Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of cultivation and induction conditions on β-lactamase production in Acinetobacter calcoaceticus

Summary The formation and localization of the -lactamase of Acinetobacter calcoaceticus CCM 5593 is strongly affected by cultivation and induction conditions. Optimal parameters for enzyme yield are cultivation on minimal salts medium with acetate (10 g·1^–1) as carbon source and addition of yeast extract (5–10 g·l^–1), induction by cefotaxime (50g·ml^–1) immediately after inoculation and growth for 24 h at 25° C. The strain forms a basal level of -lactamase constitutively [70 units (U)·g^–1]. Nearly all of this was found to be cell-bound. However, -lactamase activity additionally produced after induction (up to 500 U·g^–1 wet bacteria) was located in the culture medium (up to 96%). This unusual localization is a special feature of A. calcoaceticus and is not attributed to cell lysis. Offprint requests to: P. Borneleit     

Acute lymphoblastic leukemia in adult identical twins

The development of acute lymphoblastic leukemia (c-ALL) in identical twins is reported. The first born had ALL in 1982 and bone marrow transplantation was performed in first complete remission (CR) from his healthy twin-brother the same year. The bone marrow donor developed ALL in 1985; he received an autologous bone marrow transplantation in first CR in 1986. Unfortunately, both patients relapsed in 1986. Cytogenetic studies of the first born revealed multiple chromosomal abnormalities and a marker chromosome whereas the second patient had a Philadelphia chromosome. Genetic reasons or exposure to leukemogenic agents may be responsible for the onset of these leukemias.     

Steroid-binding site of human and rabbit sex steroid binding protein of plasma: fluorescence characterization with equilenin

The interaction of the estrogen d-3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one (equilenin) with the human and rabbit sex steroid binding proteins (hSBP and rSBP, respectively) has been investigated by using fluorescence and absorption spectroscopy. Equilenin competes for the binding of 5 alpha-dihydrotestosterone. The calculated binding constant of equilenin for rSBP is 1.9 X 10(7) M-1 at 4 degrees C, which can be compared with the binding constant of 5.7 X 10(7) M-1 reported for hSBP [Ross, J.B.A., Torres, R., & Petra, P.H. (1982) FEBS Lett. 149, 240]. The results of fluorescence quenching experiments with the collisional quenchers KI and acrylamide indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site. The fluorescence excitation spectra of SBP-equilenin complexes are similar to the absorption spectra of equilenin in low-dielectric solvents. The fluorescence emission of the SBP-equilenin complexes, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents or complexed with beta-cyclodextrin (blue shifts) but similar to the red shift produced by addition of the proton acceptor triethylamine to equilenin in cyclohexane. These data indicate that the steroid-binding site of hSBP and rSBP is a nonpolar cavity containing a proton acceptor that participates in a specific interaction, possibly a hydrogen bond, with the 3'-hydroxyl group of the bound steroid.     

Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques

Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females).     

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.     

DNA Polymorphism of the major histocompatibility class I genes and their association with serologically defined haplotypes

DNA of unrelated persons as well as members of families that were totally or partially homozygous or completely heterozygous on the loci of the major histocompatibility class I genes has been isolated from peripheral blood lymphocytes and blot hybridized with the class I pseudogene pHLA 12.4 probe. The autoradiographic DNA patterns were discussed and compared with well-defined serological features. Positive associations with serologically typed alleles had been demonstrated for HLA-A1,11 ; -A2; -A3; -B7; -B14; -B35;-Bw41; and -Cw5.     

Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.)

A virus inhibiting protein (VI) was isolated from spinach (Spinacia oleracea L.). The VI inhibited infections of test plants with plus- and minus-strand RNA viruses. Inoculation of both local lesion and systemic hosts with TMV in the presence of varying amounts of the VI resulted in typical dose response curves for the number of local lesions or the amount of virus respectively. The lowest concentration of VI leading to a significant reduction in the number of local lesions was 0.06 μg/ml. The VI was found to inhibit local lesion formation only when applied within 2–3 h p.i. but still reduced the number of local lesions when applied up to 9 h prior to virus inoculation. The antiviral activity could be attributed to a protein of molecular weight 29,000 dalton with an isoelectric point of 10.3. Its activity was destroyed by heating for 30 min to 70°C. These characteristics resemble those of other virus inhibiting proteins described for members of the order Caryophyllales such as the Phytolacca inhibitor against which a serological relationship was obtained.