Thiolysis of poly(3-hydroxybutyrate) with polyhydroxyalkanoate synthase from Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) is synthesized from 3-hydroxybutyryl-CoA by polyhydroxyalkanoate synthase and hydrolyzed by PHB depolymerase. In this study, we focused on the reverse reaction of polyhydroxyalkanoate synthase, and propose the possibility that PHB can be degraded through a novel process, that is thiolysis of PHB with CoASH. Polyhydroxyalkanoate synthase of Ralstonia eutropha was incubated with 14C-labeled PHB and CoASH. The reaction mixture was fractionated by HPLC and then analyzed with a scintillation counter. The analysis revealed 3-hydroxybutyryl-CoA to be a product of the reaction. When NADP+ and acetoacetyl-CoA reductase were added to the reaction mixture, an increase in absorbance at 340 nm was observed. Native PHB inclusion bodies from R. eutropha also showed thiolytic activity. This is the first indication that polyhydroxyalkanoate synthase catalyzes both the synthesis and degradation of PHB, and that native PHB inclusion bodies has thiolytic activity.     

Polyhydroxybutyrate accumulation by a Serratia sp.

A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbon-limitation in continuous culture. About 1.3 mmol citrate were consumed per 450 mg biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry wt. The inclusion bodies were stained with Sudan Black and Nile Red (NR), suggesting a lipid material, which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with NR fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increasing the overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.     

Structural and metabolic correlation for Bacillus megaterium ACBT03 in response to colchicine biotransformation

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.     

Structural and metabolic correlation for <Emphasis Type="Italic">Bacillus megaterium</Emphasis> ACBT03 in response to colchicine biotransformation

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.     

Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase

An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2(Reu)) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2(Reu). The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1(Reu)). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.     

Microscopic observations of the early development of Pleurotus pulmonarius fruit bodies

From observations made by light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy we conclude that the expansion of the young fruit body of Pleurotus pulmonarius involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix (ECM), the components of which must be under the control of the hyphae which the ECM surrounds. However the ECM in these fruit bodies is a dilute material. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae. Observations of the hyphal and fruit body structures with a range of conventional microscopic techniques are crucial to complement the information obtained through physiological and molecular studies for understanding the cellular changes that occur during mushroom development.     

The fine structure of the neurons in the rat substantia nigra

Three distinct neurons were identified in the substantia nigra of the rat using Golgi, light, and electron microscopic techniques. A large neuron, found in the pars reticulata, is characterized by well-developed RER, a tubular cytoplasmic inclusion, and somatic and dendritec thorns. A medium-sized neuron, found in the pars compacta, has an eccentric nucleus, distinct Nissl bodies, and an inclusion composed of whorls of concentric cisternae. A small neuron, found in both nigral regions, contains a highly invaginated nucleus, fibrous nuclear inclusion, and paucity of cytoplasmic organelles. Its axon synapses around other nigral dendrites. The presence of these neurons was correlated with the efferent projections and function of the substantia nigra.     

Identity of Holmes Positive Bodies with Electron Microscopically Demonstrable Nucleolus-Like Bodies in Neuronal Cytoplasm

By light microscopic observation of mouse brain stained by Holmes' silver method deeply stained cytoplasmic inclusion bodies were seen in almost all nerve cells of the locus coeruleus. Electron microscopy of tissue samples from floating Vibratome sections stained by Holmes' silver method demonstrated that the nucleolus-like bodies in the cytoplasm were densely impregnated with gold particles. Hence, it was confirmed that the cytoplasmic inclusion bodies of paraffin sections stained by Holmes' method are identical to the so-called nucleolus-like bodies seen in electron microscopic studies.     

斜带髭鲷外周血嗜中性粒细胞核内包涵体的超微结构

目的在对斜带髭鲷（Hapalogenys nitens）外周血细胞结构进行透射电镜观察时发现嗜中性粒细胞存在大量的核内包涵体,系统研究了这些核内包涵体的超微结构,以探讨其来源和形成过程。方法应用电镜技术对这些核内包涵体的超微结构进行研究。结果斜带髭鲷外周血嗜中性粒细胞的核内包涵体可分为假包涵体和真包涵体两种类型,包涵体中的内含物来自胞质。胞核首先是以核膜内陷的方式将胞质及其各种有形成分包绕进核内而在核质外层形成具有双层膜包裹的典型假包涵体,随后假包涵体双层膜降解消失而转化成无被膜包裹的真包涵体,即核内糖原包涵体。结论假包涵体是形成真包涵体的开始阶段。随着假包涵体向真包涵体的转变,包涵体内含物的组成及其超微结构也出现了显著变化。     

