Ontogenetic development of S-antigen- and rodopsin immunoreactions in retinal and pineal photoreceptors of Xenopus laevis in relation to the onset of melatonin-dependent color-change mechanisms

Summary In Xenopus laevis Daud., the ontogenetic occurrence of two photoreceptor-specific proteins, S-antigen and rod-opsin, was investigated and correlated to the maturation of the neurohormonal effector system involved in melatonin-dependent color-change mechanisms. Tadpoles ranging from stage 12 to 57 (Nieuwkoop and Faber 1956) were fixed in Zamboni's or Bouin's solution. Frozen or paraffin sections of either total heads or dissected brains and eyes were prepared and treated with highly specific antisera against S-antigen and rod-opsin. In the retina, immunoreactive S-antigen and rod-opsin were first demonstrated in a few centrally located photoreceptors at stage 37/38. Photoreceptors of the peripheral (iridical) portions of the retina gradually became immunoreactive during further development. As in the retina, the first S-antigen-immunoreactive photoreceptors in the pineal complex appeared at stage 37/ 38. At this and all later stages investigated rod-opsin immunoreactivity was restricted to a few dot-like structures resembling developing pineal outer and inner segments. In most animals rod-opsin immunoreactivity was completely absent from the pineal complex. The analysis of retinal proteins with the immunoblotting technique (Western blot) revealed that the S-antigen antibody bound to a 48-kDa protein and the rod-opsin antibody to a 38-kDa protein. The body lightening reaction was determined with the aid of the melanophore index in larvae fixed in light or darkness, respectively. Aggregation of melanophore melanosomes in darkness (the melatonin-dependent primary chromatic response) first occurred at stage 37/38 when melanophores started to differentiate and became pigmented. These results indicate that in Xenopus laevis (i) the molecular mechanisms of photoreception develop simultaneously in retina and pineal complex; (ii) most pineal photoreceptors differ from retinal rods in that they contain immunoreactive S-antigen but essentially no immunoreactive rod-opsin; and (iii) the differentiation of phototransduction processes coincides with the onset of melatonin-dependent photoneuroendocrine regulation of color-change mechanisms.Supported by USUHS protocol C07049 (MDR) and the Deutsche Forschungsgemeinschaft (HWK)     

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

Summary After introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.     

Inhibition of photosynthetic reactions by light

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m^-2 red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO₂-dependent O₂ evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI photosystem I - PSII photosystem II     

Genetic Candidate Variants in Two Multigenerational Families with Childhood Apraxia of Speech

Childhood apraxia of speech (CAS) is a severe and socially debilitating form of speech sound disorder with suspected genetic involvement, but the genetic etiology is not yet well understood. Very few known or putative causal genes have been identified to date, e.g., FOXP2 and BCL11A. Building a knowledge base of the genetic etiology of CAS will make it possible to identify infants at genetic risk and motivate the development of effective very early intervention programs. We investigated the genetic etiology of CAS in two large multigenerational families with familial CAS. Complementary genomic methods included Markov chain Monte Carlo linkage analysis, copy-number analysis, identity-by-descent sharing, and exome sequencing with variant filtering. No overlaps in regions with positive evidence of linkage between the two families were found. In one family, linkage analysis detected two chromosomal regions of interest, 5p15.1-p14.1, and 17p13.1-q11.1, inherited separately from the two founders. Single-point linkage analysis of selected variants identified CDH18 as a primary gene of interest and additionally, MYO10, NIPBL, GLP2R, NCOR1, FLCN, SMCR8, NEK8, and ANKRD12, possibly with additive effects. Linkage analysis in the second family detected five regions with LOD scores approaching the highest values possible in the family. A gene of interest was C4orf21 (ZGRF1) on 4q25-q28.2. Evidence for previously described causal copy-number variations and validated or suspected genes was not found. Results are consistent with a heterogeneous CAS etiology, as is expected in many neurogenic disorders. Future studies will investigate genome variants in these and other families with CAS.     

Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus

We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the γ phosphate of triphosphate-terminated RNA at a rate of 1 s⁻¹. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.     

The wingless gene is required for embryonic brain development in Drosophila

 We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate. Received: 7 October 1997 / Accepted: 30 December 1997     

X-ray structure of the cambialistic superoxide dismutase from Propionibacterium shermanii active with Fe or Mn

The first structure of a cambialistic superoxide dismutase (SOD) from Propionibacterium shermanii exhibiting similar activity with iron and with manganese was solved at a resolution of 1.6?Å and 1.9?Å respectively. Surprisingly, no obvious differences between the two SODs were observable. The protein crystallises as a homo dimer in the asymmetric unit. Because of the crystallographic symmetry, it forms a tetramer. Structures of both the manganese and the ferric form were solved using molecular replacement techniques and multiple isomorphous replacement. The tertiary structure is similar to that of the other superoxide dismutases, the metal being fivefold coordinated by three histidines, one aspartate and one water molecule. The second shell of residues consists of hydrophobic amino acids, histidines and two water molecules, which are assumed to be involved in both the catalytic activity and structural stability of this superoxide dismutase. This shell may also be responsible for the cambialistic behaviour. This work shows that the reason for the metal specificity is not trivial, although minor alterations in the metal environment might be responsible for this behaviour.     

1α,25-Dihydroxyvitamin D3; its role for homeostasis of keratinocytes

Epidermal homeostasis is influenced by a number of hormones and regulative growth factors that are maintained by a tightly regulated balance between cell proliferation, cell differentiation, and cell death. Vitamin D is one of those regulatory factors. It has recently been demonstrated that 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) takes part in the regulation of the cell cycle by multiple and complex functions. This review discusses 1α,25(OH)₂D₃ and its analogues in connection with the renewal of epidermal keratinocytes as well as the molecular mechanisms underlying terminal differentiation or rather programmed cell death. Furthermore, interest is focused on the possible clinical application of vitamin D₃ analogues.