Physics of the Preparation and Observation of Specimens that Involve Cryoprocedures

With this introductory chapter we attempt a synthesis of old and new knowledge of the physical principles that govern cryomethods. Interface phenomena determine the increase or decrease of the number of particles observed in frozen-hydrated suspensions because they occupy the air-liquid interface according to their specific balance of hydrophobic versus hydrophilic properties. Biological macromolecules are surrounded by organised water, the hydration shell, that prevents them from sticking to each other. Partial or complete removal of these hydration shells by freeze-drying or freeze-substitution leads to collapses or aggregations. The solvent-induced aggregation is usually decreased by prior cross-linking with adequate chemical fixatives. A new finding is that aggregations are also decreased with lower temperatures. This al lows us, for example, to preserve DNA-containing plasma from coarse aggregation even in cases where it has not been previously crosslinked. When rapidly freezing a physiologically homogeneous population of bacterial cells without added cryoprotectants we find a 10-20 μm thick layer of cells without ice crystal formation. In deeper layers an increasing proportion of cells exhibits crystallization damage, although some cells are still well preserved. Treatment of cells with aqueous solutions of OsO4 and/or uranyl acetate leads to 10-20 % (w/w) heavy metal deposit. Direct staining of sections of resin embedded material results in 10 times more deposit. The location of these deposited metals is at first unknown and is best visualized through a comparison with totally unstained material. Sufficient contrast is achieved with the dark field, or the ratio-contrast mode of imaging. The latter has the advantage of giving less weight to thickness variations than does the former. When observing fully unstained thin sections by ratio contrast the influence of the surface reliefs is thereby virtually eliminated. With CTEM the dark field mode requires too great a dose to use it successfully on frozen-hydrated material. With STEM this is possible for both dark field and ratio contrast

Low Temperature Embedding

The Lowicryl resins K4M and HM20 are methacrylate/acrylate based formulations which can re used for embedding biological material at low temperature in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. The resins are applicable over a very extended temperature range, approximately 210°K to 340°K. Even lower temperatures down to ca. 190°K can be reached with two new resins, K11M and HM23. Test embeddings of unfixed material after freeze-substitution have given promising results which could re useful for imnunocytochemical labeling. Lipid extraction is small or absent when the two new resins are used in combination with freeze-substitution

A 12-year-old girl with absent radial pulse: arterial thoracic outlet syndrome with subclavian artery aneurysm and thrombosis of the brachial artery

Brachial arterial occlusion is rare in children and adolescents. Once a traumatic cause is excluded, the differential diagnosis consists of a variety of rare conditions. We report the case of a 12-year-old girl whose presenting symptoms—an absent radial pulse and Raynaud's phenomenon of the right hand—could be easily mistaken for a vasculitis. She was found to have arterial thoracic outlet syndrome with right subclavian artery compression and aneurysm formation caused by an anomalous first rib and consecutive thromboembolic occlusion of the brachial artery. The diagnosis and differential diagnosis of this condition are reviewe

The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC): IUPHAR Review 19.

Acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC) are both members of the ENaC/degenerin family of amiloride-sensitive Na(+) channels. ASICs act as proton sensors in the nervous system where they contribute, besides other roles, to fear behaviour, learning and pain sensation. ENaC mediates Na(+) reabsorption across epithelia of the distal kidney and colon and of the airways. ENaC is a clinically used drug target in the context of hypertension and cystic fibrosis, while ASIC is an interesting potential target. Following a brief introduction, here we will review selected aspects of ASIC and ENaC function. We discuss the origin and nature of pH changes in the brain and the involvement of ASICs in synaptic signalling. We expose how in the peripheral nervous system, ASICs cover together with other ion channels a wide pH range as proton sensors. We introduce the mechanisms of aldosterone-dependent ENaC regulation and the evidence for an aldosterone-independent control of ENaC activity, such as regulation by dietary K(+) . We then provide an overview of the regulation of ENaC by proteases, a topic of increasing interest over the past few years. In spite of the profound differences in the physiological and pathological roles of ASICs and ENaC, these channels share many basic functional and structural properties. It is likely that further research will identify physiological contexts in which ASICs and ENaC have similar or overlapping roles

