Large Inclusion Bodies Research Articles

Response of bacterial and viral communities to nutrient manipulations in seawater mesocosms

Changes in natural bacterial and viral assemblages were studied in seawater meso- cosms manipulated with inorganic (nitrate + phosphate) and inorganic + organic (glucose) nutrient additions. As inferred from the gel band patterns obtained by DGGE, only moderate changes within the bacterial community took place when mineral nutrients were added alone. Supplementing the mineral nutrients with glucose in excess of what the bacteria could consume led, however, to major changes in band patterns. Based on fluorescence in situ hybridisation (FISH), the major bacterial response was identified as an increase in the population of γ-Proteobacteria with a smaller response in α -Proteobacteria. Sequencing of bands from the DGGE gels indicated that glucose + mineral nutri- ents led to a Vibrio-dominated bacterial community. A specific FISH probe was designed from a band sequence affiliated to Vibrio splendidus, and linked a large-celled bacterial morphotype to the DGGE-gel bands dominating in glucose-amended mesocosms. A similar difference in the response of the viral populations among treatments was demonstrated using pulsed field gel electrophoresis (PFGE). The number of bands on DGGE gels and PFGE gels were similar (mean ratio 0.98). We suggest an interpretation of these results where coexistence of nutrient-competing bacterial hosts is controlled by viral lysis. We also suggest that the success of large bacteria in glucose-replete treat- ments was not based on superior glucose-utilisation abilities, but rather on an advantage in competi- tion for limiting mineral nutrients derived from the combination of a large cell surface with a low cellular content of the limiting element, possible for cells with large C-rich inclusion bodies.

Characterization of Cytoplasmic α-Synuclein Aggregates: FIBRIL FORMATION IS TIGHTLY LINKED TO THE INCLUSION-FORMING PROCESS IN CELLS

The alpha-synuclein fibrillation process has been associated with the pathogenesis of several neurodegenerative diseases. Here, we have characterized the cytoplasmic alpha-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of alpha-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloid-like alpha-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves non-fibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.

Reovirus core protein mu2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules.

Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.

Herpes Simplex Encephalitis in a Domestic Rabbit (Oryctolagus cuniculus)

An adult domestic rabbit showing neurological signs was subjected to euthanasia. At necropsy, macroscopical lesions were absent. Histopathologically, extensive lesions were seen, particularly in the cerebral cortex. Non-suppurative meningitis was present and there was lymphocytic and plasmacytic perivascular cuffing in the neuropil. The cerebral cortex showed extensive segmental neuronal and glial necrosis. Within the necrotic areas, large amphophilic intranuclear inclusion bodies were present in neurons and glial cells. Immunohistochemically, neurons and glial cells in the affected areas were labelled by polyclonal antibodies against both herpes simplex virus-1 (HSV-1) and HSV-2. The agent was classified as HSV-1 by polymerase chain reaction analysis. This is only the second reported natural case of herpes simplex infection in a rabbit.

Effects of cigarette smoke exposure on the ultrastructure of the golden hamster parathyroid gland.

Cigarette smoking has been identified as one of the risk factors to induce osteoporosis. However, we find no study on the morphology of the parathyroid gland under smoking exposure. We studied the ultrastructure of the parathyroid gland, lung and femur of the golden hamster exposed to cigarette smoke. Four-week-old male hamsters were housed in a plastic case (48x31x30 cm) and were exposed to cigarette smoke for 12 weeks, 5 minutes exposure, 4 times a day, 4 days a week. There were no differences in serum calcium level and the whole bone mineral density between the control and the smoke-exposed groups. In the parathyroid gland of the smoke-exposed animals, the Golgi complexes associated with many prosecretory granules were well developed and many secretory granules were located near the plasma membrane. Large lipid-like inclusion bodies were observed in the alveolar macrophages of the smoke-exposed animals. The femur morphology showed a wider area of resorbing surface in the smoke-exposed group than in the control group. From these findings, it is conceivable that the secretory activity of the parathyroid gland was stimulated with cigarette smoke exposure.

