Quantitative Electron Microscopic Study Research Articles

Accumulation of Globotriaosylceramide in Podocytes in Fabry Nephropathy Is Associated with Progressive Podocyte Loss.

In males with classic Fabry disease, the processes leading to the frequent outcome of ESKD are poorly understood. Defects in the gene encoding α-galactosidase A lead to accumulation of globotriaosylceramide (GL3) in various cell types. In the glomerular podocytes, accumulation of GL3 progresses with age. Of concern, podocytes are relatively resistant to enzyme replacement therapy and are poorly replicating, with little ability to compensate for cell loss. In this study of 55 males (mean age 27 years) with classic Fabry disease genotype and/or phenotype, we performed unbiased quantitative morphometric electron microscopic studies of biopsied kidney samples from patients and seven living transplant donors (to serve as controls). We extracted clinical information from medical records and clinical trial databases. Podocyte GL3 volume fraction (proportion of podocyte cytoplasm occupied by GL3) increased with age up to about age 27, suggesting that increasing podocyte GL3 volume fraction beyond a threshold may compromise survival of these cells. GL3 accumulation was associated with podocyte injury and loss, as evidenced by increased foot process width (a generally accepted structural marker of podocyte stress and injury) and with decreased podocyte number density per glomerular volume. Worsening podocyte structural parameters (increasing podocyte GL3 volume fraction and foot process width) was also associated with increasing urinary protein excretion-a strong prognosticator of adverse renal outcomes in Fabry disease-as well as with decreasing GFR. Given the known association between podocyte loss and irreversible FSGS and global glomerulosclerosis, this study points to an important role for podocyte injury and loss in the progression of Fabry nephropathy and indicates a need for therapeutic intervention before critical podocyte loss occurs.

Scopoletin intervention in pancreatic endoplasmic reticulum stress induced by lipotoxicity.

Endoplasmic reticulum (ER), a dynamic organelle, plays an essential role in organizing the signaling pathways involved in cellular adaptation, resilience, and survival. Impairment in the functions of ER occurs in a variety of nutritive disorders including obesity and type 2 diabetes. Here, we hypothesize that (scopoletin) SPL, a coumarin, has the potential to alleviate ER stress induced in vitro and in vivo models by lipotoxicity. To test this hypothesis, the ability of SPL to restore the levels of proteins of ER stress was analyzed. Rat insulinoma 5f (RIN5f) cells and Sprague Dawley rats were the models used for this study. Groups of control and high-fat, high-fructose diet (HFFD)-fed rats were treated with either SPL or 4-phenylbutyric acid. Status of ER stress was enumerated by quantitative RT-PCR, Western blot, electron microscopic, and immunohistochemical studies. Proximal proteins of ER stress inositol requiring enzyme 1 (IRE1), protein kinase like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) were reduced in the β-cells by SPL. The subsequent signaling proteins X-box binding protein 1, eukaryotic initiation factor2α, activating transcription factor 4, and C/EBP homologous protein were also suppressed in their expression levels when treated with SPL. IRE1, PERK signaling leads to c-Jun-N-terminal kinases phosphorylation, a kinase that interrupts insulin signaling, which was also reverted upon scopoletin treatment. Finally, we confirm that SPL has the ability to suppress the stress proteins and limit pancreatic ER stress which might help in delaying the progression of insulin resistance.

The summer distribution of coccolithophores and its relationship to water masses in the East China Sea

Coccolithophores are calcifying phytoplankton belonging to the phylum Haptophyta, and are thought to play an important role in the marine carbon cycle through the production and export of organic carbon and calcite. A quantitative scanning electron microscope study of coccolithophores was conducted to survey the spatial distribution of coccolithophore communities in surface water covering the continental shelf of the East China Sea (ECS) during the summer of 2014. The abundance of haptophytes was also revealed by fluorescence in situ hybridization with an 18S rRNA-specific probe. The abundance of coccolithophores in the surface-water ranged from 3.7 to 82.8 cells mL−1, contributing a mean 7.0 % to haptophyte abundance. Four distinct coccolithophore groups (A, B, C, and D) were categorized based on similarities of the coccolithophore communities among the stations. Group A was located in the north of the ECS in the Yellow Sea Mixed Water area, where coccolithophore abundance was low. Group B was dominated by Gephyrocapsa oceanica and was located in the south of the ECS in the Taiwan Warm Current. Group C represented the inner shelf assemblages, with Emiliania huxleyi as the major dominant species, and was influenced by the Changjiang discharges. Group D represented the Kuroshio assemblages, with communities dominated by E. huxleyi and Umbellosphaera tenuis in the outer shelf region. These results suggest that coccolithophores tend to migrate with the water masses in the ECS during the summer.

