Phylogenetic relationships between annual and perennial species of Helianthus: evolution of a tandem repeated DNA sequence and cytological hybridization experiments.

The amplification and chromosomal localization of tandem repeated DNA sequences from Helianthus annuus (clone HAG004N15) and the physical organization of ribosomal DNA were studied in annual and perennial species of Helianthus. HAG004N15-related sequences, which did not show amplification in other Asteraceae except for Viguiera multiflora, were redundant in all the Helianthus species tested, but their frequency was significantly higher in perennials than in annuals. These sequences were located at the ends and intercalary regions of all chromosome pairs of annual species. A similar pattern was found in the perennials, but a metacentric pair in their complement was not labelled. Ribosomal cistrons were carried on two chromosome pairs in perennials and on three pairs in annuals except for H. annuus, where rDNA loci were on four pairs. No difference was observed between cultivated H. annuus and its wild accessions in the hybridization pattern of the HAG004N15 and ribosomal probes. These findings support the hypothesis that the separation between annual and perennial Helianthus species occurred through interspecific hybridization involving at least one different parent. However, GISH in H. annuus using genomic DNA from the perennial Helianthus giganteus as blocking DNA failed to reveal different genomic assets in annual and perennial species.

Characterization and chromosomal organization of satellite DNA sequences in Picea abies.

Three clones containing satellite DNA sequences were selected from a randomly sheared genomic DNA library of Picea abies (clones PAF1, PAG004P22F (2F), and PAG004E03C (3C)). PAF1 contained 7 repeats that were 37-55 bp in length and had 68.9%-91.9% nucleotide sequence similarity. Two 2F repeats were 305-306 bp in length and had 83% sequence similarity. Two 3C repeats were 193-226 bp in length and had a sequence similarity of 78.6%. The copy number per 1C DNA of PAF1, 2F, and 3C repeats was 2.7 x 10(6), 2.9 x 10(5), and 2.9 x 10(4), respectively. In situ hybridization showed centromeric localization of these sequences in two chromosome pairs with PAF1, all pairs but one with 2F, and three pairs with 3C. Moreover, PAF1 sequences hybridized at secondary constrictions in six pairs, while 2F-related sequences were found at these chromosome regions only in four pairs. These hybridization patterns allow all chromosome pairs to be distinguished. PAF1-related repeats were contained in the intergenic spacer (IGS) of ribosomal cistrons in all six nucleolar organizers of the complement, while sequences related to 2F were found on only one side of the rDNA arrays in four pairs, showing structural diversity between rDNA regions of different chromosomes.

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence.

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.

Effect of furosemide on the rat submandibular gland kallikrein secretion.

The effects of furosemide and Captopril were studied in normals and nephrectomized rats. Different doses of furosemide (5 to 50 mg/kg) increased the saliva kallikrein activity of submaxillary gland perfused with pilocarpine. Rats injected with captopril (10 mg) increased the blood flow of the gland, but did not modify the blood pressure. After furosemide (50 mg/kg) and captopril (10 mg), a decrease in arterial blood pressure was observed. The results suggest a release of glandular kallikrein which is secreted from the gland directly into the vascular compartment. On the other hand, rats sialodectomized showed no alterations in blood pressure in response to both drugs. These data suggest that submaxillary gland kallikrein play a role in regulating blood flow of the gland and blood pressure, at least in our experimental conditions.

Insulin binding and degradation in short time incubation at 37 C.

