Comparative analysis of the localization and mobility of retrotransposons in sibling species Drosophila simulans and Drosophila melanogaster]

The distribution of four retrotransposon families (MDG1, MDG3, MDG4 and copia) on polytene chromosomes of different (from 9 to 15) Drosophila simulans strains is studied. The mean number of MDG1 and copia euchromatic hybridization sites (3 sites for each element) is drastically decreased in D. simulans in comparison with D. melanogaster (24 and 18 sites respectively). The mean number of MDG3 sites of hybridization is 5 in D. simulans against 12 in D. melanogaster. As for MDG4 both species have on the average about 2-3 euchromatic sites. The majority of MDG1 and copia and about a half of MDG3 euchromatic copies are localized in restricted number of sites (hot spots) on D. simulans polytene chromosomes. In D. melanogaster these elements are scattered along the chromosomes though there are some hot spots too. It appears that euchromatic copies of MDG1 and copia are considerably less mobile in D. simulans in contrast to D. melanogaster. Some common hot spots of retrotransposon localization in D. simulans and D. melanogaster were earlier described as intercalary heterochromatin regions in D. melanogaster. The level of interstrain variability of MDG4 hybridization sites is comparable in both species. Comparative blot-analysis of adult and larval salivary gland DNA shows that MDG1 and copia are situated mainly in euchromatic regions of D. melanogaster chromosomes. In D. simulans genome they are located mainly in heterochromatic regions underreplicated in salivary gland polytene chromosomes. There are interspecies differences in the distribution of retrotransposons in beta-heterochromatic chromosome regions.     

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.     

Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO₃⁻ uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO₃⁻ leaves) or 40 mm NO₃⁻ solution (high-NO₃⁻ leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO₃⁻-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO₃⁻ and high-NO₃⁻ leaves, indicating that NO₃⁻ had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO₃⁻ compared with low NO₃⁻ leaves showed evidence that NO₃⁻ enhanced the protein kinase activity. These results support the concept that NO₃⁻, or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).     

Nuclear magnetic tomography in the diagnosis of spinal cord cysts]

Magnetic resonance tomography (MRT) was used in examinations of 172 patients with spinal cord abnormalities. Thirty-eight cysts were detected: 25 in syringomyelia, 5 in intramedullary tumors, 4 posttraumatic and 4 postoperational ones. Based on the MRT patterns, two types of syringomyelia were distinguished, the occlusive and idiopathic ones. The size and site of cyst, as shown by the MRT, did not conform to its neurologic symptoms. A characteristic feature of tumorous cysts was a highly intensive signal originating from their contents. Traumatic cysts were detected against the background of diffuse atrophy of the cord. Computer-aided tomography did not give full-value information in such cases and therefore could not be recommended in cases with suspected intramedullary cysts.     

Ossification of the posterior longitudinal ligament]

The authors described 21 patients aged 37 to 83 with ossification of the posterior longitudinal ligament (10 with ischemic myelopathy, 6 with aftermaths of a trauma of the cervical spine, and 5 with the radicular syndrome on the basis of vertebral osteochondrosis. X-ray signs of three types of ossification of the posterior longitudinal ligament were defined and specified: linear, curved and mixed. The linear type was more frequently observed in the cervical spine whereas the curved type was more frequent in the lumbar spine.     

Homeosis in the mouse induced by a null mutation in the Hox-3.1 gene.

We have replaced the Hox-3.1 coding sequence with the E. coli lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Homozygous mice were born alive, but most of them died within a few days. In the trunk region of homozygotes, several skeletal segments were transformed into the likeness of more anterior ones, as observed in Drosophila with loss-of-function homeotic mutations. The most obvious transformations were the attachment of the 8th pair of ribs to the sternum and the appearance of a 14th pair of ribs on the 1st lumbar vertebra. The pattern of beta-galactosidase activity was identical in heterozygotes and homozygotes and reflected faithfully the Hox-3.1 expression pattern. Thus, the mutation modified the identity, rather than the position, of embryonic cells that would normally express Hox-3.1.     

Contribution of colostral fat to thermogenesis and glucose homeostasis in the newborn pig.

This study was designed to determine the effects of colostral fat on energy metabolism, fat oxidation and glucose homeostasis in newborn pigs maintained during the first 29h of life at thermal neutrality (34 degrees C) or in the cold (21 degrees C). Piglets were intragastric fed normal colostrum (NFC, 6% fat) or colostrum deprived of fat (LFC, less than 1% fat). A total of 21 meals of 15 to 18g colostrum/kg birthweight was given at 65- to 70-min intervals. Feeding NFC resulted in a higher amount of retained fat with the highest value being obtained in the 34 degrees C group (P less than 0.01). Fat oxidation represented 47% of the absorbed fat in NFC-fed piglets at 34 degrees C; it was 4.5 fold higher in piglets fed NFC than in those fed LFC (P less than 0.01), and 1.8 fold higher at 21 degrees C than at 34 degrees C (P less than 0.01). At both temperatures, feeding LFC resulted in a lower energy balance (P less than 0.01), whereas nitrogen balance was not affected by temperature and colostrum treatments. At 29 hours of age, rectal temperature was the lowest in piglets fed LFC at 21 degrees C (P less than 0.05). Postnatal enhancement of fat metabolism in relation to environmental and nutritional conditions was evidenced at the tissue level through an adaptation of lipoprotein lipase and cytochrome oxidase activities, especially in the red rhomboideus muscle and the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of thyroparathyroidectomy and thyroxine replacement on CU and ZN cellular distribution and on the metallothionein level and induction in rats

Thyroid hormones are involved in copper and zinc distribution in rat tissues. We examined the influence of thyroparathyroidectomy (TPTY) and of a replacement therapy by T4 on Cu and Zn organ distribution. MT levels were also measured both in basal conditions and after induction by cadmium. The results confirm that a lack of T4 modified Cu and Zn in serum and tissues. In serum, TPTY increased Cu (+15%) and ceruloplasmin (+18%), and decreased Zn (−18%). In tissues, Cu was altered in liver (+13%), kidney (−24%), heart (−16%) duodenum (−18%), and Zn in liver (+25%) and kidney (−10%). The soluble fractions (100,000 g supernatant) were mainly affected in liver and kidney, and the subcellular fractions in heart and duodenum. MT levels were modified in basal conditions only in liver (+57%) and kidney (−36%). T4 administration partially prevented the effect of TPTY on both elements and MT concentrations. Therefore, no evidence is provided for a direct role of T4 in the metabolism of MT in a way comparable to the effects of glucocorticoids. However, MT could mediate the consequences of TPTY on metal distribution in certain organs, such as liver and kidney.