of the recent literature on amber microfossils indicates several protist-like inclusions that are unlikely to have a biological origin have already been described as real fossil protists. This is problematic in that it will bias our understanding of protist evolution.     

Production and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions

This work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems.     

缅甸琥珀内含物形态结构在X射线下的可辨识性

【目的】产自缅甸北部胡康河谷的缅甸琥珀形成于白垩纪中期。其艺术价值很高,同时其内含物的生物多样性程度也很高,故其科学价值也不可估量。显微CT能够提供化石(琥珀)内部解剖结构的高分辨率断层图像,故该方法日渐成为目前琥珀研究中的常用方法之一。然而在可见光下可见的琥珀内的生物结构,在X射线下却有不同的结果,这与现生研究材料在显微CT下的表现非常不同。本研究对产自胡康河谷的9块缅甸琥珀进行显微CT检测,试图对这个特殊的现象进行较为系统的解读。【方法】利用数码相机(Nikon 5200D)拍摄琥珀照片,并用Helicon Focus 5.3软件合成。通过显微CT技术扫描琥珀和计算机断层重建技术重建出缅甸琥珀内含物的三维结构形态。【结果】显微CT检测结果主要分为3种:完全无衬度、部分结构有衬度和整体结构有较好衬度。本研究对有较好衬度的琥珀内含物进行了三维重建,展示了琥珀内含物的外部和内部三维结构。【结论】琥珀内含物的可见光成像和X射线成像不存在一一对应关系,其原因和琥珀保存的好坏以及琥珀的密度差、琥珀围岩之间的对比度差异有关。琥珀形成和埋藏过程中的物理和化学变化非常复杂,其机理的探究也更为复杂和困难,本文对这个现象的主要类型做了较为初步的阐述,后续研究需要更为全面的选样和更为严格的实验设计才能够最终解决这个埋藏学上的难题。     

Intermembrane inclusions induced by anoxia in heart and skeletal muscle mitochondria.

Heart and skeletal muscle from rats of different ages were incubated in vitro in an oxygen-free medium supplied with substrates in order to investigate the effect of anoxia on muscle fine structure, particulary on the mitochondria. In skeletal muscle fibers anoxia has been found to induce changes similar to those previously described in ischemic muscles in vivo namely giant mitochondria, apparently derived by mitochondrial fusion, and intermembrane inclusions with a paracrystalline structure. The plate-like inclusions are mostly located in the intracristal spaces and are closely associated to cristal membranes even in markedly swollen mitochondria. Identical inclusions have been observed in cardiac muscle cells following anoxic injury, whereas they are never found in non-muscle cells such as endothelia, fibroblasts and nerve fibers. Cardiac and skeletal muscle fibers from newborn rats maintained in an oxygen-free medium show mitochondrial swelling but no intermembrane inclusions. The different response of mitochondria from developing vs adult striated muscle to anoxia may be due to changes during postnatal development in the quality or quantity of the protein component(s) involved in paracrystal formation.     

Interaction between inclusions embedded in membranes.

We calculate the membrane-induced interaction between inclusions, in terms of the membrane stretching and bending moduli and the spontaneous curvature. We find that the membrane-induced interaction between inclusions varies nonmonotonically as a function of the inclusion spacing. The location of the energy minimum depends on the spontaneous curvature and the membrane perturbation decay length, where the latter is set by the membrane moduli. The membrane perturbation energy increases with the inclusion radius. The Ornstein-Zernike theory, with the Percus-Yevick closure, is used to calculate the radial distribution function of inclusions. We find that when the spontaneous curvature is zero, the interaction between inclusions due to the membrane deformation is qualitatively similar to the hard-core interaction. However, in the case of finite spontaneous curvature, the effective interaction is dramatically modified.     

Isolated cilia and crystalloid inclusions in murine bone marrow stromal cells

Bone marrow stromal cells, especially reticular cells and macrophages, are thought to have important functions in the hematopoietic inductive microenvironment (HIM). In order to define the details of their fine structure, the femoral bone marrow of 50 C 57 BL mice was examined by electron microscopy after fixation by vascular perfusion. Almost all the structures of the murine bone marrow stromal cells were similar to those previously reported. Murine bone marrow reticular cells had some isolated cilia (with a frequency of 6.0%) of 9 + 0 pattern in their axoneme. The murine bone marrow macrophages had crystalloid inclusions (CI) that revealed characteristic periodic substructure. These periodic structures could be classified into four types (types A-D). Type A inclusions had a rather amorphous structure and were the most common (with a frequency of 61.9%). Type B inclusions had a periodic lamellar structure oblique to the long axis of the CI itself at intervals of about 3.5 nm. Type C inclusions had a periodic lattice structure at intervals of about 3.7 nm, and were the least frequent (with a frequency of 6.0%). Type D inclusions had periodic laminated structure parallel to the long axis of CI at intervals of about 3.5 nm. Although the true significance of isolated cilia of murine bone marrow reticular cells and the periodic internal structures of CI in murine bone marrow macrophages remains unknown, they must not be ignored as only incidental findings: they may be useful to the understanding of the relationship between function and structure in future research on HIM.     

