Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

Hot Melt “Corner Point Method” for Attaching Large Plastic Sections to Glass Slides

We describe a fast method for firm attachment of large plastic sections to glass slides with EVA-copolymers, commonly known as hot melt sticks. Solid hot melt sticks dissolve slowly in xylene to form an adhesive gel within 6 hours. Small drops of hot melt gel are applied to the corners of the sections and surrounding slide surface at ambient or elevated temperatures. The gel sticks to both the plastic and the glass slides. The hot melt “corner point method” prevented detachment of sections in staining procedures. As an additional technique, we suggest the use of hot melt adhesive for attaching plastic specimen blocks to wooden blocks or metallic specimen holders.     

Detection of Radio-Elements in Histological Slides by Coating with Stripping Emulsion—The “Strip-Coating” Technic

The “strip-coating” technic offers some improvement of the published methods for the autographic detection of Tadio-elements in sections by stripping films. Reproducible results are easily obtained with Ilford Half Tone Stripping Plate Emulsion. The method does not lend itself readily to the preparation of the large number of slides usually required for biological research.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

Cellular “bauplans”: Evolving unicellular forms by means of Julia sets and Pickover biomorphs

The universe of cellular forms has received scarce attention by mainstream neo-Darwinian views. The possibility that a fundamental trait of biological order may consist upon, or be guided by, developmental processes not completely amenable to natural selection was more akin to previous epochs of biological thought, i.e. the “bauplan” discussion. Thirty years ago, however, Lynn and Tucker studied the biological mechanisms responsible for defining organelles position inside cells. The fact that differentiated structures performing a specific function within the eukaryotic cell (i.e. mitochondrion, vacuole, or chloroplast) were occupying specific positions in the protoplasm was the observational and experimental support of the ‘morphogenetic field’ notion at the cellular level. In the present paper we study the morphogenetic field evolution yielding from an initial population of undifferentiated cells to diversified unicellular organisms as well as specialized eukaryotic cell types. The cells are represented as Julia sets and Pickover biomorphs, simulating the effect of Darwinian natural selection with a simple genetic algorithm. The morphogenetic field “defines” the locations where cells are differentiated or sub-cellular components (or organelles) become organized. It may be realized by different possibilities, one of them by diffusing chemicals along the Turing model. We found that Pickover cells show a higher diversity of size and form than those populations evolved as Julia sets. Another novelty is the way that cellular organelles and cell nucleus fill in the cell, always in dependence on the previous cell definition as Julia set or Pickover biomorph. Our findings support the existence of specific attractors representing the functional and stable form of a differentiated cell—genuine cellular bauplans. The configuration of the morphogenetic field is “attracted” towards one or another attractor depending on the environmental influences as modeled by a particular fitness function. The model promotes the classical discussions of D’Arcy Thompson and the more recent views of Waddington, Goodwin and others that consider organisms as dynamical systems that evolve through a ‘master plan’ of transformations, amenable to natural selection. Intriguingly, the model also connects with current developments on mechanobiology, highlighting the informational–developmental role that cytoskeletons may play.     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

Basophilic “Dark Cells:” Optimal Conditions for Toluidine Blue Staining

Differential staining of basophilic “dark cells” of epithelia in thick epoxy sections with toluidine blue is critically affected by fixation and embedding conditions. Optimal conditions are described.     

Apparatus for Bone Sectioning

A new type of apparatus for cutting 100-200µ sections of bone is described. This apparatus consists of an especially designed bone vise and a mounted circular saw with three directional movement controls. The saw is driven by one D.C. motor and its vertical movement assembly is driven by another. Their speeds can be regulated individually. The bone sections may be made serially at intervals of 0.3 to 0.5 mm. and bones as large as 2 cm. in diameter or longitudinal slabs of larger bones may be sectioned.     

