The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.     

Substructure of inner mitochondrial membranes of myocardial cells as shown by cryo-ultramicrotomy

Summary The substructure of the inner mitochondrial membranes has been studied by cryo-ultramicrotomy under conditions during which denaturation of proteins by treatment with chemical solutes has been totally avoided. In such preparations, the inner membrane has a substructure consisting of globular subunits. These subunits have an average diameter of ca. 20Å–ca. 62Å and are fairly regularly spaced. Intracristal space is absent in the unstained, freeze-dried preparations, whereas a space of ca 40Å is seen in preparations lightly treated by OsO₄-vapour. It is concluded that the subunits of the inner mitochondrial membranes probably consist either of single protein molecules or of complexes of protein molecules.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

Immunohistochemistry of epithelial cell markers in normal and pathological colon mucosa

Summary The purpose of this study was to examine whether formal-dehyde-fixed tissue may afford reproducible and reliable immunhistochemical results when carcinoembryonic antigen (CEA), secretory component (SC), and epithelial IgA are evaluated semiquantitatively in normal and pathological colon specimens. Proximate tissue samples were processed by routine formalin fixation and by a cold-ethanol fixation method, respectively, and the immunofluorescence intensities obtained for the three antigens were scored. After formalin fixation SC and epithelial IgA were generally undetectable and also the staining for CEA was markedly reduced compared with that seen after ethanol fixation. Significant antigenic unmasking was obtained by enzyme treatment of the formalin-fixed tissue sections-resulting in enhanced staining for SC and epithelial IgA but not consistently so for CEA. With this modification scores from duplicate tissue samples processed by the two methods showed significant correlations for all the three epithelial markers; small amounts of CEA and epithelial IgA, and especially SC, nevertheless remained undetectable after formalin fixation. This result should be taken into account when epithelial markers are applied in studies of premalignant lesions of the colon where minor changes in the antigen pattern may be of diagnostic importance.     

Solar heating of mammals: Observations of hair transmittance

The transmission of solar radiation through animal coats by diffuse scattering is well known, but the part played by hair structure has not been examined. Transmittance of light through single guard hairs of 6 marine and 5 terrestrial mammals was measured through a microscope and values ranging from 0.29 to 0.94 were found. The hair transmittance was negatively correlated with pigmentation and medullation. Stepwise regression analysis indicates that solar heating at skin level is correlated with both hair transmittance and coat reflectance besides being coupled with coat thickness.     

Accuracy and repeatability of moose (<Emphasis Type="Italic">Alces alces</Emphasis>) age as estimated from dental cement layers

Optimal research and management of species with age structure often depends on estimates of age-specific population parameters, which in turn depends on reliable methods for age determination. By counting annuli in the cementum of incisor root tips from 51 known-age moose (Alces alces) between 1 and 12 years old, we examined the variation in accuracy and repeatability of age estimates provided by three research technicians with different experiences of aging moose. The most experienced technician estimated the moose age correctly in up to 90% of the cases, while the technician with no prior experience estimated age correctly in up to 73% of the cases. The medium-experienced technician achieved a lower accuracy (up to 53%), indicating that experience alone is not sufficient if the basic training or lack of routine checks against other colleagues or a known-age material are not undertaken. The percentage moose aged within ±1 year from correct age was significantly higher in all technicians (94–98%). After the generally high accuracy, we also found high repeatability (0.980–0.994) within technicians. We conclude that this method of age-determination provides reliable estimates that can be used by management and research to gain information about age-specific patterns in moose populations. However, to obtain estimates of high accuracy the technicians should be well trained, have gained the necessary experience, and most preferably, have access to a sample of teeth from known-age moose.