Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 µm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

Development in Aspergillus.

The genus Aspergillus represents a diverse group of fungi that are among the most abundant fungi in the world. Germination of a spore can lead to a vegetative mycelium that colonizes a substrate. The hyphae within the mycelium are highly heterogeneous with respect to gene expression, growth, and secretion. Aspergilli can reproduce both asexually and sexually. To this end, conidiophores and ascocarps are produced that form conidia and ascospores, respectively. This review describes the molecular mechanisms underlying growth and development of Aspergillus.

Heterogeneity in liquid shaken cultures of Aspergillus niger inoculated with melanised conidia or conidia of pigmentation mutants.

Black pigmented conidia of Aspergillus niger give rise to micro-colonies when incubated in liquid shaken medium. These micro-colonies are heterogeneous with respect to gene expression and size. We here studied the biophysical properties of the conidia of a control strain and of strains in which the fwnA, olvA or brnA gene is inactivated. These strains form fawn-, olive-, and brown-coloured conidia, respectively. The ΔolvA strain produced larger conidia (3.8 µm) when compared to the other strains (3.2-3.3 µm). Moreover, the conidia of the ΔolvA strain were highly hydrophilic, whereas those of the other strains were hydrophobic. The zeta potential of the ΔolvA conidia in medium was also more negative when compared to the control strain. This was accompanied by the near absence of a rodlet layer of hydrophobins. Using the Complex Object Parametric Analyzer and Sorter it was shown that the ratio of individual hyphae and micro-colonies in liquid shaken cultures of the deletion strains was lower when compared to the control strain. The average size of the micro-colonies of the control strain was also smaller (628 µm) than that of the deletion strains (790-858 µm). The size distribution of the micro-colonies of the ΔfwnA strain was normally distributed, while that of the other strains could be explained by assuming a population of small and a population of large micro-colonies. In the last set of experiments it was shown that relative expression levels of gpdA, and AmyR and XlnR regulated genes correlate in individual hyphae at the periphery of micro-colonies. This indicates the existence of transcriptionally and translationally highly active and lowly active hyphae as was previously shown in macro-colonies. However, the existence of distinct populations of hyphae with high and low transcriptional and translational activity seems to be less robust when compared to macro-colonies grown on solid medium.

Geometrically nonlinear dynamic model for a hexagonal lattice.

It is shown that angular stiffness in the hexagonal lattice model plays a significant role in the geometrical nonlinear terms in the equations of the continuum limit. A geometrically nonlinear discrete model is formulated for the hexagonal lattice by considering the interaction of two sublattices. An asymptotic procedure is developed in order to obtain the nonlinear coupled equations of motion in the continuum limit of the discrete model. An interaction of longitudinal and shear plane strain waves is studied by using the solutions of the obtained equations.

[Intermittent vomiting in a patient with superior mesenteric artery syndrome]. / Intermitterend braken van een patiënte met het A.-mesenterica-superiorsyndroom.

A 51-year-old woman presented with nausea, vomiting and weight loss. The diagnosis of superior mesenteric artery syndrome was established by CT and upper-gastrointestinal contrast radiography. This revealed a characteristic dilatation of the first and second parts of the duodenum and an abrupt cutoff in the third part due to vascular compression. The obstruction disappeared when the patient was placed in the left lateral recumbent position. The thin habitus of this patient probably played an important role in the development of the syndrome. She was given dietary and positioning advice and within 4 months relief of symptoms was accompanied by a weight gain of 4 kg.

Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.

Involvement of microbodies in penicillin biosynthesis.

Penicillium chrysogenum strains were constructed which express a mutant acyltransferase lacking the putative targeting signal for microbody proteins. The mutated enzyme was located in vacuoles and in neighbouring cytoplasm. Although acyltransferase was expressed in vivo and was active in vitro, the mutants did not produce penicillin. The results demonstrate the involvement of microbodies in penicillin production.

Isolated mitral valve replacement with the Kay-Shiley disc. valve. Acturial analysis of the long term results.

During a five-year period the Kay-Shiley (K and T series) prosthesis was used for 83 isolated mitral valve replacements. There were 14 early deaths, for a 17.28 per cent mortality rate. Survival determined by the actuarial method revealed a 6 year cumulative survival rate of 39.8 per cent. Thromboembolism was a significant problem in this series, with 33 patients experiencing a total of 55 embolic events. This represented a rate of 24.7 emboli per 1,000 patient months at risk. From our experience, it is concluded that the Kay-Shiley prosthesis is associated with a high incidence of thromboembolism and late death.

Long-term prednisone followed by thymectomy in myasthenia gravis.

The present series of thirty patients has led us to certain conclusions concerning the management and treatment of patients with myasthenia gravis. The use of cholinesterase inhibitors alone is reserved for those patients with purely ocular myasthenia whose deficits can be satisfactorily corrected with those agents. Some of those with ocular involvement may be disabled; and in light of our excellent results with that small group, as well as similar findings presented by Fischer et al., patients with disabling or refractory ocular myasthenia should be considered for treatment with prednisone. All other patients with myasthenia are given a course of oral corticosteroids (prednisone) initially at high doses, with subsequent tapering to maintenance, alternate-day low-dose therapy. Cholinesterase inhibitors are used as needed while the patient is receiving corticosteroids. We now anticipate that patients will exhibit sustained improvement within the first two weeks, reaching maximal improvement at about three months. Exacerbations of myasthenic weakness may occur in the early phases of treatment. Such exacerbations have been commonly mild and occur with a mean onset at 5 days, and have a mean duration of 6 days. Most patients have been able to tolerate an alternate-day schedule of prednisone therapy when maintenance levels were achieved. The effective maintenance dose has been determined as the smallest dose of prednisone which allows the patient to maintain maximal improvement. Following the establishment of maximal improvement, patients have been considered for thymectomy. In our experience, the sternum-splitting procedure has been tolerated extremely well by patients exhibiting marked imporvement or remission while on corticosteroids. In those patients where thymectomy is contraindicated, irradiation of the thymus might be considered. Patients are continued on maintenance steroid therapy following surgery for a period of time that has been arbitrary. Currently, we consider an attempt to discontinue steroids at approximately one year reasonable. Should the patient relapse after discontinuation of the medication, oral corticosteroid treatment is reinitiated. Consideration is given to the possibility of recurrent thymus in patients who repeatedly fail to maintain a remission when steroids have been stopped. Our experience has not permitted us to draw firm conclusions concerning how long a time high-dose daily steroid treatment should be continued in patients who show no favorable response to that therapy. Other modes of treatment, such as courses of parenteral ACTH, methyl prednisolone, dexamethazone, or antimetabolites might be considered if there is no response after 12 weeks of high-dose, daily corticosteroid therapy.