The possibilities and prospects of obtaining high-resolution information (below 30 Å) on biological material using the electron microscope: Some comments and reports inspired by an EMBO workshop held at Gais, Switzerland, October 1973

Commercially available electron microscopes routinely provide resolution of some 2-4 Å, as determined on the spacing of crystalline lattices of certain stable, small-molecular substances. On biological material either macromolecules or macromolecular assemblies— ‘biologically significant' details below some 20 Å have hitherto not been observed.we consider as ‘biologically significant' those structural details observed or contained in electronmicrographs which are consistent with, or confirmed by, other data obtained from biochemical or functional experiments or by other physical methods (optical, magnetic, electric

Incidental findings of mass lesions on neuroimages in children

Increasing use of neuroimaging in children has led to more incidental findings of CNS mass lesions, the management of which is uncertain. The authors' aims in this study are to describe these mass lesions and their evolution, as well as to discuss the management options and determine the prevalence of incidental CNS mass lesions at their pediatric clinic. A retrospective study was undertaken in children with primary CNS tumors who were younger than 18 years old and were admitted to the University Children's Hospital of Zurich, Switzerland, between January 1995 and December 2010. In 19 (5.7%) of 335 patients with newly diagnosed CNS tumors, the diagnosis of a CNS mass lesion was an incidental finding. Reasons for obtaining neuroimages in these 19 patients were head trauma (in 6 patients); research protocols (in 3); nasal/orbital malformations (in 2); endocrinological and psychiatric evaluations (in 2); and vertebral bone anomaly without neurological signs, absence seizures, congenital ataxia, recurrent vomiting, developmental delay, and "check-up" at the explicit request of the parents (in 1 patient each). Seven patients underwent immediate surgery for low-grade glioma (4 patients) and craniopharyngioma, ependymoma, and choroid plexus papilloma (1 patient each); and 12 were treated conservatively or were observed. Ten of 12 conservatively treated patients remained stable (median follow-up time 1.8 years) and the other 2 underwent delayed surgery because of tumor progression (medulloblastoma in one patient and fibrillary astrocytoma in the other). Clinicians are increasingly challenged by the discovery of incidental CNS mass lesions. A subgroup of such lesions (with typical imaging patterns such as tectal glioma and dysembryoplastic neuroepithelial tumor) can be monitored conservatively, clinically, and radiographically. Future prospective studies are needed to define optimal management strategies based on larger collections of natural histories, as well as to assess the true prevalence of incidental CNS mass lesions

The Role of Dysregulated Glucose Metabolism in Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and also one of the most poorly understood. Other health issues that are affecting women with increasing frequency are obesity and diabetes, which are associated with dysglycemia and increased blood glucose. The Warburg Effect describes the ability of fast-growing cancer cells to preferentially metabolize glucose via anaerobic glycolysis rather than oxidative phosphorylation. Recent epidemiological studies have suggested a role for hyperglycemia in the pathogenesis of a number of cancers. If hyperglycemia contributes to tumour growth and progression, then it is intuitive that antihyperglycemic drugs may also have an important antitumour role. Preliminary reports suggest that these drugs not only reduce available plasma glucose, but also have direct effects on cancer cell viability through modification of molecular energy-sensing pathways. This review investigates the effect that hyperglycemia may have on EOC and the potential of antihyperglycemic drugs as therapeutic adjuncts

Extracellular Matrix Proteins and Tumor Angiogenesis

Tumor development is a complex process that relies on interaction and communication between a number of cellular compartments. Much of the mass of a solid tumor is comprised of the stroma which is richly invested with extracellular matrix. Within this matrix are a host of matricellular proteins that regulate the expression and function of a myriad of proteins that regulate tumorigenic processes. One of the processes that is vital to tumor growth and progression is angiogenesis, or the formation of new blood vessels from preexisting vasculature. Within the extracellular matrix are structural proteins, a host of proteases, and resident pro- and antiangiogenic factors that control tumor angiogenesis in a tightly regulated fashion. This paper discusses the role that the extracellular matrix and ECM proteins play in the regulation of tumor angiogenesis