Photoaffinity labeling of the Ah receptor

A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected for radiosynthesis with 125I (specific activity 2176 Ci/mmol, equilibrium dissociation constant, KD = 0.76 nM). Following incubation of this 125I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultraviolet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:1 ratio; 3) co-incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a similar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed.     

Measurement of capillary pressure in humans using a venous occlusion method

The venous occlusion technique was used to measure capillary pressure in the forearm and foot of man over a wide range of venous pressures. In six recumbent subjects venous pressure (Pv) in the forearm (mean +/- SE) was 9.3 +/- 1.4 mmHg and the venous occlusion estimate of capillary pressure (Pc) was 17.0 +/- 1.6 mmHg, whereas in another six subjects Pv in the foot was 17.1 +/- 1.2 mmHg and Pc was 23.4 +/- 2.5 mmHg. Venous pressure in the limbs was increased either by changes in posture or by venous congestion with a sphygmomanometer cuff. On standing Pv in the foot increased to 95.2 +/- 1.5 mmHg and Pc rose to 112.8 +/- 3.1 mmHg. The relationship established between venous pressure and capillary pressure in the forearm is Pc = 1.16 Pv + 8.1, whereas in the foot the relationship is Pc = 1.2 Pv + 1.6. The magnitude and duration of the changes in capillary pressure were also recorded during reactive hyperemia. The venous occlusion method of measuring capillary pressure is simple and easily applied to studies in humans.     

pH-dependent processes in proteins

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.     

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

Single Doses of Acrylamide Reduce Retrograde Transport Velocity

Abstract: Single doses of acrylamide (0–1.3 mmol/kg) produced a dose-dependent decrease in the transport of ¹²⁵I-tetanus toxin to the perikarya of sensory neurons in dorsal root ganglia and motor neurons in ventral spinal cord. Acrylamide was a more potent inhibitor of retrograde transport in sensory axons than in motor axons. Substantially greater doses of N,N '-methylene-bis-acryl-amide, a reportedly non-neurotoxic analog of acrylamide, were required to alter the axonal transport of ¹²⁵I-tetanus toxin. Velocity of retrograde transport was assessed by determining the position of the leading edge of transported¹²⁵I-tetanus toxin at times following single doses of acrylamide. Acrylamide reduced the velocity of ¹²⁵I-tetanus toxin transport in a dose-dependent manner by up to 75%. No change in neuronal uptake of ¹²⁵I-tet-anus toxin was detected. It is concluded that single doses of acrylamide produce profound alterations in retrograde transport which precede the appearance of structural changes in affected nerve fibers.     

The stability of the yeast plasmid pJDB248 depends on growth rate of the culture

Summary The stability of the plasmid pJDB 248 has been measured in theS. cerevisiae strain S150-2B growing in a chemostat under conditions of glucose limitation. It was found that reducing the growth rate of the culture led to a more rapid loss of the plasmid from the cells.     

The pH dependence of hydrogen exchange in proteins

The static accessibility modified discrete charge model for electrostatic interactions in proteins is extended to the prediction of the pH dependence of hydrogen exchange reactions. The exchange rate profiles of buried amide protons are shown to follow the calculated pH dependence of the electrostatic component of protein stability. Rate profiles are calculated for individual buried amide protons in ribonuclease S and bovine pancreatic trypsin inhibitor. The electrostatic free energy of stabilization of the protein and the energy required to bring the catalytic ion to an exchange site are expressed as an apparent, pH-dependent contribution to the activation energy. Changes in the electrostatic stabilization of the proteins affect the calculated exchange rate for buried amide protons by more than 1000, while local field effects raise or lower the predicted exchange rates by less than 100. The pH dependence of exchangeable protons at the protein surface, such as the C-2 imidazole protons, is shown to follow the estimated energy required to introduce the catalytic ion at the exchange site. These calculations are discussed in terms of current models for proton exchange which incorporate the dynamic nature of the structure to explain exchange data from the interior of a protein.     

Neutral and Acid Sphingomyelinases of Rat Brain: Somatotopographical Distribution and Activity Following Experimental Manipulation of the Dopaminergic System In Vivo

The activities of neutral, magnesium-stimulated, and acid sphingomyelinases were measured in five regions of rat brain. Neutral enzyme activity was 2-3-fold higher in striatum than in parietal cortex and 13-fold higher than in cerebral white matter. Acid sphingomyelinase activity was more evenly distributed throughout these regions. Striatal neutral sphingomyelinase activity was not affected by treatment of rats with reserpine or haloperidol and was reduced (16%) by 6-hydroxydopamine. Striatal acid sphingomyelinase was unaffected by reserpine and 6-hydroxydopamine, and was increased (17%) by haloperidol. We conclude that neutral, magnesium-stimulated sphingomyelinase activity differs in various regions of rat brain and is particularly enriched in the corpus striatum. However, it appears to be a constitutive component of tissue rather than a readily modulated regulatory element of the catecholaminergic system.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Growth and lipolysis of rat adipose tissue: effect of age, body weight, and food intake

The purpose of the present work was to study age- and weight-controlled rats to determine which is the primary factor in reducing the lipolytic response of free fat cells and which has the greater effect on the ratio of fat cells to nonfat cells in adipose tissue. The method for estimating fat cell and nonfat cell numbers is based on the analysis of adipose tissue and fat cell DNA and lipid. In adequately fed rats, epididymal adipocyte hyperplasia is complete between 9 and 14 wk of age. Chronic underfeeding delays, but does not eliminate, normal fat cell hyperplasia and is accompanied by a net loss in the nonfat cell population. During 9-14 wk of age, rat epididymal adipose tissue enlarges mainly through adipocyte hypertrophy. Total fat cells from the epididymal adipose tissue of control rats represent only 20-23% of the total cell population. Chronic underfeeding increases the percentage of fat cells in the fat pad from 23 to 28%. Noradrenaline-stimulated lipolysis is proportional to fat cell numbers but is inhibited when fat cell lipid increases to over 80% of fat pad wet weight. Rat age is apparently not primarily responsible for the decreased noradrenaline-stimulated lipolysis in fat cells of 350-g rats in vitro.