The Effect of Size and Taper of Apical Preparation in Reducing Intra-Canal Bacteria: A Quantitative SEM Study.

Bacteria and their byproducts are major etiologic factors in endodontic diseases. Prevention or reduction of root canal bacterial contamination is the main aim of endodontic treatment. The purpose of this in vitro study was to evaluate the effect of size and taper of master apical file (MAF) in reducing bacteria from the apical third of the curved canals using a quantitative scanning electron microscope (SEM) study. Eighty-nine human mandibular first molars with curved MB canals (20(º)-35(º)) were divided into one control group (n=5) (without rotary instrumentation) and 6 experimental groups (n=14). The canals were prepared using RaCe rotary files to the MAF sizes 25/0.04, 25/0.06, 30/0.04, 30/0.06, 35/0.04 and 35/0.06, in groups 1 to 6, respectively. All the experimental groups were finally rinsed with 2 mL of 17% EDTA followed by 3 mL of 5.25% NaOCl. The mesial roots were split longitudinally. Remaining bacteria in the apical third of MB canals were evaluated using SEM (2000×). Data analysis was performed using one way ANOVA with Tukey's post hoc test. The level of significance was set at 0.05. All the experimental groups showed significant bacterial reduction (P<0.001). Although the greater size and/or taper resulted in decrease in bacteria, differences between the groups were not significant. Based on this in vitro study the MAF #25/0.04 had no significant difference compared to other groups with greater apical size/taper; all groups could effectively reduce intra-canal bacteria.

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.

Synaptic Reorganization in the Adult Rat's Ventral Cochlear Nucleus following Its Total Sensory Deafferentation

Ablation of a cochlea causes total sensory deafferentation of the cochlear nucleus in the brainstem, providing a model to investigate nervous degeneration and formation of new synaptic contacts in the adult brain. In a quantitative electron microscopical study on the plasticity of the central auditory system of the Wistar rat, we first determined what fraction of the total number of synaptic contact zones (SCZs) in the anteroventral cochlear nucleus (AVCN) is attributable to primary sensory innervation and how many synapses remain after total unilateral cochlear ablation. Second, we attempted to identify the potential for a deafferentation-dependent synaptogenesis. SCZs were ultrastructurally identified before and after deafferentation in tissue treated for ethanolic phosphotungstic acid (EPTA) staining. This was combined with pre-embedding immunocytochemistry for gephyrin identifying inhibitory SCZs, the growth-associated protein GAP-43, glutamate, and choline acetyltransferase. A stereological analysis of EPTA stained sections revealed 1.11±0.09 (S.E.M.)×109 SCZs per mm3 of AVCN tissue. Within 7 days of deafferentation, this number was down by 46%. Excitatory and inhibitory synapses were differentially affected on the side of deafferentation. Excitatory synapses were quickly reduced and then began to increase in number again, necessarily being complemented from sources other than cochlear neurons, while inhibitory synapses were reduced more slowly and continuously. The result was a transient rise of the relative fraction of inhibitory synapses with a decline below original levels thereafter. Synaptogenesis was inferred by the emergence of morphologically immature SCZs that were consistently associated with GAP-43 immunoreactivity. SCZs of this type were estimated to make up a fraction of close to 30% of the total synaptic population present by ten weeks after sensory deafferentation. In conclusion, there appears to be a substantial potential for network reorganization and synaptogenesis in the auditory brainstem after loss of hearing, even in the adult brain.

Loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress

Parkinson's disease (PD) is a common neurodegenerative disorder thought to be associated with mitochondrial dysfunction. Loss of function mutations in the putative mitochondrial protein PINK1 (PTEN-induced kinase 1) have been linked to familial forms of PD, but the relation of PINK1 to mammalian mitochondrial function remains unclear. Here, we report that germline deletion of the PINK1 gene in mice significantly impairs mitochondrial functions. Quantitative electron microscopic studies of the striatum in PINK1(-/-) mice at 3-4 and 24 months revealed no gross changes in the ultrastructure or the total number of mitochondria, although the number of larger mitochondria is selectively increased. Functional assays showed impaired mitochondrial respiration in the striatum but not in the cerebral cortex at 3-4 months of age, suggesting specificity of this defect for dopaminergic circuitry. Aconitase activity associated with the Krebs cycle is also reduced in the striatum of PINK1(-/-) mice. Interestingly, mitochondrial respiration activities in the cerebral cortex are decreased in PINK1(-/-) mice at 2 years compared with control mice, indicating that aging can exacerbate mitochondrial dysfunction in these mice. Furthermore, mitochondrial respiration defects can be induced in the cerebral cortex of PINK1(-/-) mice by cellular stress, such as exposure to H(2)O(2) or mild heat shock. Together, our findings demonstrate that mammalian PINK1 is important for mitochondrial function and provides critical protection against both intrinsic and environmental stress, suggesting a pathogenic mechanism by which loss of PINK1 may lead to nigrostriatal degeneration in PD.

A qualitative and quantitative electron microscopic study of differences in adrenomedullary adrenaline cells between golden hamsters and rats, with special reference to the Golgi apparatus

Ultrastructural features of adrenomedullary adrenaline (A) cells in golden hamsters and rats were investigated qualitatively and quantitatively with special reference to the Golgi apparatus. The A cells displayed a characteristic follicular arrangement, with each cell showing structural polarity in hamsters, but not apparently in rats. In hamsters, the Golgi apparatus of A cells was larger (t-test: P<0.001) and more frequently showed large and complexly organized structures (chi(2)-test: P<0.005) compared with that of rats. Quantitative analysis of the Golgi apparatus revealed differences in the size and numerical density of Golgi vesicles in relation to the animal species and region. Two-way analysis of variance (ANOVA) confirmed species difference in the size of coated vesicles (P<0.005) and interaction between species and region concerning the size of smooth-clear vesicles (P<0.01) and numerical density of granular vesicles (P<0.05). One-way ANOVA revealed regional differences in the size and numerical density of smooth-clear vesicles in rats and hamsters (P<0.01 approximately 0.001), and in the numerical density of coated vesicles in hamsters (P<0.05). Data were further analyzed by Tukey-Kramer's method. These and other reported results suggest that, in hamster A cells, the Golgi apparatus has different structural, molecular, and functional mechanisms which are at least partly related to the distinct cellular polarity and higher concentration of peptide hormones in secretory vesicles, and that in rat A cells, in contrast, loading secretory vesicles with A during the post-Golgi stage is predominant. In conclusion, the Golgi apparatus in hamster A cells shows markedly different ultrastructural features compared with that in rat A cells.

Postnatal development of the cholinergic innervation in the dorsal hippocampus of rat: Quantitative light and electron microscopic immunocytochemical study

Choline acetyltransferase (ChAT) immunocytochemistry was used to examine the distribution and ultrastructural features of the acetylcholine (ACh) innervation in the dorsal hippocampus of postnatal rat. The length of ChAT-immunostained axons was measured and the number of ChAT-immunostained varicosities counted, in each layer of CA1, CA3, and dentate gyrus, at postnatal ages P8, P16, and P32. At P8, an elaborate network of varicose ChAT-immunostained axons was already visible. At P16, the laminar distribution of this network resembled that in the adult, but adult densities were reached only by P32. Between P8 and P32, the mean densities for the three regions increased from 8.4 to 14 meters of axons and 2.3 to 5.7 million varicosities per cubic millimeter of tissue. At the three postnatal ages, the ultrastructural features of ChAT-immunostained axon varicosities from the strata pyramidale and radiatum of CA1 were similar between layers and comparable to those in adult, except for an increasing frequency of mitochondria (up to 41% at P32). The proportion of these profiles displaying a synaptic junction was equally low at all ages, indicating an average synaptic incidence of 7% for whole varicosities, as previously found in adult. The observed junctions were small, usually symmetrical, and made mostly with dendritic branches. These results demonstrate the precocious and rapid maturation of the hippocampal cholinergic innervation and reveal its largely asynaptic nature as soon as it is formed. They emphasize the remarkable growth capacities of individual ACh neurons and substantiate a role for diffuse transmission by ACh during hippocampal development.