Histological and morphometric changes in untraumatised rabbit skeletal muscle treated with deep transverse friction

Deep transverse friction (DTF) is used in clinical practice and by its nature it may cause muscle injury. This study investigates the morphologic and morphometric changes in untraumatised rabbit skeletal muscle treated with DTF.Method: 16 New Zealand white rabbits were studied. The right vastus lateralis muscle was used as a control and the left vastus lateralis was treated with DTF. Muscle biopsies were taken 10 min, 24 h and 48 h after 1 treatment, 48 h after 2 treatments and 48 h and 6 days after 3 treatments. Treatments were 48 h apart. Biopsies were prepared for light microscopy and tissue morphometry.Results: After 1 DTF treatment, intracellular and extracellular oedema was noted. Contraction bands seen throughout the fibres suggested severe mechanical trauma to the muscle. 48 hours after 1, 2, and 3 treatments, the muscle appeared to be recovering with reduced oedema, and the contraction banding was limited to small focal areas throughout each fibre. Six days after the last treatment, the myofibers, although normal in diameter, showed small focal areas of super contraction and large internalised inclusion bodies composed of a pool of myofilaments or whorls of membranous material. Morphometry showed oedema to be maximal immediately after treatment.Conclusion: DTF causes a severe but reversible injury to untraumatized myofibers. Its possible mode of action in treatment of injured muscle requires further investigation.

Formation and Removal of α-Synuclein Aggregates in Cells Exposed to Mitochondrial Inhibitors

Mitochondrial dysfunction has been associated with Parkinson's disease. However, the role of mitochondrial defects in the formation of Lewy bodies, a pathological hallmark of Parkinson's disease has not been addressed directly. In this report, we investigated the effects of inhibitors of the mitochondrial electron-transport chain on the aggregation of alpha-synuclein, a major protein component of Lewy bodies. Treatment with rotenone, an inhibitor of complex I, resulted in an increase of detergent-resistant alpha-synuclein aggregates and a reduction in ATP level. Another inhibitor of the electron-transport chain, oligomycin, also showed temporal correlation between the formation of aggregates and ATP reduction. Microscopic analyses showed a progressive evolution of small aggregates of alpha-synuclein to a large perinuclear inclusion body. The inclusions were co-stained with ubiquitin, 20 S proteasome, gamma-tubulin, and vimentin. The perinuclear inclusion bodies, but not the small cytoplasmic aggregates, were thioflavin S-positive, suggesting the amyloid-like conformation. Interestingly, the aggregates disappeared when the cells were replenished with inhibitor-free medium. Disappearance of aggregates coincided with the recovery of mitochondrial metabolism and was partially inhibited by proteasome inhibitors. These results suggest that the formation of alpha-synuclein inclusions could be initiated by an impaired mitochondrial function and be reversed by restoring normal mitochondrial metabolism.

Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment.

Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein. The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein. However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear. Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1). Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies. Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form. Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1. Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells.

Distribution of rabies antigen in infected brain material: determining the reliability of different regions of the brain for the rabies fluorescent antibody test

To assist in making recommendations for sampling of brains for the fluorescent antibody test (FAT), a study was conducted to determine the regions of the brain where rabies antigen is found most reliably. Each identifiable part of 252 rabies-positive brains of various species was re-tested using routine FA tests. It was found that there was frequent variation in the quantity of antigen between regions of the brain. The thalamus, pons and medulla were the most reliable parts of the brain as they were positive in all specimens tested. The cerebellum, hippocampus and different parts of the cerebrum were negative in, respectively, 4.5, 4.9 and 3.9–11.1% of positive brains. It is recommended that specimens for rabies diagnosis must include the brain stem. The structure of choice would be the thalamus as it was positive in all specimens and had the most frequent prevalence (97.8%) of abundant antigen. These findings contradict many old studies that state that the hippocampus should be the structure of choice for rabies diagnosis. The current data demonstrate that the reason for the old recommendations is that the hippocampus has the highest frequency of large inclusion bodies, as the reliability of the histological tests used previously depended on inclusion body size.

Methylosarcina fibrata gen. nov., sp. nov. and Methylosarcina quisquiliarum sp.nov., novel type 1 methanotrophs.

Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized. Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters. Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies. The individual cells of each strain were sometimes motile and had differing morphologies. Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils. Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology. AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T. Both strains utilized only methane and methanol as carbon sources. In stationary phase, the cells of each strain swelled in size and formed cysts. Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances. 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values. By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp. However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains. On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen. nov., harbouring two species, Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.

Pathological Findings in Two Fin Whales (Balaenoptera physalus) with Evidence of Morbillivirus Infection

Two immature female fin whales stranded on the Belgian and French coastlines, were examined post mortem. The main gross findings were massive parasitic infestation, associated with a large thrombus in one whale, and severe emaciation. Microscopical investigations revealed multinucleated syncytia with large intranuclear inclusion bodies in various tissues, and positive immunolabelling for morbillivirus antigens. Other evidence of morbillivirus infection was provided by the demonstration of specific viral structures in syncytia and in cell cultures, and the detection of neutralizing antibodies to canine distemper virus. To the authors» knowledge, this is the first firm report of morbillivirus infection in baleen whales.

How neurotoxic glutamate is released in stroke

True retroviruses are not known in plants; however, plant pararetroviruses (caulimoviridae) share many retroviral properties, replicating by transcription in the nucleus followed by reverse transcription in the cytoplasm. Pararetroviruses have circular DNA genomes that do not integrate into the host genome, and display several unique expression strategies. Typical of plant pararetroviral pregenomic RNA is a highly structured leader of about 600nt long that is bypassed by scanning ribosomes. Caulimoviruses and Soymoviruses have a further interesting translation mechanism: at least six of the seven open reading frames are translated via polycistronic translation mediated by a specific transactivator (TAV), which modifies the translation complex. TAV also forms large intracellular inclusion bodies, which are the site of translation and virus assembly.

Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B.

Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters (gap and eno) were tested and found to be less effective than the original celZ promoter. By screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau3A1 fragments, one of which was previously sequenced in Z. mobilis. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.

Platyspondylic lethal skeletal dysplasia, San Diego type, is caused by FGFR3 mutations

The platyspondylic lethal skeletal dysplasias (PLSDs) are a heterogeneous group of short-limb dwarfing conditions. The most common form of PLSD is thanatophoric dysplasia (TD), which has been divided into two types (TD1 and TD2). Three other types of PLSD, or TD variants (San Diego, Torrance, and Luton), have been distinguished from TD. The most notable difference between TD and the variants is the presence of large rough endoplasmic reticulum (rER) inclusion bodies within chondrocytes of the variants. We examined 22 cases of TD variants for the presence of missense mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. All 17 cases of the San Diego type (PLSD-SD) were heterozygous for the same FGFR3 mutations found in TD1. No mutations were identified in the Torrance and Luton types. Large inclusion bodies were found in all 14 cases of PLSD-SD. Similar inclusion bodies were present in two of 72 TD1 cases, but not in 39 controls. The material retained within the rER stained only with antibody to the FGFR3 protein. The radiographic and morphologic differences between TD and PLSD-SD may be a consequence of other genetic factors, perhaps in the processing of mutant FGFR3 molecules within the rER. The presence of rER inclusion bodies cannot reliably discriminate between closely related skeletal dysplasias.

An ultrastructural study of age-related changes in mouse olfactory epithelium.

Transmission and scanning electron microscopic examinations were undertaken to detail changes in the olfactory epithelium (OE) resulting from the ageing process. Samples were prepared from 3% glutaraldehyde/1% formaldehyde perfused mice aged 6 months and 29-30 months. Compared to OE from young adults, a number of striking changes were apparent in tissue from older animals. The most obvious of these were extensive local accumulations of large inclusion bodies, totally disrupting the normal morphology of such affected areas of olfactory epithelium. Even in areas where these deposits were absent, other significant signs of ageing were noted in the seemingly unaffected OE of all older mice studied. Quantitative analysis of semi-thin resin sections revealed that the OE of aged mice was significantly reduced in thickness, and with significantly fewer olfactory sensory neurons, irrespective of whether or not inclusions were present. In addition, pale cells, which have been suggested to be a form of degenerating cells, were more abundant in aged OE. The straight, sharp boundary between respiratory and olfactory epithelia which is seen in young adults became irregular and disrupted with age due to an intermingling of the respiratory and olfactory cells. Such structural alterations may parallel olfactory dysfunction reputed to occur in older mammals.

