Kinetic analysis of short-term effects of alpha-agonists on gluconeogenesis in isolated rat hepatocytes

Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10(-5) M) as alpha-adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol-3-phosphate dehydrogenase and glycerol kinase.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.     

Elevation of the number of cell-surface insulin receptors and the rate of 2-deoxyglucose uptake by exposure of 3T3-L1 adipocytes to tolbutamide

Sulfonylurea compounds are hypoglycemic agents which by unknown mechanisms alter the amount of insulin receptor and the rate of glucose utilization in tissues exposed to the drugs. In this study the effects on insulin binding and uptake of 2-deoxyglucose by 3T3-L1 adipocytes were assessed after maintaining cell monolayers for 1-3 days in medium containing different concentrations of the sulfonylurea, tolbutamide. The amount of 125I-insulin bound by treated monolayers gradually increased to values 150-250% of those of control monolayers after 2-3 days of exposure to 1.5 mM tolbutamide. Such increases in insulin binding capacity arose primarily from an increase in receptor number and not from an alteration in the affinity of the receptor for insulin. Concomitant with the changes observed for the insulin receptor, tolbutamide-treated monolayers expressed 1.5-2-fold higher rates of uptake of 2-deoxyglucose relative to control monolayers at concentrations of insulin between 0 and 10(-10) M. This study thus demonstrates the responsiveness of adipocytes to tolbutamide and also establishes the usefulness of 3T3-L1 cells as a model system in which to study the mechanism of tolbutamide action, both as it relates to the use of sulfonylurea compounds in clinical applications and as possible probes for perturbing and studying relatively uncharacterized regulatory pathways controlling receptor level and biological responses to insulin.     

Repopulation kinetics in irradiated mouse lip mucosa: the relative importance of treatment protraction and time distribution of irradiations

The repopulation kinetics of the irradiated lip mucosa of mice has been investigated. Split-dose experiments showed that, in this tissue, repopulation starts within 3 days after the first irradiation and increases exponentially within 10 days. To assess the relative importance of protraction and distribution of irradiations as a function of time, 10 fractions were given in (1) 3 days (three irradiations per day with a 4-hr interval), (2) 11 days (daily fractions), or (3) two short courses, each consisting of five fractions given in 1.5 days separated by a rest period of 8 days, with an overall time of 11 days. The results show that by protracting the treatment from 3 to 11 days (with daily irradiations) repopulation accounts for recovery of approximately 13 Gy. Delivering the radiation in two short courses separated by a rest period leads to an additional recovery of approximately 5 Gy. The most plausible explanation for this observation is that repopulation is much more efficient during the rest period between the two courses than during continuous daily irradiation. Although the regimen of two short courses with a rest period spares the acute reaction, it will not enhance the late tolerance. Before thorough knowledge about the repopulation kinetics of the tumors can be gained, caution should be observed for indiscriminate use of split-course multiple-fraction-per-day (MFD) regimens for treating various tumors.     

Anaerobic digestion of high-strength acidic whey in a pH-controlled up-flow fixed film loop reactor

Summary A fixed film loop reactor was developed for the stabilization of undiluted sour whey. Porous clay beads were used to immobilize the population. The fermentation system was self-supporting with the aid of a pH-titrator. Within 2 months; the loading increased automatically to its maximum of 14 kg COD (chemical oxygen demand)/m³ per day. Parallel to this, the bacterial film was formed on the surface of the support material. For a pH of 6.7 the steady state was reached at a hydraulic retention time of 5 days equivalent to a loading of 14 kg COD/m³ per day. An amount of 5.6 m³ biogas was produced per m³ digester content and day and the COD-reduction was 95%. The pH-controlled whey addition led to only minor disturbances when overloading or oxygenation occured and a fast recovery of methanogenesis was observed. The economics of anaerobic whey digestion compared with conventional whey utilization is estimated by a simple cost/benefit calculation.     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.     

Human DNA sequences exhibiting gamete-specific hypomethylation.

Three human DNA sequences have been cloned from DNA regions which are strikingly undermethylated in sperm, highly methylated in adult somatic tissues, and methylated to an intermediate extent in tissues of extraembryonic origin. It is proposed that some such DNA sequences may function specifically early in embryogenesis or during gametogenesis. They may be subsequently extensively methylated in the embryonic cell lineage and methylated to a lesser extent in extraembryonic tissues in order to allow embryogenesis to proceed.