血管性痴呆―アルツハイマー病との接点

Cerebrovascular disease and Alzheimer's disease (AD) are the most frequent causes of dementia in the elderly. Pathological backgrounds for vascular dementia (VaD) are heterogenous. Cerebral white matter is mostly involved in dementia due to small-vessel disease and Binswanger's diease. In Binswanger's disease, a quantitative electron microscopic study showed the reduction of nerve fibers, oligodendrocytes and astrocytes in the white matter. These changes are associated mainly with lipohyalinosis (angionecrosis) of small intraparechymal arteries as well as atherosclerosis of main cerebral arteries. The changes in arterioles are believed to cause blood-brain barrier impairments, activation of microglia, and increased production of cytokines and free radicals, all of which may lead to white matter damage. Clinical, pathological and neuroimaging features are different between typical cases with VaD and AD. However, recent studies have reported that VaD has also characteristic features of AD, including hippocampal atrophy, the reduction in acetylcholine concentration in the bain, and possible beneficial effect of acetylcholine esterase inhibitors. Like VaD, AD also shows carebral white matter changes and changes in cerebral blood flow and oxygen metaboloism. Epidemiological studies have reported that almost all risk factors for cerebrovascular disease increased the risk of AD. Further clinicopathological investigation will enhance our understanding of the relation of VaD to AD and our capability to prevent and treat VaD and AD.

Summer and winter distribution and malformation of coccolithophores in the East China Sea

A quantitative scanning electron microscope study of coccolithophores was sampled during the summer and winter from the surface waters over the continental shelf of the East China Sea (ECS) was conducted. In all, only ten coccolithophorid taxa were observed during the investigation periods. Emiliania huxleyi (Lohmann) Hay et Mohler and Gephyrocapsa oceanica Kamptner were predominant among the coccolithophorid flora during both seasons. E. huxleyi increased its relative abundance seaward, while G. oceanica concentrated in inner shelf area. These two species accounted for over half of the coccolithophores in the middle shelf of the ECS. These dominant taxa are eurythermal, occurring in patchy and “bull‘s-eyes” distributions in both the summer and winter, respectively. Special emphasis has been paid to the overall distribution and intra-species frequency of malformed cells of the two dominant species, as well as other minor species. Malformed cells occurred so frequently in both seasons that the frequency amounted to as much as 80% in both dominant species. The malformation was manifested largely by corroded defects. A comparison of the malformed distribution with the hydrological and nutrient parameters suggests that the malformation was caused mainly by a deficiency of nitrate in the ambient waters.

Synaptic loss following depletion of noradrenaline and/or serotonin in the rat visual cortex: a quantitative electron microscopic study

Biogenic amines have a trophic-like role for the formation and the maintenance of synapses in the CNS. We examined the changes in the number of synaptic profiles in the developing and adult rat visual cortex following selective depletion of noradrenaline and/or serotonin. By the drug-induced decreases in levels of noradrenaline or serotonin between 1 and 2 weeks after birth, the number of synaptic profiles was decreased by 29–55% compared with that of control animals. The magnitude of reduction in the number of synaptic profiles was virtually the same following simultaneous depletion of both noradrenaline and serotonin compared with the depletion of noradrenaline or serotonin alone. Later in the developmental period, the function of noradrenaline and serotonin in facilitating synapse formation and maintenance became less prominent than that in younger animals. In the control animals, the number of axosomatic synapses was the highest at around 2 weeks after birth, and decreased with development. The number of axodendritic synapses was the highest between 2 and 7 weeks after birth, and decreased to 50% at 11 weeks after birth. These data demonstrate that synapses in the rat visual cortex are overproduced during the early developmental period. We suggest that both serotonin and noradrenaline are necessary for synapse formation during the early stages of development of the rat visual cortex.

