Potentiation of γ-Aminobutyric Acid-Mediated Chloride Flux by Pentobarbital and Diazepam but Not Ethanol

The influx of 36Cl- into cerebral cortical and cerebellar microsacs from ICR mice and Sprague-Dawley rats was studied in incubations lasting 3 s, 500 ms, or 21 ms. In the 3-s assay, 10-40 mM ethanol did not affect either basal or gamma-aminobutyric acid (GABA)-mediated Cl- flux, at any GABA concentration tested. Only at a concentration of 600 mM did ethanol potentiate Cl- flux in both mouse and rat preparations. Ethanol (20 mM) also did not affect the significant potentiation of GABA-mediated flux produced by 50 microM pentobarbital or 2 microM diazepam in ICR mouse microsacs. In 21- and 500-ms incubations (quench-flow method), 50 microM pentobarbital significantly potentiated GABA-mediated Cl- flux in rat cortical microsacs, but 10-50 mM ethanol did not. These studies suggest that some as yet unrecognized factor is essential for ethanol enhancement of GABA-mediated Cl- flux, as reported by others in brain homogenates and in tissue culture.     

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.     

Dynamic ion-ion and water-ion interactions in ion channels.

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels.     

Thyrotropin-secreting pituitary adenoma: a case report.

We report a 44-year-old male with a thyrotropin (TSH)-secreting pituitary adenoma. Based serum free triiodothyronine (FT3, 12.1 pmol/l) and free thyroxine (FT4, 28 pmol/l) were increased with normal basal TSH (3.1 mU/l). There was impaired TSH response to thyrotropin releasing hormone (TRH) test. Serum TSH was suppressed to 59% of the basal level after oral administration of 1.4 mg 3,3'-5-triiodothyroacetic acid (triac), whereas no suppression was observed after 75 micrograms daily administration of triiodothyronine (T3). Serum concentrations of alpha-subunit of TSH (TSH-alpha) and TSH-alpha/TSH molar ratio were high, being 1.95 micrograms/l, and 4.4, respectively. Pituitary CT and MRI scan showed the presence of a macroadenoma in the anterior lobe of the pituitary gland. Histopathology of the excised pituitary confirmed the diagnosis of a TSH-producing adenoma. A positive correlation between TSH and FT3 (r = 0.66, P less than 0.01) or FT4 (r = 0.54, P less than 0.01) was observed in serial sera obtained before and after operation.     

Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.

The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.     

The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase.

X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size.

The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.     

Primary structure of human thromboxane synthase determined from the cDNA sequence.

Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase-I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450.     

Subgroups of Tcr α chains and correlation with T-cell function

T-cell receptor (Tcr) chains are classified into four subgroups (I, II, III, and miscellaneous) based on the amino acid residues at positions 61 and 62. Subgroup I has Gly Phe at these positions, subgroup II has Arg Phe, subgroup III has Arg Leu, and subgroup miscellaneous has several other combinations. Variability plots for subgroups I, II, and III sequences show higher values around positions 93–103, 105, 108, 111, 113, and 115, suggesting that these positions may interact with the processed antigen molecules. Smaller peaks are present at various other regions which may bind the major histocompatibility complex class I or II molecules. The patterns of variability within one subgroup are similar for all species, for human alone, and for mouse alone. These subgroup patterns appear much less complicated than patterns for sequences in all subgroups taken together, implying that subgroups may be related to Tcr functions. Among 83 mouse chains, 15 are from cytotoxic cells and 40 from helper cells. Of the 15 from cytotoxic cells, 11, 2, 0, and 2 are in subgroups I, II, III, and miscellaneous; and of the 40 from helper cells, 9, 16, 12, ans 3 are in subgroups I, II, III, and miscellaneous, respectively. Thus, a correlation between sequence and function of Tcr chains seems possible. Address correspondence and offprint requests to: M. Schiffer.