Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Intracortical connectivity of layer VI pyramidal neurons in the somatosensory cortex of normal and barrelless mice.

In mice, barrels in layer IV of the somatosensory cortex correspond to the columnar representations of whisker follicles. In barrelless (BRL) mice, barrels are absent, but functionally, a columnar organization persists. Previously we characterized the aberrant geometry of thalamic projection of BRL mice using axonal reconstructions of individual neurons. Here we proceeded with the analysis of the intracortical projections from layer VI pyramidal neurons, to assess their contribution to the columnar organization. From series of tangential sections we reconstructed the axon collaterals of individual layer VI pyramidal neurons in the C2 barrel column that were labelled with biocytin [controls from normal (NOR) strain, 19 cells; BRL strain, nine cells]. Using six morphological parameters in a cluster analysis, we showed that layer VI neurons in NOR mice are distributed into four clusters distinguished by the radial and tangential extent of their intracortical projections. These clusters correlated with the cortical or subcortical projection of the main axon. In BRL mice, neurons were distributed within the same four clusters, but their projections to the granular and supragranular layers were significantly smaller and their tangential projection was less columnar than in NOR mice. However, in both strains the intracortical projections had a preference for the appropriate barrel column (C2), indicating that layer VI pyramidal cells could participate in the functional columnar organization of the barrel cortex. Correlative light and electron microscopy analyses provided morphometric data on the intracortical synaptic boutons and synapses of layer VI pyramidal neurons and revealed that projections to layer IV preferentially target excitatory dendritic spines and shafts.

Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Correlative microscopy.

In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array tomography. More and more efforts are put in either converting a fluorescence label into an electron dense product or preserving the fluorescence throughout preparation for the electron microscopy. Here, we will review successful protocols and where possible try to extract common features to better understand the importance of the individual steps in the preparation. Further the new instruments and software, intended to ease correlative light and electron microscopy, are discussed. Last but not least we will detail the approach we have chosen for correlative microscopy.

Ultrastructural observation of the airways of recovered and susceptible pigs after inoculation with Mycoplasma hyopneumoniae

To determine the morphological differences in the epithelium of the airways of recovered and susceptible pigs after Mycoplasma hyopneumoniae challenge, twenty-four 4-week-old M. hyopneumoniae-free pigs were intratracheally inoculated with 107ccu/ml of a pure low-passaged culture of the P5722-3 strain of M. hyopneumoniae challenge material. Eight pigs (group I) were challenged at the beginning of the experiment and rechallenged 3 months later. Group II pigs were also challenged at the beginning of the experiment and necropsied 3 months later. Group III pigs were challenged at the same time as the rechallenge of group I pigs. Eight nonchallenged pigs served as controls (group IV). Three days after the second challenge of group I and the first challenge of group III, and every 3 and 4 days thereafter, two pigs from each group were euthanatized by electrocution and necropsied. Samples of bronchi and lung tissue were examined using light and electron microscopy (SEM and TEM). Macroscopic lesions were observed in the lungs of all group III pigs (average = 4.74%) and were characterized by purple-red areas of discoloration and increased firmness affecting the cranioventral aspect of the lungs. Macroscopic lesions of pneumonia in groups I and II were minimal (less than 1%). There were no gross lesions of pneumonia in control (group IV) pigs. Microscopic lesions were characterized by hyperplasia of the peribronchial lymphoid tissue and mild neutrophilic infiltrates in alveoli. Electron microscopy showed patchy areas with loss of cilia and presence of leukocytes and mycoplasmas in bronchi of susceptible pigs (group III). The bronchial epithelium of rechallenged (group I), recovered (group II), and control (group IV) pigs was ultrastructurally similar indicating recovery of the former two groups. Although mycoplasmas were seen among cilia, a second challenge on pigs of group I did not produce another episode of the disease nor did it enhance morphological changes, suggesting that those pigs could become carriers of M. hyopneumoniae.

Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)

(Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)). Colleters are secretory structures formed by a parenchymatic axis with vascular bundles, bound by a layer of secretory palisade-like epidermis. Some studies regarding the structure of colleters have focused on secretory cells structure, but not distinguished the secretory and senescent phases. Generally, in mucilage-secreting cells such as colleters, the endoplasmic reticulum and Golgi apparatus are involved in secretion production and transport. In these study, colleters structure of Bathysa gymnocarpa K. Schum. and B. stipulata (Vell.) C. Presl. (Rubiaceae) were determined in two phases: a secretory phase and a senescence one. Samples were collected and processed by usual light and electron microscopy techniques. Studied colleters are constituted by an epidermal palisade layer and a central axis formed by parenchymatic cells with rare vascular traces. During the secretory phase, epidermal cells presented a dense cytoplasm, small vacuoles, enhanced rough and smooth endoplasmic reticulum, and a Golgi apparatus close to large vesicles. During the senescence phase epidermal cells presented a disorganized membrane system. No intact organelles or vesicles were observed. The outer cell wall exhibited similar layers to that observed during the secretory phase. The senescent phase is easily defined by the morphology of the colleters, but not well defined at subcellular level. Our research suggests that programmed cell death starts on secretory phase. However, more evidences are needed to evaluate the phenomena.

Techniques of radioautography for medical and biological research

Standard techniques for radioautography used in biological and medical research can be classified into three categories, i.e., macroscopic radioautography, light microscopic radioautography and electron microscopic radioautography. The routine techniques used in these three procedures are described. With regard to macroscopic radioautography, whole body radioautography is a standard technique which employs freezing and cryosectioning and can demonstrate organ distributions of both soluble and insoluble compounds. In contrast, in light and electron microscopic radioautography, soluble and insoluble techniques are separated. In order to demonstrate insoluble labeled compounds, conventional chemical fixations such as formalin for light microscopy or buffered glutaraldehyde and osmium tetroxide for both light and electron microscopy followed by dehydration, embedding and wet-mounting applications of radioautographic emulsions can be used. For the demonstration of soluble labeled compounds, however, cryotechniques such as cryofixation, cryosectioning, freeze-drying, freeze-substitution followed by dry-sectioning and dry-mounting radioautography should be employed both for light and electron microscopy. The outlines of these techniques, which should be utilized in various fields of biological and medical research, are described in detail

Morphometric and ultrastructural analysis of different pituitary cell populations in undernourished monkeys

Undernutrition elicited by a low-protein diet determines a marked reduction of hypophyseal activity and affects the function of the respective target organs. The objective of the present investigation was to study the ultrastructural and quantitative immunohistochemical changes of the different pituitary cell populations in undernourished monkeys that had been previously shown to have significant changes in craniofacial growth. Twenty Saimiri sciureus boliviensis monkeys of both sexes were used. The animals were born in captivity and were separated into two groups at one year of age, i.e., control and undernourished animals. The monkeys were fed ad libitum a 20% (control group) and a 10% (experimental group) protein diet for two years. Pituitaries were processed for light and electron microscopy. The former was immunolabeled with anti-GH, -PRL, -LH, -FSH, -ACTH, and -TSH sera. Volume density and cell density were measured using an image analyzer. Quantitative immunohistochemistry revealed a decrease in these parameters with regard to somatotrophs, lactotrophs, gonadotrophs and thyrotrophs from undernourished animals compared to control ones. In these populations, the ultrastructural study showed changes suggesting compensatory hyperfunction. On the contrary, no significant changes were found in the morphometric parameters or the ultrastructure of the corticotroph population. We conclude that in undernourished monkeys the somatotroph, lactotroph, gonadotroph, and thyrotroph cell populations showed quantitative immunohistochemical changes that can be correlated with ultrastructural findings.

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.

Experimental chronic entrapment of the sciatic nerve in adult hamsters: an ultrastructural and morphometric study

Entrapment neuropathy is a group of clinical disorders involving compression of a peripheral nerve and interference with nerve function mostly through traction injury. We have investigated the chronic compression of peripheral nerves as an experimental procedure for detecting changes in ultrastructural nerve morphology. Adult hamsters (Mesocricetus auratus, N = 30) were anesthetized with a 25% pentobarbital solution and received a cuff around the right sciatic nerve. Left sciatic nerves were not operated (control group). Animals survived for varying times (up to 15 weeks), after which they were sacrificed and both sciatic nerves were immediately fixed with a paraformaldehyde solution. Experimental nerves were divided into segments based upon their distance from the site of compression (proximal, entrapment and distal). Semithin and ultrathin sections were obtained and examined by light and electron microscopy. Ultrastructural changes were qualitatively described and data from semithin sections were morphometrically analyzed both in control and in compressed nerves. We observed endoneurial edema along with both perineurial and endoneurial thickening and also the existence of whorled cell-sparse structures (Renaut bodies) in the subperineurial space of compressed sciatic nerves. Morphometric analyses of myelinated axons at the compression sites displayed a remarkable increase in the number of small axons (up to 60%) in comparison with the control axonal number. The distal segment of compressed nerves presented a distinct decrease in axon number (up to 40%) comparatively to the control group. The present experimental model of nerve entrapment in adult hamsters was shown to promote consistent histopathologic alterations analogous to those found in chronic compressive neuropathies.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Systematics and floral evolution of the Dialiinae (Caesalpinioideae), a diverse lineage of tropical legumes