Quantitative Transmission Electron Microscopic Study of Dental Plaque - An In Vivo Study with Different Mouthrinses

Only a few studies of dental plaque in transmission electron microscopy (TEM) have been performed. The authors have applied TEM for morphometric analysis of dental plaque. Wearing acrylic splints with bovine enamel pieces on the buccal surface of teeth 36 and 46, 21 volunteers rinsed twice a day with a solution of chlorhexidine digluconate, amine/stannous fluoride, or sodium chloride. After 24, 48, and 72 h each a piece of enamel was taken and quantitatively analyzed by TEM for plaque thickness and composition. Similar results were obtained for plaque thickness after rinsing with chlorhexidine digluconate or amine/stannous fluoride. Numerical differences at the counts of cocci or bacilli after rinsing with chlorhexidine digluconate or amine/stannous fluoride did not always have statistical significance.

Specificity of cortical synaptic connectivity: emphasis on perspectives gained from quantitative electron microscopy.

This report traces the historical development of concepts regarding the specificity of synaptic connectivity in the cerebral cortex as viewed primarily from the perspective of electron microscopy. The occurrence of stereotypical patterns of connection (e.g., contrasting synaptic patterns on the surfaces of spiny vs. non-spiny neurons, the general consistency with which axonal pathways impinge on and originate within specific cortical areas and layers, triadic synaptic relationships) implies that cortical connectivity is highly structured. The high degree of order characterizing many aspects of cortical organization is mirrored by an equally ordered arrangement of synaptic connections between specific types of neurons. This observation is based on quantitative electron microscopic studies of synapses between identified neurons and from the results of correlative anatomical/electrophysiological investigations. The recognition of recurring synaptic patterns and responses between specific neurons has generated increased support for the notion of specificity of synaptic connections at the expense of randomness, but the role of specificity in cortical function is an unresolved question. At the core of cortical processing lie myriad possibilities for computation provided by the wealth of synaptic connections involving each cortical neuron. Specificity, by limiting possibilities for connection, can impose an order on synaptic interactions even as processes of dynamic selection or synaptic remodeling ensure the constant formation and dissolution of cortical circuits. These operations make maximal use of the richness of cortical synaptic connections to produce a highly flexible system, irrespective of the degree of randomness or specificity that obtains for cortical wiring at any particular time.

Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats: a quantitative electron microscopic immunogold study

Transcellular transport of energy substrates across the vascular endothelial cells of the brain is accomplished by integral membrane carrier proteins, such as the glucose transporter GLUT1 and the monocarboxylic acid transporter MCT1. The abundance of these proteins may vary depending on age and nutritional status. In this study we compared the expression of MCT1 in cerebral cortex of suckling and adult rats to determine whether the former, which use considerably more monocarboxylates such as lactate and ketone bodies as fuel than do older rats, correspondingly express more MCT1 than adults. Using electron microscopic immunogold methods, we found that 17-day old suckling rat pups had 25 times more MCT1 labeling in the membranes of capillary endothelial cells than adults. This transporter was nearly equally distributed in luminal and abluminal membranes with less than 10% of the immunogold particles in the endothelial cytoplasmic compartment. The suckling rats also had 15 times more immunogold particles associated with pericyte membranes and 19 times heavier labeling of membranes associated with astrocytic end feet adjacent to microvessels. Neuropil and choroid plexus were lightly labeled. Some MCT1-positive astrocyte and neuron cell bodies were observed, suggesting active synthesis of MCT1 by these cells. The potential for regulation of expression of MCTs by dietary or other factors may have important consequences for the progression and treatment of cerebrovascular disorders and other diseases.

The small-cell variant of mycosis fungoides. A clinicopathological and quantitative electron microscopic study on 14 patients.