The Stability Of So-Called Axonal Acid Phosphatase As Determined By Experiments In Its “Stainability”

Since the histochemical method for exhibiting acid phosphatase in bodily tissues is said to depend upon the enzyme acting on suitable substrates, it is possible to test its stability by various tests. It has been found that the background element or elements, whatever they may b e, concerned with the “staining” properties of the reaction are very stable and somewhat resistant to destruction. So-called acid phosphatase in spinal cord axons has not been inactivated by subjecting it to various fixing solutions, changes in temperature and pH, relatively prolonged post-mortem autolysis nor by well known enzyme inactivators. It is believed that the “staining” reaction may be dependent fundamentally on other factors than enzymatic activity.     

Aromatase immunoreactivity in the rat brain: Gonadectomy-sensitive hypothalamic neurons and an unresponsive “limbic ring” of the lateral septum-bed nucleus-amygdala complex

The aromatase (estrogen synthetase) enzyme catalyzes the conversion of androgens to estrogens in peripheral tissues, as well as in the brain. Our study aimed at comparing the brain distribution of aromatase-immunoreactive neurons in male and female, normal and gonadectomized rats. Light microscopic immunostaining was employed using a purified polyclonal antiserum raised against human placental aromatase. Two anatomically separate aromatase-immunoreactive neuronal systems were detected in the rat brain: A “limbic telencephalic” aromatase system was composed by a large population of labeled neurons in the lateral septal area, and by a continuous “ring” of neurons of the laterodorsal division of the bed nucleus of stria terminalis, central amygdaloid nucleus, stria terminalis, and the substantia inominata-ventral pallidum-fundus striati region. The other, “hypothalamic” aromatase system consisted of neurons scattered in a dorsolateral hypothalamic area including the paraventricular, lateral and dorsomedial hypothalamic nuclei, the subincertal nucleus as well as the zona incerta. In addition, a few axon-like processes (unresponsive to gonadectomy) were present in the preoptic-anterior hypothalamic complex, the ventral striatum, and midline thalamic regions. No sexual dimorphism was observed in the distribution or intensity of aromatase-immunostaining. However, 3 days, 2, 3, 8, 16, or 32 weeks after gonadectomy, aromatase-immunoreactive neurons disappeared from the hypothalamus, whereas they were still present in the limbic areas of both sexes. The results indicate the existence of two distinct estrogen-producing neuron systems in the rat brain: (1) a “limbic ring” of aromatase-labeled neurons of the lateral septum-bed nucleus-amygdala complex unresponsive to gonadectomy; and (2) a sex hormone-sensitive “hypothalamic” aromatase neuron system.     

Immunofluorescent and ultrastructural studies of “sarcomeric” units in stress fibers of cultured non-muscle cells

Immunofluorescence and electron microscopy were used to study the organization of actin, myosin and α-actinin in the “sarcomeric” units of the stress fibers of a selected non-muscle cell type. The results of indirect immunofluorescence confirm that myosin and α-actinin are periodically distributed in discrete units along stress fibers and demonstrate that they are alternately spaced. This relationship is required by a sarcomere model of stress fiber construction. A comparison between immunofluorescent and EM images of stress fibers confirms that α-actinin is confined to Z line-like dense bodies, myosin to the spaces in between. The most intriguing result is that by immunofluorescence a periodic distribution of actin can be detected in some fibers. This may indicate that even actin is periodically distributed in non-muscle “sarcomeres”.     

Comparing a range of impellers for “stirring as foam disruption”

The effectiveness of a range of impellers for “stirring as foam disruption” (SAFD) is assessed in a vessel of 0.72 m diameter and an aspect ratio of 2:1. Measurement of power drawn by the impeller achieving SAFD and of the three-dimensional flow field close to the dispersion surface are both used to explain the findings along with the global gas hold-up. A large radial flow Rushton turbine can disrupt foam at a great height but requires high power. Down-pumping hydrofoils are only effective when the ungassed liquid height is below the level of the impeller employed to disrupt foam. Up-pumping hydrofoils are the most effective because their flow pattern gives rise to high velocities across the dispersion surface, which are able to entrain foam in the downflow generated at the walls.     

