Characterization of two enzyme proteins catalyzing NADP+/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 from swine kidney

Two proteins (pI 4.8 and 5.8) capable of catalyzing NADP⁺/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 but not at C-11 have been purified to homogeneity from swine kidney. Both proteins exhibited identical molecular weight and subunit size. Similar amino acid composition, antigenic determinants, and coenzyme and substrate specificity were also found. The molecular weight of the enzyme as determined by gel filtration was 29,000. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel gave a value of 29,500 indicating the presence of a single polypeptide chain. Either enzyme protein utilized a variety of prostaglandins (PGS) as substrates. PGA₁-glutathione conjugate and PGB₁ were found to be the best substrates for prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase activities, respectively. For prostaglandins having dual reactive groups in a single molecule, the rate of oxidation of PGF_2α at C-15 was comparable to that at C-9, whereas the rate of reduction of 15-keto-PGE₂ at C-15 was far greater than that at C-9.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

黑龙江省东宁县山区蜱类的生态调查

我国东北地区有的蜱种已证实是传播森林脑炎、北亚蜱传斑疹伤寒的媒介。近年来,国内文献又报导了从东北牡丹江林区发现了莱姆病(Lyme Disease)病人,病人都有被婢咬史,并从全沟硬蜱(Ixodes persulcatus)的中     

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.     

Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and [35S]methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.     

Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.

To gain insight into the mechanism of facilitated sugar transport and possible mechanisms by which glucose transporter intrinsic activity might be altered, we have investigated conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding and by the transporter inhibitor, cytochalasin B. Changes in the ability of thermolysin to digest glucose transporters present in erythrocyte ghosts were used to monitor conformational changes of the glucose transporter. The degree of protease digestion was determined by the amount of undigested glucose transporter remaining after the protease treatment, as assessed in Western blots using the glucose transporter specific monoclonal antibody 7F7.5. D-Glucose, the physiological substrate of the transporter, increased the transporter's susceptibility to cleavage by thermolysin. Nontransportable glucose analogues which bind specifically to either an internal or external glucose transporter sugar binding site also altered susceptibility of the transporter to thermolysin. Both methyl and propyl glucoside, which preferentially bind the internal sugar site, increased thermolysin susceptibility of the glucose transporter in a manner similar to that of D-glucose. In contrast, 4,6-O-ethylideneglucose, which preferentially binds the external sugar site, protected the transporter from thermolysin digestion. These results suggest that sugar binding to internal and external sugar sites induces distinct conformational changes and that the observed D-glucose effect on the susceptibility of the glucose transporter to thermolysin is due to D-glucose at equilibrium predominantly forming a complex with the internal sugar site. The protection from cleavage by thermolysin caused by external sugar binding is attenuated by the addition of an internally binding sugar.(ABSTRACT TRUNCATED AT 250 WORDS)     

The conventional short-circuiting technique under-short-circuits most epithelia

Summary The relationships among ion current, membrane potential difference, and resistance of an epithelium are studied. The short-circuit technique introduced by Ussing and Zerahn does not completely short circuit the epithelium if the series resistance parallel to the cell layer between the voltage electrodes is not properly compensated. The residual potential difference across the epithelial cell layer in the short-circuit state is proportional to both the measured short-circuit current and the resistance of the diffusion barriers not compensated. In the conventionally short-circuited small intestinal mucosa the villus and crypt areas are hypo-polarized to different degrees rather than simultaneously hyper- and hypo-polarized. Short-circuiting the whole tissue reduces but does not abolish the passive net ion movement across the tissue. Measurements of the electrical properties of the whole and denuded rat distal small intestine in HCO₃-Ringer solution containing 10mm glucose reveal that the measured short-circuit current has under-estimated approximately 33% of the true short-circuit current and that the passive net Na flux from serosa to mucosa and Cl flux from mucosa to serosa are not negligible in the short-circuit state.     

Release of arachidonate from diglyceride in human platelets requires the sequential action of a diglyceride lipase and a monoglyceride lipase

Release of arachidonate from 2-arachidonyl diglyceride by human platelet microsomes was investigated. Diglycerides labeled with ¹⁴C-stearate at sn-1 and with ³H-arachidonate at sn-2 were used as a substrate for microsomal diglyceride lipase. Diglyceride was deacylated first at sn-1 as evidenced by the accumulation of 2-arachidonyl monoglyceride but not of 1-stearoyl monoglyceride. Subsequent release of arachidonate from monoglyceride required the action of a monoglyceride lipase. Studies on substrate specificity indicated that diglyceride lipase utilized 2-arachidonyl diglyceride as the best substrate.     

Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis.

Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers. Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli. Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal. Moreover, with phage RNA these concentrations increased the synthesis of large polypeptides. We conclude that binding of gentamicin to its first site causes inhibition but little misreading; binding to additional site(s) partly reverses the inhibition by first-site binding and markedly stimulates misreading, and the misreading appears to favor "readthrough" of termination codons. In the third phase (greater than 100 micron) synthesis is slowed again but the pattern of misreading does not appear to be altered; this effect need not involve a specific further action on the ribosome.     

Hydrolase production by Aspergillus niger in solid-state cultivation

Summary A production of macerating enzymes which liquefy and hydrolyze the mandarin orange peel was studied in a solid state cultivation of Aspergillus niger on wheat bran substrate. Solid state cultivation in a 2 drum fermenter capable of interchangeable operation under dynamic or static conditions were carried out maintaining the moisture content of the substrate at 32, 39, 46, 56, 67, and 74%. Biomass grown on the solid substrate was estimated on the basis of a constant value of glucosamine content of A. niger, 50 mg glucosamine/g cell. A linear relationship between oxygen uptake rate and growth rate observed in all the experiments gave an oxygen growth yield, Y_X/O, of 28.5 g cell/mol O₂. The rate of macerating enzyme formation was also in proportion to the growth rate irrespective of the difference of the moisture content of the substrate.The enzyme accumulation on the solid substrate, the growth rate and oxygen uptake rate were maximum when the moisture content of the substrate was maintained at ca. 56% ascending from 32 to 56 and descending from 56 to 74.