Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

Occurrence of two distinct succinate thiokinases in animal tissues

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Organstruktur und Zellfunktion in explantiertem und später auto-retransplantiertem Schilddrüsengewebe

Zusammenfassung Schilddrüsengewebe von erwachsenen Kaninchen wurde in heterologem Medium in Rollröhrchen gezüchtet und 3 Wochen bis 4 1/2 Monate später auf das Spenderkaninchen rückverpflanzt. Hier blieben die Transplantate von 1 bis zu 8 Monaten. Die histologischen Befunde unmittelbar vor und nach Abschluß der Transplantation wurden miteinander verglichen. Unter beiden Lebensbedingungen, in vitro und im Transplantat, produzierten die Schilddrüsenzellen massenhaft Sekret, das in seinem färberischen Verhalten dem Schilddrüsenkolloid gleicht. Es wird für unwahrscheinlich gehalten, daß das Sekret biochemisch vollwertiges Schilddrüsenkolloid darstellt. Das Sekret wurde in großen Mengen intrazellulär gestapelt und führte schließlich zum Zellverfall.In den Transplantaten bestand nur dann Follikelanordnung, wenn diese auch noch in der Ausgangskultur vorhanden war. Die Follikel waren in jungen Transplantaten zunächst recht gut von Kapillaren umsponnen, doch verödeten diese später wieder. Alte Transplantate gingen schließlich genau so wie die gefäßlosen Gewebekulturen und wie alte, von vorn herein gefäßlos bleibende Transplantate durch intrazelluläre Sekretstapelung zugrunde. Es wird vermutet, daß in vitro der Mangel an thyreotropem Hormon zu dieser Fehlsteuerung führte und daß die Zellen auch im Transplantat nicht mehr auf das nun zur Verfügung stehende thyreotrope Hormon mit Ausschleusung des Sekrets reagieren konnten, weil dieser Mechanismus schon vorher in vitro pathologisch verändert worden war.Durch die Vorzüchtung wurde das Autotransplantationsergebnis erheblich verschlechtert.Eine maligne Entartung trat während der Züchtung in vitro nicht ein.     

Photoadaptation in marine Arctic diatoms

Summary Photoadaptation in some marine Arctic diatoms has been studied. Thalassiosira antarctica, Nitzschia delicatissima and Chaetoceros furcellatus were grown at-0.5°C and various irradiances and continuous light. Growth and cellular chlorophyll were followed during transitional phase after the algae had been transferred from one irradiance to another. Adaptation time for cellular chlorophyll was linearly related to the gradient in irradiance, and adaptation to transfer from high to low light was faster than from low to high light. Adaptation time was found to be species dependent, and Arctic diatoms growing at low temperature seemed to adapt as fast as temperate species.Contribution No. 243 from Trondhjem Biological Station     

Neuronal Intermediate Filament Expression During Neurite Outgrowth from Explanted Goldfish Retina: Effect of Retinoic Acid

Regulation of the goldfish neuronal intermediate filament proteins ON1 and ON2 was investigated in a retinal explant system. The synthesis of these proteins in explanted retina decreased with increasing time in culture, despite continuing neurite outgrowth. Thus, ON1/ON2 neurofilament expression is regulated independently from neurite outgrowth. During regeneration of the goldfish optic nerve in vivo, the expression of these proteins increased during the later phase of the process, when growing axons make contact with the optic tectum. The declining synthesis of ON1 and ON2 during neurite outgrowth in culture suggests that factors extrinsic to the retina are necessary to support synthesis of these proteins. Treating retinal explants with retinoic acid stimulated the synthesis of the ON1/ON2 proteins in a dose-dependent manner. This stimulation was effective during a period of declining synthesis of the ON1/ON2 proteins, restoring their synthesis towards initial levels of expression. These results show that retinoic acid serves as a modulator of neurofilament expression in this in vitro model of nerve regeneration.     

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.     

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.     

Appomattoxia ancistrophora gen. et sp. nov., a new Early Cretaceous plant with similarities to Circaeaster and extant Magnoliidae

A new genus and species of fossil angiosperm (Appomattoxia ancistrophora) is established based on well-preserved fruiting units and associated pollen from the Early Cretaceous (Early or Middle Albian) Puddledock locality in the Potomac Group sequence of Virginia, eastern North America. Fruiting units are small, unilocular, and with a single, pendulous, orthotropous seed. The fruit surface is characterized by densely spaced unicellular spines with hooklike tips, which probably functioned in biotic dispersal. Pollen grains adhering to the stigmatic area of many specimens are monocolpate and tectate with granular to columellate infratectal structure, and are similar to dispersed grains assigned to Tucanopollis and Transitoripollis. Comparison of fossil Appomattoxia ancistrophora with extant plants reveals an unusual combination of characters that includes similarities with some magnoliid taxa, particularly Piperales (Piperaceae, Saururaceae) and Laurales (Chloranthaceae), as well as the monotypic ranunculid family Circaeasteraceae. Appomattoxia ancistrophora differs from extant Piperales in having a pendulous rather than erect ovule, and differs from extant Circaeaster in details of the fruit wall, as well as the presence of monosulcate rather than tricolpate pollen.