Le clade Dialiinae représente l’une des premières lignées de la sous-famille Caesalpinioideae des Leguminosae. Il se compose de 17 genres (environ 90 espèces), avec des taxons qui sont répandus dans toutes les régions tropicales du monde. Morphologiquement, le groupe comprend un assemblage divers de taxons qui peut représenter une «phase expérimentale» dans l’évolution florale des légumineuses. Différents représentants du clade présentent de la poly-, mono-, et asymétrie, et semblent avoir subi un haut degré de perte d’organe, produisant, dans certains cas, des fleurs extrêmement réduites qui sont à peine reconnaissables comme appartenant à la famille des légumineuses. Afin d’obtenir une image plus claire de l’évolution florale du clade Dialiinae, une phylogénie bien résolue et bien soutenue est nécessaire. Dans le but de créer une telle phylogénie, un total de 37 échantillons d’ADN des Dialiinae a été séquencé pour deux régions chloroplastiques, soit rps16 et trnL. De plus, une étude morphologique complète a été réalisée. Un total de 135 caractères végétatifs et reproductifs a été évalué pour 79 espèces de Dialiinae et pour quatre groupes externes. Les analyses phylogénétiques ont d’abord été effectuées sur un groupe restreint de taxons pour lesquels les trois types de données étaient disponibles. Les nœuds fortement soutenus de cette phylogénie ont ensuite été utilisés comme contrainte pour une seconde analyse de parcimonie avec les données morphologiques d’un ensemble plus important de taxons. Les caractères morphologiques ont été optimisés sur l’un des arbres les plus parcimonieux de cette seconde analyse. Un certain nombre de nouvelles relations au niveau de l’espèce ont été résolues, créant une image plus claire quant à l’évolution de la forme florale dans le temps, particulièrement pour les genres Labichea et Dialium. En plus de leur morphologie florale mature diverse, les Dialiinae sont également très variables dans leur ontogénèse florale, affichant à la fois la perte et la suppression des organes, et présentant une variété de modes d’initiation d’organes. Afin de construire une image plus complète du développement floral et de l’évolution dans ce clade, l’ontogénèse florale de plusieurs espèces non documentées à ce jour a été étudiée. La série complète du développement a été compilée pour six espèces de Dialiinae; quatre de Dialium, ainsi que Poeppigia procera et Mendoravia dumaziana. Le mode et le moment de l’initiation des organes étaient pour la plupart uniforme pour toutes les espèces de Dialium étudiés. Tant pour ce qui est des gains ou des pertes d’organes chez Dialium, une tendance est apparente – l’absence d’organe abaxial. Que ce soit pour les sépales ou les étamines, les gains se produisent toujours en position médiane adaxiale, tandis que les étamines et les pétales perdus sont toujours les organes les plus ventraux. Les taxons étudiés ici illustrent le manque apparent de canalisation du développement observé chez les Caesalpinioideae. Cette plasticité ontogénétique est le reflet de la diversité morphologique au niveau des fleurs tel qu’observée dans l’ensemble de la sous-famille. Une des espèces de Dialiinae, Apuleia leiocarpa, produit une inflorescence andromonoïque, une caractéristique qui est unique en son clade et rare dans les légumineuses dans son ensemble. La microscopie optique et électronique ont été utilisées pour entreprendre une étude détaillée de la morphologie florale de ce taxon. On a constaté que tandis que les fleurs hermaphrodites produisent un seul carpelle et deux étamines, les fleurs staminées produisent trois étamines sans toutefois montrer signe de développement du carpelle. Les inflorescences semblent produire près de quatre fois plus de fleurs staminées que de fleurs hermaphrodites, lesquelles occupent toujours la position centrale de l’inflorescence cymeuse. Ce ratio élevé mâle/bisexuel et la détermination précoce du sexe chez Apuleia sont rares chez les Caesalpinioideae, ce qui suggère que l’andromonoecie se développe dans ce genre comme un moyen d’accroître la dispersion du pollen plutôt qu’en réponse à des limitations de ressources.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history for Cosmos atrosanguineus

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Wearied plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls. (C) 2003 Annals of Botany Company.

Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history for cosmos atrosanguineus

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls.