Behavioural expression of D-1 receptor supersensitivity depends on previous stimulation of D-2 receptors

SKF 38393 (2 mg/kg s.c.), a reportedly selective D-1 agonist, failed to induce contralateral turning behaviour in naive rats bearing 12 days old unilateral 6-hydroxydopamine lesions. On the other hand strong contraversive turning in response to SKF 38393 was obtained if rats had been tested 2 or 7 days before with apomorphine (0.1 mg/kg s.c.) or with LY 171555 (0.2 mg/kg s.c.), a selective D-2 receptor agonist. Contraversive turning in response to SKF 38393 was blocked by a low dose (0.05 mg/kg s.c.) of the specific D-1 antagonist SCH 23390. The results indicate that the behavioural expression of D-1 receptor supersensitivity following lesion of dopaminergic neurons depends on previous exposure to a stimulation of D-2 receptors.     

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.     

The human HOX gene family.

We report the identification of 10 new human homeobox sequences. Altogether, we have isolated and sequenced 30 human homeoboxes clustered in 4 chromosomal regions called HOX loci. HOX1 includes 8 homeoboxes in 90 kb of DNA on chromosome 7. HOX2 includes 9 homeoboxes in 180 kb on chromosome 17. HOX3 contains at least 7 homeoboxes in 160 kb on chromosome 12. Finally, HOX4 includes 6 homeoboxes in 70 kb on chromosome 2. Homeodomains obtained from the conceptual translation of the isolated homeoboxes can be attributed to 13 homology groups on the basis of their primary peptide sequence. Moreover, it is possible to align the 4 HOX loci so that corresponding homeodomains in all loci share the maximal sequence identity. The complex of these observations supports and extends an evolutionary hypothesis concerning the origin of mammalian and fly homeobox gene complexes. We also determined the coding region present in 3 HOX2 cDNA clones corresponding to HOX2G, HOX2H and HOX2I.     

Sequential 1H NMR assignment and secondary structure determination of salmon calcitonin in solution

Salmon calcitonin (sCT) has been investigated by NMR at 500 MHz in a 90% DMSOd6-10% 1H2O (v/v) mixture at 278 K. All backbone and side-chain resonances of the hormone have been assigned by using high-resolution phase-sensitive two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects, the NH-alpha CH spin-spin coupling constants, and the 1H/2H exchange kinetics measured in 80% DMSOd6-20% 2H2O (v/v) at 278 K enabled prediction of the secondary structure. Overall, an extended conformation is the dominant feature of the solution, but there are clear indications for a short double-stranded antiparallel beta sheet in the central region comprising residues 12-18, connected by a three-residue hairpin loop formed by residues 14-16. Two tight turns, made by residues 6-9 and 25-28, were also identified, but no evidence was found for the presence of a regular helical segment. The beta sheet favors an amphipathic distribution of the residues, orienting the predominantly hydrophilic Ser13, Glu15, and His17 side chains above the plane of the sheet, and the predominantly hydrophobic Leu12, Gln14, and Leu16 below it. This is interpreted as the "seed" of the amphipathic alpha helix postulated to be responsible for the interaction of sCT with lipids, a situation reminiscent of the folding mechanism of signal peptides in the interaction with membranes. The possible significance of the cis-trans Pro23 isomerism is discussed.     

A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif.

The homeobox, a 183 bp DNA sequence element, was originally identified as a region of sequence similarity between many Drosophila homeotic genes. The homeobox codes for a DNA-binding motif known as the homeodomain. Homeobox genes have been found in many animal species, including sea urchins, nematodes, frogs, mice and humans. To isolate homeobox-containing sequences from the plant Arabidopsis thaliana, a cDNA library was screened with a highly degenerate oligonucleotide corresponding to a conserved eight amino acid sequence from the helix-3 region of the homeodomain. Using this strategy two cDNA clones sharing homeobox-related sequences were identified. Interestingly, both of the cDNAs also contain a second element that potentially codes for a leucine zipper motif which is located immediately 3'' to the homeobox. The close proximity of these two domains suggests that the homeodomain-leucine zipper motif could, via dimerization of the leucine zippers, recognize dyad-symmetrical DNA sequences.     

pH measurements in ultranarrow immobilized pH gradients

It is possible to measure pH values in immobilized pH gradients (IPG) when the polyacrylamide matrix is made to contain an additional, carrier ampholyte-generated pH gradient. After an IPG run, 5 mm gel segments, along the separation axis, are cut and eluted in 300 microliter of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 microliter) are eluted in 900 microliter of 100 mM KCl and the pH assessed with a differential pH meter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert delta pH values into an actual pH curve. The reproducibility of the system is better than +/- 0.05 pH units, with a ca. 15% error over a 0.3 pH unit span. In ultranarrow pH gradients, it is imperative to use mixtures of all commercially available carrier ampholytes, so as to smoothen conductivity and buffering capacity gaps. By the present method, it is also possible to convert a wide (2-3 pH unit) carrier ampholyte interval into a narrow (0.2-0.3 pH unit) one.     

Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline-Induced Status Epilepticus. Effect of Pretreatment with α-Methyl-p-Tyrosine and p-Chlorophenylalanine

The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline-induced seizures were studied. A fourfold increase in cerebral FFA occurred 3-4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic-clonic convulsion. However, a 13- to 17-fold increase in arachidonic acid was seen approximately 5-6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl- and arachidonoyl-DG were also accumulated. The drug alpha-methyl-p-tyrosine was found to (a) potentiate the bicuculline-stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl- and arachidonoyl-DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p-chlorophenylalanine, but the drug had no effect on the bicuculline-induced changes. Hydrolysis of phospholipids enriched in stearoyl-arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.     

Oxidant damage of normal and glucose 6-phosphate dehydrogenase (G6PD)-deficient red blood cells is enhanced by iron-EDTA complex

A combination of divicine (an aglycone from the fava bean beta-glucoside vicine) and ascorbate results in a marked production of ethylene from methional, as a probable indication of OH radical formation. Addition of iron-EDTA to this oxidising system enhances the ethylene production significantly. The enhancing effect of iron-EDTA is also observed when both normal and Glucose 6-phosphate dehydrogenase (G6PD)-deficient red cells are exposed to the divicine-ascorbate system. Moreover, iron-EDTA magnifies other consequences of oxidant damage afforded by divicine-ascorbate or by ascorbate alone on the target red cells, such as depletion of reduced glutathione, formation of methemoglobin, stimulation of hexose monophosphate shunt activity and lipid peroxidation. Although the biochemical changes induced by this oxidative system are not remarkably different in normal and in G6PD-deficient red cells, the extra-damaging effect of chelated iron might be important in the mechanism of hemolysis.