Studies on insulin-receptor binding in a short time incubations at 37 C have shown that neither internalization nor receptor-mediated insulin degradation are demonstrable during the first minutes. In the present study insulin receptor binding at 37 C in short time incubation periods was studied in mouse-hepatocytes, simultaneously determinating the proportion of degradation due to the cell activity. Degradation in the incubation buffer after cell separation was abolished during the experiment (900 sec) by a careful wash of the cells. 7.5 cells/ml were incubated with a tracer concentration (14.17 pM) of 125I-insulin and a pharmacological concentration (16.6 microM) of native insulin plus tracer. In the case of tracer insulin, 50% binding was reached in 55 sec and steady state in 160 sec. Once reached, steady state persisted along the experimental time. Binding follows a second order kinetics with k+1: 5 649 X 10(6) M-1 sec-1. In the presence of pharmacological insulin there is competitive inhibition of the tracer which reduces to zero the percent of binding. Binding increases along the time taking positive values, and the slope of binding versus time intersects the abscissa at 102 sec (r: 0.864). As long as binding of the tracer takes place, no degradation occurs until 635 sec, when a degradation slope abruptly appears (r: 0.722). Dissociation studies were followed previous incubation at 37 C during 200 sec with tracer and pharmacological doses. Specific dissociation follows a monoexponential kinetics with k-1: 3 067 X 10(-3) sec-1 and t 1/2: 226 sec. Eighty percent of bound insulin is dissociated with no changes in the slope (r: 0.820), thus suggesting that insulin-receptor binding in the present experimental conditions is basically a reversible process. No degradation was observed during dissociation, which demonstrates that insulin-receptor binding does not degrade insulin if internalization is not performed. At steady state, competitive inhibition curves showed two components: high and low affinity. Doses of 1.66 microM produce a 98% inhibition in the binding of 125I-insulin. The high affinity slope shows two components in the physiological range of insulin concentrations. The first one of very high affinity has a dissociation constant Ko: 7 075 X 10(-10), and a binding capacity of 1.5 X 10(-10). This study demonstrates that, with physiological concentrations of insulin, internalization is the only mechanism of insulin degradation in mouse-hepatocytes.

Liver cells binding with high insulin doses at 37 C.

Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100% oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg% glucose, 1% bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53% of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.

Insulin binding and degradation in short time incubation at 37 C.

Studies on insulin-receptor binding in a short time incubations at 37 C have shown that neither internalization nor receptor-mediated insulin degradation are demonstrable during the first minutes. In the present study insulin receptor binding at 37 C in short time incubation periods was studied in mouse-hepatocytes, simultaneously determinating the proportion of degradation due to the cell activity. Degradation in the incubation buffer after cell separation was abolished during the experiment (900 sec) by a careful wash of the cells. 7.5 cells/ml were incubated with a tracer concentration (14.17 pM) of 125I-insulin and a pharmacological concentration (16.6 microM) of native insulin plus tracer. In the case of tracer insulin, 50

binding was reached in 55 sec and steady state in 160 sec. Once reached, steady state persisted along the experimental time. Binding follows a second order kinetics with k+1: 5 649 X 10(6) M-1 sec-1. In the presence of pharmacological insulin there is competitive inhibition of the tracer which reduces to zero the percent of binding. Binding increases along the time taking positive values, and the slope of binding versus time intersects the abscissa at 102 sec (r: 0.864). As long as binding of the tracer takes place, no degradation occurs until 635 sec, when a degradation slope abruptly appears (r: 0.722). Dissociation studies were followed previous incubation at 37 C during 200 sec with tracer and pharmacological doses. Specific dissociation follows a monoexponential kinetics with k-1: 3 067 X 10(-3) sec-1 and t 1/2: 226 sec. Eighty percent of bound insulin is dissociated with no changes in the slope (r: 0.820), thus suggesting that insulin-receptor binding in the present experimental conditions is basically a reversible process. No degradation was observed during dissociation, which demonstrates that insulin-receptor binding does not degrade insulin if internalization is not performed. At steady state, competitive inhibition curves showed two components: high and low affinity. Doses of 1.66 microM produce a 98

inhibition in the binding of 125I-insulin. The high affinity slope shows two components in the physiological range of insulin concentrations. The first one of very high affinity has a dissociation constant Ko: 7 075 X 10(-10), and a binding capacity of 1.5 X 10(-10). This study demonstrates that, with physiological concentrations of insulin, internalization is the only mechanism of insulin degradation in mouse-hepatocytes.

Liver cells binding with high insulin doses at 37 C.

Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100

oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg

glucose, 1

bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53

of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.