Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm.
Lower DMZ of early gastrula didn't show any axial-mesoderm (notochord and somitic mesoderm) but lateral mesoderm (mesenchyme, mesothelium, or blood cells) in combinant with non-competent ventral ectoderm, while combinant with competent ectoderm was found to have well-differentiated axial-mesoderm with deutero-spinocaudal neurals. The axial-mesoderms have origin in the ectoderm. Uninvaginated DMZ of middle gastrula also showed difference in mesodermal differentiation between competent and non-competent ectoderms; axial-mesoderm differentiation was much better in competent than in non-competent. The axial-mesoderm originated from the uninvaginated DMZ. Archenteron roof of late gastrula showed regional difference in mesodermal differentiation in both combinants with competent and non-competent. The present study further demonstrated that there was regionality in promoting effect of induced neurectoderm on axial-mesoderm differentiation of invaginated archenteron roof.
The present experiments suggest that the cranio-caudal and dorso-ventral axis formations of amphibian mesoderm are finally determined by sequential and reciprocal interactions between the mesodermal anlage and the overlying ectoderm. It should be also shown that lower DMZ acts to trigger a series of the sequential interactions during primary embryonic induction.     

Evolution of the primary and secondary structures of the E1a mRNAs of the adenovirus

In this paper we investigate and compare (evolutionary) patterns in the primary and secondary structure of four homologous E1a mRNAs of the adenovirus. Our main results are as follows: (1) The similarity of the coding regions of the mRNA sequences reflects both similarity in function (i.e., oncogenicity) and evolutionary divergence. (2) The similarity of the leader and the trailer regions reflects host specificity (i.e., human or simian) and must therefore arise from convergence. (3) Minimal energy foldings of the mRNAs show similar secondary structures (in particular around the splice sites). The conservation of pre-mRNA secondary structure shows that mRNAs are subject to selection constraints in addition to those associated with proteins. (4) The conserved secondary (helical) structures consist of nonhomologous subsequences, i.e., shifts have occurred. The observed shifts near the splice sites seem to be the simplest way of dealing with the dual constraints.      

Complete sequences of the rRNA genes of Drosophila melanogaster

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.      

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

A 13C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-13C]glycine, L-[3-13C]alanine and L-[3-13C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK1/Cl4 cells, a renal epithelial cell line, incubated with either [2-13C]glycine L-[3-13C]alanine, or D,L-[3-13C]aspartic acid were investigated by 13C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of gamma-glutamyltransferase activity and glutathione synthetase activity in LLC-PK1 cells, as is evident from enrichment of reduced glutathione. Time courses showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of L-alanine and 60% of L-aspartic acid was utilized during the same period. 13C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK1 cells. These uptake experiments indicated that glycine, alanine and aspartic acid are transported into Cl4 cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% when labelled L-alanine was the only carbon source for LLC-PK1/Cl4 cells. Experiments with labelled D,L-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.     

Codominant expression of a mutation affecting the cAMP-dependent protein kinase catalytic subunit in somatic cell hybrids of LLC-PK1 cells

The LLC-PK1 mutant cell lines FIB4 and FIB6 are affected in the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) such that they possess less than 10% parental activity. However, by Western blot analysis they were shown to possess normal levels of C subunit protein. Somatic cell hybrids were derived between mutant and LLC-PK1 cells, and examined for complementation of the cAMP-PK lesion. Codominant expression of mutant and normal alleles was observed, in that somatic cell hybrids between FIB4 and LLC-PK1, and between FIB6 and LLC-PK1 cells, exhibited cAMP-PK activity 60-75% that of LLC-PK1 cells, intermediate between mutant and normal parental cell lines. The cAMP-PK of the FIB6 x LLC-PK1 and FIB4 x LLC-PK1 hybrids was examined by ion exchange chromatography. In contrast to the FIB6 and FIB4 mutants which lack an active Type I cAMP-PK, the hybrids retained levels of active Type I cAMP-PK greater than 30% that of LLC-PK1, concomitant with the retention of catalytic activity. It was concluded that the loss of Type I kinase in the FIB6 and FIB4 mutants is most likely a consequence of the lesion in the cAMP-PK C subunit. All somatic cell hybrids examined showed levels of cAMP-PK C subunit (as determined by Western blot analysis), and in vivo regulation of cAMP-PK activation (in response to hormonal or nonreceptor-mediated stimulation of adenylate cyclase), completely comparable to those of the parental LLC-PK1 cells. Hence, no aberrant regulation of either cAMP-PK subunit levels or cAMP-PK activities was evident in the somatic cells hybrids. All data were consistent with the hypothesis that FIB4 and FIB6 contain a structural mutation affecting the cAMP-PK catalytic subunit that is expressed phenotypically in the presence of the normal allele.     

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]     

Sabellaria wilsoni sp.n. (Polychaeta, Sabellariidae) from the southeast coast of Brazil

A new species of Sabellaria is described from the southeast coast of Brazil and compared with related species known from South or North America.