“Harpoon” Model for Cell–Cell Adhesion and Recognition of Target Cells by the Natural Killer Cells

The mechanism of recognition by natural killer (NK) cells is still unknown. A dynamic model is formulated describing recognition or NK-sensitive target cells (TCs) by NK cells of NK-like cells. This model does not assume the presence of the specific NK-receptor(s) on the membrane of NK cells and corresponding specific ligands on the NK-sensitive TCs. We suggest: (1) the expression of various kinds of “non-NK receptors” and corresponding ligands (counter-receptors) on the plasma membrane of the same NK cell and, possibly, of TCs (e.g. LFA-1 and ICAM-1-ICAM3, CD2 and LFA-3; receptors for TNF and corresponding ligand etc.); (2) the presence of multiple disorders in the organization of “extracellular matrix-surface membrane-submembrane cytoskeleton” assembly of the NK-sensitive TCs; (3) non-specific primary linking of NK cell with TCs, which induces a transfer of vesicles or membrane fragments from the NK surface to the target cell surface (and perhaps vice versa). These processes may also permit the transfer of many types of receptor and counter-receptor molecules from the surface of one conjugated cell to another by vesicles or membrane fragments. After transferral through the intercellular cleft, the free receptors and counter-receptors will be localized on both cell surfaces at the contact region between conjugated cells. By this model the NK cell can “harpoon” the TC and enhance the binding forces between cells up to the critical level and then switch on killing mechanisms for the TC. By means of this “harpoon” model of cell recognition, it seems possible to explain the nature of the wide polymorphism of TCs which are sensitive to the effect of NK and NK-like cells. A mathematical model of the NK cell cytotoxic reaction is described. The model describes many nonlinear peculiarities of the cytotoxic process and predicts some new phenomena. We suggest new approaches of manipulation of cell membranes which can transform NK-resistant target cells in NK sensitive cells and vice versa.     

Facial symmetry and judgements of apparent health: Support for a “good genes” explanation of the attractiveness–symmetry relationship

The “good genes” explanation of attractiveness posits that mate preferences favour healthy individuals due to direct and indirect benefits associated with the selection of a healthy mate. Consequently, attractiveness judgements are likely to reflect judgements of apparent health. One physical characteristic that may inform health judgements is fluctuating asymmetry as it may act as a visual marker for genetic quality and developmental stability. Consistent with these suggestions, a number of studies have found relationships between facial symmetry and facial attractiveness. In Study 1, the interplay between facial symmetry, attractiveness, and judgements of apparent health was explored within a partial correlation design. Findings suggest that the attractiveness–symmetry relationship is mediated by a link between judgements of apparent health and facial symmetry. In Study 2, an opposite-sex bias in sensitivity to facial symmetry was observed when judging health. Thus, perceptual analysis of symmetry may be an adaptation facilitating discrimination between potential mates on the basis of apparent health. The findings of both studies are consistent with a “good genes” explanation of the attractiveness–symmetry relationship and problematic for the claim that symmetry is attractive as a by-product of the ease with which the visual recognition system processes symmetric stimuli.     

“Adaptive” changes in the behaviour of parasitized animals: A critical review

Changes in host behaviour following infection with parasites are frequently reported in the literature, and are often hypothesized to be adaptive for either host or parasite. However, investigators of such phenomena often use the “adaptation” label for host behavioural changes based on their intuition and not on rigorous criteria. Alterations in host behaviour following infection can only be considered adaptive if they satisfy certain conditions: (1) they must be complex; (2) they must show signs of a purposive design; (3) they are more likely to be adaptations if they have arisen independently in several lineages of hosts or parasites; and (4) they must be shown to increase the fitness of either the host or the parasite. A survey of published examples of host behavioural changes indicates that while some are spectacularly complex and are extremely well-fitted to their presumed function, most are simple increases or decreases in an activity performed prior to infection. There are some suggestions of convergent evolution in behavioural change in distantly related host or parasite groups but more evidence is needed. Finally, most known behavioural changes have not been demonstrated to lead to fitness gains in either hosts or parasites. Few known examples satisfy more than two of the above criteria, and, in general, the adaptive function of changes in host behaviour following infection is in need of more solid proof.     

