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.     

The human alpha-fetoprotein gene. Sequence organization and the 5' flanking region

The human alpha-fetoprotein (AFP) gene was isolated into three overlapping clones in bacteriophage lambda vectors and its sequence organization analyzed by restriction endonuclease mapping and nucleotide sequencing. The human AFP gene is about 20 kilobase pairs long and contains 15 exons and 14 introns. The overall organization of the human AFP gene is similar to that of the mouse AFP gene, with all but two exons showing identical sizes. Nucleotide sequences at all exon/intron junctions display similarity to the consensus boundary sequence (Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349-383), with the GT-AG rule applied to the splicing point. The cap site maps 44 nucleotides upstream from the translation initiation site. The "TATA box" is located 27 nucleotides upstream from the putative cap site and is flanked by sequences with dyad symmetry. The TATA box can thus be placed in the loop portion of a possible stem-loop structure formed by intrastrand base-pairing. Other characteristic nucleotide sequences in the 5' flanking region include a CCAAC pentamer, a 14-base pair (bp) enhancer-like sequence, and a 9-bp sequence homologous to the glucocorticoid responsive element. A long (90 bp) direct repeat and several alternating purine/pyrimidine sequences are also present in the 5' flanking region. A 736-bp sequence of the 5' flanking region adjacent to the cap site of the human AFP gene shows a 61% similarity with the corresponding region of the mouse AFP gene. There are two Alu family sequences and two poly(dT-dG) repeats in the human AFP gene that show different distribution patterns from those in the mouse AFP gene.     

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000-43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.     

Genetic variability in the European minnow,Phoxinus phoxinus (L.)

An electrophoretic study of genetic variation at 11 loci was performedfor a population of European minnows, Phoxinus phoxinus (L.). Ten loci, EST-1 ^*, EST-2 ^* EST-3 ^*,GPD-1 ^*,GPD-2 ^*,GPI-1 ^*,GPI-2 ^*,MPI ^*,6PGD ^* and PGM ^* were polymorphic. IDH ^*wasmonomorphic. The mean number of heterozygotic loci over all 176 fish was 3.05 ± 0.104(SE). Observed mean heterozygosity was 0.28±0.058(SE) and expected mean heterozygosity was 0.27±0.054(SE). EST-2 ^*, EST-3 ^* andPGM ^* were not in Hardy-Weinberg equilibrium. Length,condition, parasite numbers or male breeding characters, i.e. red colorationand tubercles, were not influenced by single enzyme loci.     

Sucrose biosynthesis in Dunaliella

Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.     

Single amino acid substitutions in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus. Structural and spectral effects.

To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct energy transfer from the B800-850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.     

Influence of melatonin treatment on human circadian rhythmicity before and after a simulated 9-hr time shift.

The hormone melatonin is currently proposed by some investigators to be an efficient means for decreasing the impairing effects of jet lag. Eight healthy male subjects, aged 20 to 32, underwent a 9-hr advance shift in the isolation facility of our institute during two periods each of 15 days' duration. In a double-blind, crossover design, subjects took either melatonin or placebo at 1800 hr local time for 3 days before the time shift and at 1400 hr for 4 days afterwards. The time shift was simulated on days 7 and 8 by shortening the sleep period by 6 hr and the following wake period by 3 hr. Body temperature was recorded every 90 min, and urine was collected at 3-hr intervals all day and night. Melatonin treatment enhanced the resynchronization speed of some, but not all, hormone and electrolyte excretion rates for several days after the time shift. The adaptation speed of the temperature rhythm significantly increased during one postshift day. In addition, the circadian temperature rhythm had a significantly higher amplitude under melatonin treatment than under placebo after the time displacement. For the placebo group, the rhythm of 6-hydroxymelatoninsulfate excretion exhibited an advance shift in five subjects, whereas the other three showed a delay shift, and adjustment did not achieve more than one-half of the expected value within 8 days. A significantly different adjustment could be observed in the melatonin-treated group: Seven subjects underwent an advance shift of the expected 9 hr within an average of 8 days. The results suggest that melatonin treatment can accelerate resynchronization of the melatonin excretion rhythm after eastward time zone transitions. The improvement is not, however, sufficiently great that we can recommend melatonin for the alleviation of jet lag.