Small-cell variants of Sézary syndrome and mycosis fungoides (MF) have been described. However, in these studies the nuclear area of the small-cell variant of MF (SC-MF) as compared to histological classical MF (CL-MF) was not characterized objectively by quantitative electron microscopy. In a 14-year follow-up period, of a total of 76 patch/plaque stage MF patients seen in the Department of Dermatology of the University Hospital Utrecht, 14 (18%) had an infiltrate composed of atypical lymphocytes characterized by a distinctly smaller cell diameter and smaller, hyperchromatic, deeply indented nuclei as compared to the usual cell type of MF. The aim of the investigation was to confirm this observation objectively using quantitative electron microscopy (morphometry) and to define SC-MF as compared to CL-MF. The study was performed on the 14 patients with SC-MF, and 10 patients with clinical and histological CL-MF and 4 patients with chronic eczema. Electron micrographs of sections obtained from each biopsy were analysed by computer to produce the following data: a nuclear contour index (NCI), the mean nuclear area (MNA), the mean nuclear area of the cells above the 75th percentile (P75NA) and the percentage of cells larger than 30 microm2. The values of MNA differed significantly between patients with SC-MF and those with CL-MF (17.6 vs 23.2 microm2; P = 0.02), as did the values of P75NA (20.7 vs 27.9 microm2; P = 0.01). The NCI of the SC-MF and CL-MF patients were similar. These results are consistent with our observations that SC-MF does indeed exist.

Isolation and Characterization of Rat Liver Amphisomes: EVIDENCE FOR FUSION OF AUTOPHAGOSOMES WITH BOTH EARLY AND LATE ENDOSOMES

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.

The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages.

Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP-100/Clp family. A ClpC-deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro. Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L. monocytogenes, during the infectious process. A quantitative electron microscope study showed that, contrary to wild-type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane-bound phagosomes. Only a few mutant bacteria disrupted the phagosome membrane after 4h of incubation, then polymerized actin filaments and multiplied within the cytoplasm. The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival. The ClpC ATPase was produced in vivo during experimental infection by wild-type bacteria. The virulence of the ClpC-deficient mutant was severely attenuated in mice, with a three-log decrease in its 50% lethal dose compared with wild-type bacteria. Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues. Our results provide evidence that a general stress protein is required for the virulence of L. monocytogenes, which behaves as a virulence factor promoting intracellular survival of this pathogen.

The temporal progression of the myelination defect in the taiep rat.

The Sprague Dawley myelin mutant, the taiep rat, demonstrates a defect in CNS myelination which worsens with age and which is associated with abnormal accumulations of microtubules in oligodendrocytes. Quantitative and qualitative electron microscopic studies of myelin development and oligodendrocyte morphology were used to describe the temporal development of the defect in this mutant, in three regions of the CNS. The results indicate that the time of onset of myelination is similar in mutant and control rats, however the amount of myelin formed is reduced in the mutant, compared to controls, and there is a loss of myelin from the taiep CNS as the animals age. Thus the myelination defect in taiep has features of both hypomyelination and demyelination. Oligodendrocyte microtubule abnormalities were noted in each region of the taiep CNS at the time of onset of myelination. The earliest changes seen were close associations of oligodendrocyte microtubules with endoplasmic reticulum, with marked accumulations of microtubules filling the cytoplasm of oligodendrocytes from older taiep rats. These findings suggest that the microtubule abnormality in the taiep mutant inhibits both the initial formation and the long-term maintenance of myelin by the oligodendrocyte. In addition, there is also evidence to suggest that although the microtubule abnormality is present in oligodendrocytes throughout the taiep CNS, it results in a more marked defect in the myelination of axons of small diameter.

Effects of ischaemia and preservation on the ultrastructure of the bronchiolar epithelium. A quantitative electron microscopic study of human and canine lungs.

In ten cases of clinical human single-lung transplantation, the nontransplanted Euro-Collins-preserved contralateral lungs were examined using electron microscopy to determine the effects of ischaemia on the bronchiolar epithelium. Existing structural damage at the time of transplantation was characterized using this approach, and nine nonpreserved canine single lungs were also investigated to identify the impact of ischaemia. The study revealed a significant correlation between the duration of ischaemia and the mitochondrial surface-to-volume ratio, which can serve as a morphometric criterion for mitochondrial damage, in canine lungs. However, this correlation was not found in the human donor lungs. Further examination of human donor lungs showed slight to moderate damage to the endoplasmic reticulum and nuclear chromatin. In addition, various degrees of damage to mitochondrial structure, ranging from inconspicuous to severe, were found. The mitochondrial surface-to-volume ratio can be considered to be a suitable criterion for the quantification of ischaemic damage of the bronchiolar epithelium under experimental conditions. Ultrastructural analysis of human donor lungs revealed intact bronchiolar epithelial cell structures at the time of transplantation, reflecting adequate organ preservation with Euro-Collins solution.