Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes

We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage.     

Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata

We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.     

Preparative electrophoretic method for the purification of a hydrophobic membrane protein: subunit c of the mitochondrial ATP synthase from rat liver.

A method is described for the purification of subunit c of ATP synthase from rat liver mitochondria. After sample preparation and solvent extraction, the protein was purified to homogeneity by a single-step preparative electrophoretic procedure, using aqueous buffer and containing lithium dodecyl sulfate. The subunit is an extremely hydrophobic and insoluble protein and all solubilization attempts, using a variety of detergents, were unsuccessful except for lithium dodecyl sulfate. Buffer exchange and FPLC gel filtration removed the detergent from the purified sample, leaving the protein in a soluble form. The mammalian protein is composed of 75 amino acid residues, with a molecular mass of 7602 Da and is classified as a proteolipid. Subunit c accounts for 25 and 85% of the intralysosomal accumulation, within neurons, of storage material in juvenile and late-infantile forms of Batten's disease, respectively. This purification procedure allows access to a continuous supply of pure subunit c from a conventional source such as rat liver and preserves precious autopsy materials. The protein could be used as substrate in future proteolytic studies involving pepstatin-insensitive lysosomal proteases and for raising of more specific antibodies. The procedure could also be adapted/modified and used as a model for purifying other extremely insoluble proteins.     

Regulation of fatty acid synthesis in lactating rat mammary gland in the fed to starved transition: asynchronous control of pyruvate dehydrogenase, phosphofructokinase and acetyl-CoA carboxylase.

1. Withdrawal of food from lactating rats produced a rapid and dramatic decrease in the uptake of glucose by the mammary gland and an inhibition of the rate of fatty acid synthesis that could not be explained alone by decreased substrate supply to the tissue. 2. Within the first 6 hr starvation, fatty acid synthesis and pyruvate dehydrogenase activity were inhibited by 87 and 80%, respectively, but acetyl-CoA carboxylase activity did not change significantly. 3. Between 6 and 24 hr starvation, total and expressed activities of acetyl-CoA carboxylase decreased by 62 and 55%, respectively. 4. The ratio of fructose-6-phosphate/fructose-1,6-bisphosphate concentration in mammary tissue increased 9-fold during the first 6 hr starvation, indicating an inhibition of 6-phosphofructo-1-kinase. However, the major inhibition of this enzyme occurred between 6 and 24 hr starvation when this metabolite ratio increased a further 160-fold in parallel with increased tissue citrate concentration. 5. The increase in citrate concentration between 6 and 24 hr starvation correlated with acetyl-CoA carboxylase inactivation and ketone body accumulation in the mammary gland. 6. This study confirms the asynchronous control of three important regulatory steps in the pathway of glucose utilization and fatty acid synthesis in the lactating rat mammary gland.     

Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

Background

Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive.

Results

To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids) from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE). Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms.

Conclusion

Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.     

Cytogenetic analyses of eight species in the genus <Emphasis Type="Italic">Leptodactylus</Emphasis> Fitzinger, 1843 (Amphibia,Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Background

The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.

Results

Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.

Conclusions

Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.