Definition of microsatellite size variants forTnfa andHsp70 in autoimmune and nonautoimmune mouse strains

We have cloned and sequenced the upstream regulatory region of tumor necrosis factor (Tnfa) gene in 12 different mouse strains and identified an allelic polymorphism in the upstream regulatory region of the mouseTnfa gene. TheTNF allele found in the NZW strain is distinct from those of all otherH-2 haplotypes, supporting our previous suggestion that this allele may be associated with a regulatory or structural defect. In addition, simple tandem repeat sequences (microsatellites) within the promoter region of theTnfa gene and the 3 untranslated region of one of the members of the HSP70 family (Hsp68c clone) were utilized as genetic markers. Ten TNF size variants and twelve HSP70 variants were identified in over forty mouse strains. Using these markers in a set of congenic mice, we mapped this member of the HSP70 family to the central portion of theH-2 complex, centromeric to theTnfa gene. The NOD and NZW strains carry uniqueHSP70 alleles based on the variability in the length of this marker. These findings raise the possibility that this protein may play a role in the association of the major histocompatibility complex with these autoimmune diseases.     

Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium [corrected]

One significant problem in monitoring a culture's evolution is to assess change in cell viability. We have demonstrated that LDH release could be a good indicator of cellular damage of many cell lines, especially during shear stress or sonication. Moreover, we have found a significant correlation between the number of dead cells, determined by Trypan Blue staining, and LDH activity measurements in the supernatant of hybridoma strains, whatever the culture conditions. We have also shown that when viability is still near 100% no LDH is released even at high cell concentrations. Therefore, LDH should serve as a potential marker of cell injury and death.     

Mitogenic activity of chicken chorioallantoic fluid is temporally correlated to vascular growth in the chorioallantoic membrane and related to fibroblast growth factors

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17 x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.     

Tissue and species distribution of liver type and tumor type nuclear poly(A) polymerases

Previous studies in this laboratory have identified two distinct nuclear poly(A) polymerases, a 48 kDA tumor type enzyme and a 36-38 kDA liver type enzyme. To investigate the tissue and species specificity of these enzymes, nuclear extracts were prepared from various rat tissues, pig brain and two human cell lines. These as well as whole cell extract from yeast were probed for the two enzymes by immunoblot analysis using polyclonal anti-tumor poly(A) polymerase antibodies or autoimmune sera which contain antibodies specific for the liver type enzyme. Results indicate that both tumor and liver type enzymes are conserved across species ranging from rat to human. The yeast enzyme does not appear to be immunologically related to the liver or the tumor type poly(A) polymerase. The liver type enzyme appears to be specific for normal tissues whereas the tumor type enzyme is detected only in tissues in a "tumorigenic" state or cell lines originating from tumor tissues.     

Control by the extracellular environment of differentiation pathways in 1003 embryonal carcinoma cells: study at the level of specific intermediate filaments.

1003 is a multipotential embryonal carcinoma (EC) clonal cell line which can be induced to follow different developmental pathways by altering the composition of the culture medium. When grown in serum-containing medium the great majority of 1003 cells remain undifferentiated; they express the ECMA 7 cell-surface embryonic antigen and very low amounts of vimentin. In serum-free medium, most 1003 cells differentiate into neuroepithelial cells. The majority of these cells are still labelled with ECMA 7 antibodies. They contain higher amounts of vimentin than EC cells, but no neurofilament proteins. Neuroepithelial cells then differentiate into neurons through a stage of preneurons containing both vimentin and the 70-K neurofilament protein. Fully differentiated neurons contain 70-K neurofilament protein but no vimentin. The 200-K neurofilament protein is detected later in the neurons. Mesenchymal cells (induced by re-adding serum) express high amounts of vimentin organized in networks. Preneurons , neurons, and mesenchymal cells do not express ECMA 7 antigen.     

Well-identifiable human chromosomes Isolated from mitotic fibroblasts by a new method

Summary Metaphase chromosomes isolated from human fibroblasts by lysis of mitotic cells in the presence of the intercalating DNA-specific fluorochrome propidium iodide appear relatively long, even after exposure to vinblastine sulfate overnight. Therefore, they can be easily banded and thereby unequivocally identified. Chromosomes isolated in this way may be employed in flow analysis and sorting without loosing the inducibility of their band patterns.     

A model for the temporal organization of X- and Y-type receptive fields in the primate retina

A model is proposed for the temporal characteristics of X-and Y-type responses of ganglion cells in the primate retina. The main suggestions of the model are: (I) The X-type temporal response is determined primarily by the delay between center and surround contributions. (II) The Y-type response is generated in the inner plexiform layer by a derivativelike operation on the bipolar cell's input, followed by a rectification in the convergence of these inputs onto the Y-ganglion-cell. (III) The derivative-like operation is obtained by recurrent inhibition in the dyad synaptic structure.The X-and Y-type responses predicted by the model, for a variety of stimuli, were examined and compared with available electrophysiological recordings. Finally, certain predictions derived from the model are discussed.     

Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Effects of anti-embryonal carcinoma serum on aggregation and metabolic cooperation between teratocarcinoma cells

Effects of rabbit anti-embryonal carcinoma IgG on embryonal carcinoma cells and their differentiated derivatives were studied at different levels of cell-cell interaction. Fab fragments of anti-EC IgG were found to inhibit aggregation of the majority of EC cell lines. Two, however, were insensitive. Anti-EC Fab fragments act also on the transfer of metabolites between EC cells: the rescue of HPRT^? EC cells by HPRT⁺ EC cells in selective medium is abolished. These findings are correlated with the disappearance of tight and gap junctions from the surface of EC cells (Dunia et al., 1979). The presence of the surface structure involved in the action of anti-EC Fab fragments was tested by absorption experiments followed by decompaction test on PCC4 Aza R1 cells. All EC cell lines and two embryonic cell lines—a trophectodermal and an endodermal line—were found to bear material absorbing the decompacting activity. The results are discussed in terms of state of differentiation of the cell lines and of complexity of aggregation of embyronic cells.     

Die Wirkung von Beleuchtungsfaktoren und Lichtpunktmustern auf die n?chtliche Zugaktivit?t von Rotkehlchen(Erithacus rubecula)

Zusammenfassung 1. Die nächtliche Zugunruhe setzt auch unter konstanten Lichtbedingungen (Dauerdunkel 0,06 Lux, Dauerhell 250 Lux) etwa mit der Sonnenuntergangszeit ein und erreicht im Frühjahr etwa um 21^h ihr Maximum (etwas später bei Dauerhell).2. Während der Nacht werden vom zugaktiven Rotkehlchen in stärkerem Maße als bei Tag die dunkleren Abteilungen einer Lichtorgel aufgesucht.3. Die höchste Zugaktivität wird bei Beleuchtungsstärken von 1–10 Lux erreicht, aber auch bei völliger Dunkelheit wird noch etwa 7 % der Maximalaktivität gemessen. Sommer-Nachtaktivität erreicht ebenfalls bei ca. 5 Lux ein Maximum, erlischt aber fast völlig bei Beleuchtungsstärken unter 1 Lux.4. Bei dunklem Untergrund ist die gemessene Aktivität bei gleicher Beleuchtungsstärke deutlich geringer als bei hellem Untergrund.5. Niedrigere Tagesbeleuchtungsstärken im Versuchsraum (250 gegenüber 700 Lux) bewirken ein Ansteigen der Zugaktivität in der folgenden Nacht, bei gleichzeitig zunehmender Futteraufnahme während des Tages.6. Einzelne Lichtpunkte wirken aktivitätssteigernd unabhängig von der von ihnen bewirkten Beleuchtungsstärke. Eine größere Zahl von Lichtpunkten, gleichmäßig über die Decke verteilt, hemmt dagegen die Zugaktivität, auch wenn die dabei erreichte Beleuchtungsstärke höher ist als bei einer Diffusionsbeleuchtung, unter der dieselben Tiere eine höhere Aktivität zeigen.

---

Nocturnal migratory restlessness in the robin under different light conditions and patterns of light spots

Summary 1. Nocturnal migratory restlessness even under constant LL or DD lighting conditions (250 Lux, 0,006 Lux) starts at the time of sunset, and in spring reaches a maximum at 9 pm under constant dark, and a little later in constant light (Fig. 1).2. At night, during nocturnal restlessness, the birds tend to be more active in the darker parts of a set of differently lighted chambers than during daytime (Fig. 3 b).3. Highest migratory restlessness in spring and autumn is recorded under an illumination of 1 to 10 Lux, though even in total darkness a high level of activity persists. Nocturnal activity in summer also reaches a maximum at an illumination level of 5 Lux, but drops to zero at intensities below 1 Lux (Fig. 4 a).4. In cages painted black the nocturnal activity is lower than in white cages with the same overhead illumination intensity (Fig. 5).5. Low illumination intensity during daytime (250 against 700 Lux) causes high activity during the following night and raises diurnal food consumption considerably.6. A few single light spots (stars) on the ceiling strongly activate migratory restlessness (almost independent of their brightness), while higher numbers of evenly spaced light spots lower the birds activity even though total illumination intensity is higher than under the diffuse control lighting under which the birds show a higher amount of activity (Fig. 7, 8).