Inhibition of hepatic deiodination of thyroxine is caused by selenium deficiency in rats.

Selenium (Se) deficiency produced up to a 14-fold decrease in hepatic tri-iodothyronine (T3) production from thyroxine (T4) in vitro. The T3 production rate could not be restored by the addition of a variety of cofactors, nor by the addition of control homogenate. The impairment in hepatic T3 production observed in Se deficiency was reflected in the concentrations of thyroid hormones circulating in plasma, T4 being increased approx. 40% and T3 being decreased by 30%. However, the fall in plasma T3 concentrations was smaller than might be expected in view of the marked decreased in T3 production. Se deficiency had no measurable effect on plasma reverse-tri-iodothyronine concentrations. The data suggest that Se deficiency produces an inhibition of both 5- and 5'-deiodination, consistent with the widely held view that these reactions are catalysed by the same enzyme complex. The mechanism of inhibition appears not be mediated by changes in thiol levels, but a direct role of Se in the activity of the deiodinase complex cannot be excluded.     

Identification and characterization of two novel lymphocyte function-associated antigens, L24 and L25

We describe the function and cell distribution of two novel cell surface antigens, L24 and L25. These antigens are broadly distributed on human lymphocytes. Monoclonal antibodies specific for these molecules block lysis by Class I- and II-specific cytotoxic T lymphocytes, but do not affect any other T cell functions tested. Anti-L24 antibody immunoprecipitates a molecule composed of two disulfide-linked monomers of 140 kd each. Anti-L25 antibody immunoprecipitates three proteins of 150, 85, and 75 kd. The study of these and other function associated molecules may provide insight into mechanisms of cytotoxic T lymphocyte recognition and/or function.     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

Low-Level Microwave Irradiations Affect Central Cholinergic Activity in the Rat

Sodium-dependent high-affinity choline uptake was measured in various regions of the brains of rats irradiated for 45 min with either pulsed or continuous-wave low-level microwaves (2,450 MHz; power density, 1 mW/cm2; average whole-body specific absorption rate, 0.6 W/kg). Pulsed microwave irradiation (2-microseconds pulses, 500 pulses/s) decreased choline uptake in the hippocampus and frontal cortex but had no significant effect on the hypothalamus, striatum, and inferior colliculus. Pretreatment with a narcotic antagonist (naloxone or naltrexone; 1 mg/kg i.p.) blocked the effect of pulsed microwaves on hippocampal choline uptake but did not significantly alter the effect on the frontal cortex. Irradiation with continuous-wave microwaves did not significantly affect choline uptake in the hippocampus, striatum, and hypothalamus but decreased the uptake in the frontal cortex. The effect on the frontal cortex was not altered by pretreatment with narcotic antagonist. These data suggest that exposure to low-level pulsed or continuous-wave microwaves leads to changes in cholinergic functions in the brain.     

Characterization of specific fluorenylmethyloxycarbonyl-containing calmodulin adducts by spectroscopy and phosphodiesterase stimulation

A reagent (I, N⁴-(9-fluorenylmethyloxycarbonyl-4-amino-1-oxyl-4-succinimidyloxycarbonyl-2,2,6,6-tetramethylpiperidine)) that acylates calmodulin specifically at lysines 75 and 148 was recently described (Jackson and Puett, 1984). Chromatographic procedures are described that permit purification to apparent homogeneity of a 1 : 1 and a 2 : 1 adduct characterized by modification at just Lys 75 or at Lys 75 and Lys 148, respectively. These adducts are suitable for detailed characterization in an effort to provide information on calmodulin structure-function relationships. The adducts were incapable of, or exhibited low potency (e.g., 0.1% that of calmodulin) in, stimulating the activity of an activatable bovine brain cyclic nucleotide phosphodiesterase (3,5-cyclic AMP 5-nucleotidehydrolase, EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotational correlation times of approximately 3–6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (5–7 nsec). Thus, the nitroxide reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestingly, removal of the fluorenylmethyloxycarbonyl portion from the 1 : 1 adduct to give a deprotected 1 : 1 adduct resulted in apparent greater mobility of the probe, since the rotational correlation coefficient was found to be 1 nsec. Circular dichroic spectra were obtained over the wavelength interval 200–250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmodulin, exhibited a Ca²⁺-mediated increase in helicity, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca²⁺ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetic resonance spectra were determined in the aromatic region (6–8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide with ascorbate. The nitroxide had little effect on the chemical shifts of the two tyrosines and the single histidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound II, -N-(9-fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct at Lys 75 and a 2 : 1 adduct at Lys 75 and Lys 148. Proton NMR spectra of adducts with II were not complicated by the relaxation effects arising from adducts with I; thus more definitive assignments could be made to the upfield resonances, including the fluorene protons. Again, it was possible to conclude that adduct formation had no major effect on the tertiary structure of the protein as monitored by chemical shifts associated with various residues. We conclude that modification of just Lys 75, a residue in the long connecting helix of calmodulin, does not lead to major changes in protein conformation but does interfere with the ability of calmodulin to stimulate an activatable form of bovine brain cyclic nucleotide phosphodiesterase.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Depressed natural cytotoxicity but normal natural killer cytotoxic factor (NKCF) production by mononuclear cells derived from patients with hepatocellular carcinoma

Summary This study investigated the relation between the production of natural killer cytotoxic factors (NKCF) and the phenomenon of natural killing (NK) activity against target K562 cells. Two different models of defective NK cell activity were employed. In the first instance, cytotoxic activity of mononuclear cells (MN) derived from patients with hepatocellular carcinoma was compared to the ability of these cells to produce NKCF. Although direct cytotoxicity was considerably impaired in these patients, the ability of their MN to produce NKCF when stimulated with K562 cells was found to be normal. In the second model, MN treated with the lysosomotropic drug monensin showed considerably reduced direct cytotoxic activity, although they were capable of producing normal amounts of NKCF when activated by K562 cells. These results therefore indicate that there is no correlation between NK activity and corresponding NKCF release, and suggest that NKCF production and activity is independent of direct NK cytotoxic activity.     

p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.