Intercostal muscle biopsy studies in myasthenia gravis: clinical correlations and the direct effects of drugs and myasthenic serum.

We have found a wide range of mean MEPP amplitude in intercostal muscle biopsies from 43 patients with MG, including several values in the normal range. There was no correlation between MEPP amplitude and the severity of clinical disease as assessed by manual muscle testing or by single-fiber EMG measurements of jitter in arm muscles. Through most of these patients were in a state of clinical remission or marked improvement after treatment with prednisone, we could not attribute the difference between our results and those of others to this factor alone. The application of morphine, meperidine and aminoglycoside antibiotics to intercostal muscle in vitro confirms effects previously demonstrated in rat muscle: (1) At equal therapeutic concentrations, meperidine has greater neuromuscular blocking effects than does morphine, but neither has significant effects at concentrations achieved in the serum clinically. (2) Tobramycin, netilmicin and neomycin have varying severity and sites of action, but their effects are the same in human myasthenic muscle as in normal rat muscle. Bath application of serum from myasthenic patients produces an acute, reversible worsening of neuromuscular blockade in myasthenic muscle. Electrophysiologic measurements in intercostal biopsies from patients with MG can provide information about the basic abnormality of neuromuscular transmission in this disease and can confirm the relevance of studies made in animal muscle.

Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers.

For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, hence, labeling at electron microscope resolution does not add essential information. The scope of this paper is to compare five permeabilization methods for pre-embedding labelling for electron microscopy. We aim for a method that is easy to use and suitable for routine investigations. For our ongoing work, special attention is given to labeling of the cell nucleus. Accessibility of cytoplasmic and nuclear antigens is monitored with a set of different marker antibodies. From this investigation, we suggest that prefixation with formaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure before using a detergent (Triton X-100 or Brij 58) to permeabilize or remove the membranes. The experimental conditions for labeling should be checked first with fluorescence or fluorescence-gold markers by fluorescence microscopy. Then either ultrasmall gold particles (with or without fluorochrome) with silver enhancement or, if the ultrasmall gold particles are obstructed, peroxidase markers are advised. The most promising technique to localize scarce antigens with good contrast is the combination of a pre-embedding peroxidase/tyramide-FITC or -biotin labeling followed by an on-section colloidal gold detection.

Observations on the pathogenesis and management of pulmonary hypertension.

Experimental subclavian-pulmonary and aorta-pulmonary anastomoses were performed in attempts to produce progressively increasing pulmonary vascular changes and to observe their sequential development. At six weeks after anastomosis there was medial hypertrophy, at eight weeks total involvement of the artery, and at eleven weeks severe pulmonary vascular disease, total decrease in the number of capillaries, and complete obstruction of the lumen. These pathologic changes are compared with the normal course of events in the fetal and postnatal lung. A series of patients with patent ductus arteriosus, single ventricle, and a large ventricular septal defect were studied and categorized into three phases according to the severity of their clinical symptoms. A method for producing pulmonary artery stenosis in the laboratory as applied to a three month old infant is discussed.

The influence of photoperiod on drought induction of dormancy in Lotus scoparius.

Many species of plants in Mediterranean climate regions have evolved deciduousness, causing reduced leaf area during the long summer drought characteristic of Mediterranean climates. This summer deciduous growth form has been considered a plant adaptation in Mediterranean regions allowing survival during periods of extreme water stress. Many studies have suggested the ecological importance of this growth form but few studies have examined the physiological stimulus for deciduousness.Previous data indicate that abscission in Lotus scoparius (a mediterranean California deciduous species) is influenced by both photoperiod and water stress in a complex manner. Here the physiological basis of long day enhanced leaf fall during water stress is investigated.Examination of water potential components indicate an osmotic adjustment with incresing water stress which enables the maintenance of turgor at lower water potentials. Osmotic adjustment in plants grown under long photoperiods was greater than that in short photoperiods. Therfore, long day enhanced abscission during water stress was not due to a greater susceptability to turgor loss during long days. Rather, long day treatment caused these plants to initiate dormancy (as indicated by soluble protein concentrations) during the onset of water stress. The dormant condition could not be released by subsequent release from water stress. Apparently, Lotus scoparius has evolved a photoperiodic control (presumably through growth regulators) over the initiation of dormancy during water stress. The adaptive significance of this photoperiodic control over the leaf abscission response to water stress relates to the variable climate of Mediterranean regions.

Sarcoidosis presenting with severe thrombocytopenia and arthritis.

In this case report a patient with biopsy-proven sarcoidosis is described, presenting with joint symptoms and severe thrombocytopenia, due to immune-mediated platelet destruction.

Field-emission scanning electron microscopy of the internal cellular organization of fungi.

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.