Development of woronin bodies from microbodies inFusarium oxysporum f. sp.lycopersici

Summary Woronin bodies are cytoplasmic organelles which commonly lie near the septa in ascomycetous fungi. Although these organelles were observed nearly 100 years ago, little is known about their origin and development. The present ultrastructural investigation describes the ontogeny of Woronin bodies inFusarium oxysporum f. sp.lycopersici [Sacc.] Snyd. and Hans. In this fungus, Woronin bodies are produced by microbodies. Development of the Woronin body begins with the appearance of electron dense material within the microbody. This material aggregates adjacent to the membrane of the microbody and condenses into a single paracrystalline inclusion. Following its formation, the inclusion is gradually extruded and is eventually separated from the parent organelle by an exocytotic mechanism. After the separation, the paracrystalline inclusion is found at the septal pore. Although many recent electron microscopic studies have used various terms to designate these membrane bound organelles, inFusarium these inclusions are believed to correspond to the Woronin bodies initially described by light microscopists.     

Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate

Poly(hydroxyalkanoates) PHAs are synthesized by many bacteria as inclusion bodies and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. A method based on FT-IR was developed to carry out rapid qualitative and quantitative analysis of PHAs in Pseudomonas, when they were grown on sodium octanoate. Using absorbance of the ester band of PHAs, a rapid method was reported to distinguish PHB and PHO and to determine polymer content in intact bacteria. Relative areas in which the C=O area was normalized to the area of the peak representing the amid group (1656 cm(-1)) characteristic of bacteria were calibrated to the polymer content which was determined after solvent extraction. Polymer contents vary from 0% to 53% and depend on the nature of the bacteria. Among 27 strains of Pseudomonas belonging to the rRNA homology group I, a very low amount of bacteria were able to produce PHB. The majority of strains were able to produce a copolymer, PHO, in which the major constituent unit is 3-hydroxyoctanoate. The FT-IR results were further confirmed by gas chromatography analysis after methanolysis of polymer, but FT-IR method requires less preparation of sample than gas chromatography and it is very useful for screening a large variety of Pseudomonas.     

Passive sampler for chlorinated pesticides in estuarine waters

ABSTRACT

Monitoring organic contaminants in the marine environment and identifying their bioconcentration pathways presents great difficulties, as the analytical techniques require preconcentration steps that are time-consuming and tedious. Samplers have been developed that can concentrate extremely low levels of hydrophobic compounds, such as organochlorine pesticides, from water bodies, for direct analysis with minimal sample preparation. The passive samplers consist of polyethylene tubes filled with iso-octane that are deployed within weighted wire cages in estuaries and rivers for a period of three weeks. Hydrophobic compounds such as chlorinated pesticides are continuously partitioned from the water column into the solvent within the tube, providing an integrated sample over the duration of exposure. Pesticides are concentrated 200–300 fold in three weeks. Upon retrieval, the contents can be analysed directly by gas chromatography—electron capture detector (GC—ECD) or concentrated for analysis by gas chromatography-mass spectrometry (GC—MS). Laboratory experiments to determine the uptake rate of the compounds into the sampler enable the calculation of average water concentration over the sampling period. The samplers are cheap and easy to prepare, enabling their deployment in numerous sites over long periods if required. They are envisaged as being a quick and easy monitor for background levels of these contaminants within our waterways.     

Growth and physiological response of Arthrobacter protophormiae RKJ100 toward higher concentrations of o-nitrobenzoate and p-hydroxybenzoate

Bioremediation of sites that are heavily contaminated with pollutant chemicals is a challenge as most of the microorganisms cannot tolerate higher concentrations of toxic compounds. Only a few strains of the genus Pseudomonas have been studied for their tolerance toward the higher concentrations of aromatic pollutant compounds, a phenomenon that is accompanied by various physiological changes. In the present study we have characterized the growth response and physiological changes (adaptations) of a Gram-positive bacterium, Arthrobacter protophormiae RKJ100, toward the higher concentrations of two aromatic compounds, viz. o-nitrobenzoate (ONB) and p-hydroxybenzoate (PHB). Arthrobacter protophormiae RKJ100 could utilize 30 mM ONB and 50 mM PHB as sole sources of carbon and energy. It was capable of growth on higher concentrations of ONB (up to 200 mM) and PHB (up to 150 mM) when the cells were pre-exposed to lower concentrations of these compounds. The adaptive responses shown by the organism during growth on higher concentrations of these compounds were evident from significant changes in cellular fatty acid profiles. In addition, Bacterial Adhesion To Hydrocarbon (BATH) assay and scanning electron microscopy showed substantial increase in cell surface hydrophobicity and decrease in cell size of A. protophormiae RKJ100 when grown on ONB and PHB as compared to succinate-grown cells.     

Pre- and post-embedding immunoelectron microscopy of Ki-M1P immunoreactive germinal center macrophages using ultra-small gold probes with silver enhancement

The Ki-M1P protein is primarily detected in cells deriving from the monocyte/macrophage cell lineage. The aim of this study was to investigate the occurrence of Ki-M1P immunoreactivity in germinal center macrophages by immunohistochemical and immunocytochemical staining techniques. Ultra-small (0.8 nm) gold probes combined with silver enhancement were used as a detection system for pre- and post-embedding immunostaining both at the light and electron microscopic level. Ki-M1P-positive macrophages were observed at a constant frequency in the germinal centers of the follicles throughout the tonsillar lymphatic tissue. The specific immunostaining was localized in the cytoplasm of these cells. Electron microscopic examination demonstrated the presence of abundant lysosomes in the cytoplasm, and some of the germinal center macrophages contained phagocytosed cells (tingible bodies) showing signs of various degrees of digestion. Ki-M1P immunoreactivity, as revealed by depositions of silver-enhanced ultra-small gold probes, was confined to the periphery of the lysosomes and tingible bodies. The results obtained demonstrate that the use of silver-enhanced ultra-small gold probes is a highly sensitive and specific detection system for pre- and post-embedding immunostaining of the Ki-M1P protein, and provides, in general, a flexible system for combined light and electron microscopic examination of tissue antigens. Furthermore, in the cytoplasm of the germinal center macrophages a spatial association between the Ki-M1P protein and lysosomes and tingible bodies was observed. These findings may indicate that the Ki-M1P protein is connected with phagocytosis and/or processes related to intracellular digestion in these cells.     

Anaerobic Biodegradation of Poly-3-Hydroxybutyrate (PHB) by Sulfate Reducing Bacterium Desulfotomaculum sp.

Poly-3-hydroxybutyrate (PHB) film pieces were degraded by sulfate reducing Desulfotomaculum sp. incubated under anaerobic laboratory conditions. Degradation started with adherence of the microbial cells and followed by formation of black colonies on the film surface. Scanning electron microscopic (SEM) observations revealed the presence of bacteria and formation of small holes on the film. After 60 days of incubation at 30°C, 10 % weight loss in polymer and 13 % sulfate reduction in the medium was observed. According to gel permeation chromatography (GPC) analysis, the molecular weight of the PHB decreased after 30 days and did not decrease further at a more extended incubation period. Loss of weight of PHB does not seem to be correlated with molecular weight decrease.     

THE NORMAL AND INDUCED OCCURRENCE OF CYANOPHYCIN INCLUSION BODIES IN SEVERAL BLUE-GREEN ALGAE1

Multi-L-arginyl-poly(L-aspartic acid) [arg-poly(asp)], the polypeptide component of the cyanophycin inclusion body, is found in cells of many blue-green algae. Formation of this material can be induced by a variety of treatments including the addition of excess nitrogen-containing compounds, the addition of specific inhibitors of macromolecular synthesis, and the exclusion of sulfur or phosphorus. Knowledge of the conditions that induce the synthesis of this polypeptide has made possible an ultrastructural survey to determine the presence and cellular location of cyanophycin bodies in a variety of cyanobacteria. Data presented show that certain strains of the unicellular genus Synechococcus Nägeli do not contain arg-poly(asp) under environmental conditions that markedly increase the level of such material in other cyanobacteria. In addition many strains show spatial localization of the cyanophycin bodies under normal growth conditions, and moreover the normal pattern is retained even when massive synthesis of arg-poly(asp) is induced. Finally there is no evidence that these inclusion bodies occur in certain beggiatoan gliding bacteria.     

Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture

When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.     

Transformation of phosphorus and relevant intracellular compounds by a phosphorus-accumulating enrichment culture in the presence of both the electron acceptor and electron donor

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.     

Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16

Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.     

Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.