Unusual polyphosphate inclusions observed in a marine Beggiatoa strain

Sulfide-oxidizing bacteria of the genus Beggiatoa are known to accumulate phosphate intracellularly as polyphosphate but little is known about the structure and properties of these inclusions. Application of different staining techniques revealed the presence of unusually large polyphosphate inclusions in the marine Beggiatoa strain 35Flor. The inclusions showed a co-occurrence of polyphosphate, calcium and magnesium when analyzed by scanning electron microscopy and energy dispersive X-ray analysis. Similar to polyphosphate-enriched acidocalcisomes of prokaryotes and eukaryotes, the polyphosphate inclusions in Beggiatoa strain 35Flor are enclosed by a lipid layer and store cations. However, they are not notably acidic. 16S rRNA gene sequence-based phylogenetic reconstruction showed an affiliation of Beggiatoa strain 35Flor to a monophyletic branch, comprising other narrow vacuolated and non-vacuolated Beggiatoa species. The polyphosphate inclusions represent a new type of membrane surrounded storage compartment within the genus Beggiatoa, distinct from the mostly nitrate-storing vacuoles known from other marine sulfide-oxidizing bacteria of the family Beggiatoaceae.     

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

A new group of parasporal inclusions encoded by the S-layer gene of Bacillus thuringiensis

Bacillus thuringiensis produces various groups of active proteins, such as Cyt, Vip and Parasporin, in addition to the Cry protein. In this study we show S-layer proteins to be a new group of parasporal inclusions of B. thuringiensis . The S-layer consists of a two-dimensional lattice structure and is the outermost component of many archaeobacteria and eubacteria. The parasporal inclusion of B. thuringiensis strain CTC was found to be not a typical crystal protein encoded by the cry gene, but a proteinaceous inclusion encoded by the S-layer gene. Furthermore, the CTC-like strains (with their parasporal inclusions coded by the S-layer gene) are widely distributed and accounted for 25.4% of the B. thuringiensis strains tested. These strains constitue a new group of parasporal inclusions encoded by the S-layer gene of B. thuringiensis and shed new light on B. thuringiensis nontoxic strains.     

Isolation and characterization of polyhydroxyalkanoates inclusions and their associated proteins in Pseudomonas sp. 61-3

Two types of polyester inclusions of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] were isolated from crude extract of Pseudomonas sp. 61-3. Proteins associated with each inclusion were separated by SDS-PAGE. PHA synthase 1 (PhaC1(Ps)), PhaF(Ps), and PhaI(Ps) were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbC(Ps)) and 24-kDa unknown protein were identified from P(3HB) inclusions. The structural genes of PhaF(Ps) and PhaI(Ps) were located downstream of the pha locus. The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms. Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies.     

Glomiform and fibrillar cytoplasmic inclusions

Glomiform inclusions, also called tubular arrays in endoplasmic reticulum, are found in the epithelial cells of glandular tissues of a patient with systemic lupus erythematosus, a patient with Reye’s syndrome, and a dog. Their three dimensional structure is interpreted as a skein of contorted tubules of endoplasmic reticulum. Fibrillar inclusions found in the pancreatic acinar cells of two patients are believed to represent altered zymogen granules.     

Ultrastructure of spermiogenesis and synthesis of enigmatic cytoplasmic inclusions in the spirally shaped spermatozoon of the polychaete Spio setosa (Annelida: Spionidae)

The sperm of Spio setosa (Polychaeta, Spionidae) are known to be very unusual in form; here, spermiogenesis and the structure of the spermatozoon in this species are described by transmission electron microscopy. While spermiogenesis is similar to that described for many other polychaetes, two notable exceptions to this process include the synthesis of abundant ring‐shaped and tubular, membrane‐bounded cytoplasmic inclusions in the midpiece, and the differentiation of a spirally shaped sperm head. Spermatids develop as free‐floating tetrads in the male's coelom. A microtubular manchette does not develop during chromatin condensation and nuclear elongation, and the spiral acrosome forms as a single Golgi‐derived vesicle that migrates anteriorly to become housed in a deep anterior nuclear fossa. Early in spermiogenesis, numerous Golgi‐derived, membrane‐bounded cytoplasmic inclusions appear in the cytoplasm; these ultimately occupy the sperm midpiece only. The mature spermatozoon in the male has a 15‐μm‐long head consisting of a nucleus coiled like a spring and a spiral acrosome with differentiated substructure, the posterior two thirds of which sits in an anterior nuclear fossa. The midpiece is wider than the rest of the spermatozoon and contains 9–10 spherical mitochondria surrounding the two centrioles, as well as numerous membrane‐bounded conoid and tubular cytoplasmic inclusions. The axoneme has a 9 + 2 arrangement of microtubules. By contrast, stored sperm in the female's seminal receptacles have lost the midpiece inclusions but contain an abundance of glycogen. The function of the midpiece inclusions remains unresolved, and the significance of their absence in stored sperm within the seminal receptacle and the appearance of midpiece glycogen stores remains unclear and requires additional investigation.     

Cobalt- and chromium-containing inclusions in bacterial cells

Bacteria belonging to different taxonomic and physiological groups (members of the genera Pseudomonas, Brevibacterium, Rhodopseudomonas, and Lactococcus) are able to form intracellular cobalt- and chromium-containing magnetic inclusions. The paper deals with the structure and the intracellular localization of these inclusions and their similarity to the known noncrystalline iron-containing magnetic inclusions. The possible biological role of the magnetic inclusions is discussed.