Inclusion Body Tracheitis Associated with Avian Adenovirus in Turkeys

Nine turkey flocks with basophilic intranuclear inclusion bodies, suggestive of adenovirus, within the epithelial cells of the tracheal mucosa were studied. Respiratory signs and increased mortality occurred in turkeys between 6 and 10 wk of age from nine commercial turkey meat flocks in central California. Necropsy findings included tracheitis and occasional mild sinusitis. Histopathology of the tracheas revealed epithelial deciliation, squamous metaplasia, large basophilic intranuclear inclusion bodies within epithelial cells, and lymphoplasmatic inflammation in the mucosa. Electron microscopy of the mucosa revealed hexagonal viral particles, approximately 73 nm in diameter, consistent with adenovirus within the nuclei of epithelial cells. All tracheal sections were negative for adenovirus group II by specific immunoperoxidase staining. Adenovirus group I was isolated from the trachea. In addition, Bordetella avium, Ornithobacterium rhinotracheale, and Klebsiella pneumoniae were isolated from the tracheas of three, three, and two flocks, respectively. Statistically greater total mortality and a smaller percentage of marketed turkeys were observed in the submitted flocks than in randomly selected flocks. No significant difference was observed between the two turkey groups in market weight, feed conversion, or percentage of grade "A" turkeys.

Intracellular Location of Two Groundnut Rosette Umbravirus Proteins Delivered by PVX and TMV Vectors

The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed inNicotiana benthamianaas fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.

Overproduction of Campylobacter ferritin in Escherichia coli and induction of paracrystalline inclusion by ferrous compound.

The ferritin gene (cft) of Campylobacter jejuni was overexpressed in cells of Escherichia coli using a T7 RNA polymerase expression system. Many round particles which were the same size as the ferritin particles purified from C. jejuni were observed in the lysate of the cft-overexpressed E. coli cells. Since most of them were devoid of a central electron dense core consisting of ferric irons, the Campylobacter ferritins over-produced in E. coli seemed to be apoferritin. When large amounts of ferrous iron (supplied as FeSO4) were added to culture medium, the cft-overexpressed cells formed large inclusion bodies of paracrystalline arrays comprised of ferritin particles with central electron dense cores. The addition of ferric irons did not produce paracrystalline inclusion.

Characterization of Bacillus thuringiensis isolated from infections in burn wounds

Four strains of Bacillus thuringiensis were isolated from infections in burn wounds and from water used in the treatment of burn wounds. The strains produced large parasporal inclusion bodies composed of 141, 83, and 81 kDa protoxins. The four strains were tested for insecticidal activity against larvae of Pieris brassicae and Aedes aegypti but showed no activity; Vero cell assays for the production of enterotoxins were also negative. Attempts to classify the strains according to flagellar H-serotype showed them all to be non-flagellated. Apart from two occupational health accidents that occurred during the handling of highly concentrated B. thuringiensis fluids, this is the first report of B. thuringiensis causing non-gastrointestinal clinical infection in immunosuppressed patients.

Characterization of Bacillus thuringiensis isolated from infections in burn wounds.

Four strains of Bacillus thuringiensis were isolated from infections in burn wounds and from water used in the treatment of burn wounds. The strains produced large parasporal inclusion bodies composed of 141, 83, and 81 kDa protoxins. The four strains were tested for insecticidal activity against larvae of Pieris brassicae and Aedes aegypti but showed no activity; Vero cell assays for the production of enterotoxins were also negative. Attempts to classify the strains according to flagellar H-serotype showed them all to be non-flagellated. Apart from two occupational health accidents that occurred during the handling of highly concentrated B. thuringiensis fluids, this is the first report of B. thuringiensis causing non-gastrointestinal clinical infection in immunosuppressed patients.