Petite bodies of the “robust” australopithecines

The “robust” australopithecines are often depicted as having large and powerfully built bodies to match their massive masticatory apparatus, but until 1988 the sample of postcranial remains attributed with certainty to this group was very limited. Almost nothing was known about the body of the East African “robust” australopithecine because taxonomic attribution of the postcrania was so uncertain. The body of the South African “robust” australopithecine had to be reconstructed from about a dozen isolated fragments of postcrania. Now a partial skeleton is attributed with confidence to the East African “robust” group along with several isolated bones. The South African sample has more than tripled. Analyses of this vastly expanded sample reveal that a large portion of postcrania attributed to “robust” australopithecines from Swartkrans Member 1 (35%) are from extraordinarily small-bodied individuals similar in size to a modern Pygmy weighing as little as 28 kg. These small elements include parts from the forelimb, spine, and hindlimb. About 22% of these Swartkrans 1 “robust” australopithecines are about the same size as a modern human weighing about 43 kgs and about 43% are larger than this standard but less than or equal to a 54 kg modern human. Approximately the same pattern is true for the Swartkrans 2 hominids, but taxonomic attribution is less certain. All of the Member 3 specimens are similar in size to the 45 kg standard. The partial skeleton of the East African “robust” australopithecine (KNM-ER 1500) has hindlimb joints that would correspond to a modern human of 34 kgs although the actual weight may be 5 to 10 kgs greater judging from shaft robusticity and forelimb size. The largest postcranial element attributed with some certainty to the East African “robust” australopithecine group (the talus, KNM-ER 1464) is about the same overall size as a modern human of 54 kgs, although its tibial facet is slightly smaller. Although many previous studies have hinted at the possibility that “robust” australopithecines had relatively small bodies, the new fossils provide substantial evidence that these creatures ranged from quite small to only moderate in body size relative to modern humans. These were the petite-bodied vegetarian cousins of our ancestors. Sexual dimorphism in body size appears to be greater than that in modern humans, similar to that in Pan, and less than that in Gorilla or Pongo, although such comparisons are of limited value given the small samples, poorly known body proportions, time averaging, and many other problems.     

Fusion with enucleated fibroblasts corrects “I-cell” defect

Fusions have been carried out between fibroblasts from patients with “I-cell” disease and enucleated human fibroblasts with a single lysosomal enzyme deficiency derived from patients with G_M1-gangliosidosis, Sandhoff disease and mannosidosis. Pure cytoplasts were obtained using cytochalasin B treatment followed by fluorescence activated cell sorting. After fusion with whole “I-cells”, the cybrid populations showed a restoration of deficient lysosomal enzyme activity and also the abnormal electrophoretic pattern characteristic for the residual hexosaminidase activity in “I-cells” was found to be corrected. The results described in this paper indicate that the defective post-translational modification, which is responsible for the multiple lysosomal enzyme deficiency, can be corrected by a factor that is stable for at least three days in enucleated cells. During this period the cytoplasmic factor can act without the need of de novo synthesis but the absence of correction in in vitro experiments shows that cellular integrity is required.     

A biochemical approach toward the systematics of the Leptothorax “muscorum”Group in North America (Hymenoptera: Formicidae)

Enzyme patterns have been used to distinguish among a number of morphologically very similar ant species belonging to the Leptothorax “muscoru” group in North America. Not counting the already described species L. retractus, L. sphagnicolus and L. crassipilis, the complex apparently consists of at least three or four more different taxa.