Carbon source-dependent synthesis of SecB, a cytosolic chaperone involved in protein translocation across Escherichia coli membranes.

SecB is a cytosolic chaperone involved in protein translocation across cytoplasmic membranes in Escherichia coli. It has been shown to be required for efficient translocation of a subset of precursor proteins but is not essential for cell viability. This study investigated whether synthesis of SecB is growth rate dependent. Interestingly, the total amount of SecB synthesized in the cells was relatively small. Moreover, the levels of SecB were found to be carbon source dependent since more SecB was produced in cells grown in glycerol media than in cells grown in glucose media, regardless of the growth rate. This is in contrast to the other Sec proteins, whose synthesis is growth rate dependent and not related to glucose as a carbon source. In addition, cyclic AMP (cAMP) partially relieves the lower levels of SecB observed in glucose medium, a compensatory effect that depends on the presence of both cya and crp gene products. Thus, the glucose-dependent synthesis of SecB may be related to the cAMP-cAMP receptor protein complex-mediated activation.     

A comparison of some pharmacological actions of prostaglandin E1, 6-oxo-PGE1 and PGI2

Some pharmacological actions of prostaglandin E1 (PGE1), 6-oxo-PGE1 and PGI2 have been studied. 6-oxo-PGE1 and PGE1 relaxed guinea-pig tracheal muscle in vitro and increased nasal patency in normal volunteers and in subjects with vasomotor rhinitis whereas PGI2 produced opposite effects. All three compounds produced bronchodilatation in the anaesthetised guinea-pig and relaxed human respiratory tract muscle in vitro. PGI2 was several times more potent than either 6-oxo-PGE1 or PGE1 against ADP-induced aggregation of human and baboon platelets in vitro. Intravenous 6-oxo-PGE1 in the baboon caused an ex vivo inhibition of platelet aggregation, but the EC50 was 7.7 times that of PGI2. As a vasodepressor in the baboon 6-oxo-PGE1 and PGI2 were equipotent. Thus with the exception of the vasodepressor effect, the actions of 6-oxo-PGE1 qualitatively and quantitatively resembled those of the structurally related PGE1 rather than those of PGI2.     

Hydrolysis of bile acid conjugates by selected fungi

Summary Fungi which have been previously shown to hydrolyse glycocholic acid, with liberation of the free bile acid, have now been shown to be similarly capable of hydrolysing glycodeoxycholic acid. Sodium taurocholate, however, is much less susceptible and its hydrolysis has been demonstrated with only one of the selected fungi, Penicillium chrysogenum, growing in a medium containing the conjugate as the sole sulphur source. It is concluded that the nature of the amino acid moiety is important in determining the ease of hydrolysis of bile acid conjugates by whole cells of the fungi under test.     

Metabolism of prostaglandins in spontaneously hypertensive rats: NAD"-dependent 15-hydroxyprostaglandin dehydrogenase activity is decreased in kidney and increased in lung.

Renal, pulmonary and gastric NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase activities were determined in both spontaneously hypertensive and normotensive rats at 6 and 12 weeks of age. Renal enzyme activity in hypertensive rats was only 30–40% of that present in normotensive controls at both ages. In contract, pulmonary enzyme activity in hypertensive animals was twice as active as that in normal controls. There was no significant difference in gastric enzyme activity. NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase activity, the enzyme responsible for the conversion of vasoinactive PGF metabolites to PGE metabolites, also failed to show any difference in two types of rat kidneys. The results indicate that, in hypertension, prostaglandin inactivation is impaired in kidney but is facilitated in lung.     

Relaxation kinetics of Helix pomatia alpha-hemocyanin. Whole-half molecule reaction at pH 5.7 in 0.4 M NaCl.

Two independent relaxation kinetics methods were used to study samples of α-hemocyanin kindly furnished to us by members of the Biochemical Laboratory of the University of Groningen. A Durrum-Gibson stopped-flow apparatus was used to obtain concentration-jump data in the light-scattering mode. A recently developed pressurejump light-scattering apparatus was used to obtain completely independent data. The studies were made in 0.1 m acetate buffer at pH 5.7 containing 0.4 m NaCl, conditions under which equilibrium light-scattering studies had been reported by Engelborghs and Lontie (1973, J. Mol. Biol., 77, 577–587). In the companion paper (Kegeles, 1977, Arch. Biochem. Biophys., 180, 530–536), a model is proposed, consisting of a system containing a mixture of reactive and unreactive whole molecules, from which data are derived for the formation constant of whole molecules from halves and the fraction of material which is capable of undergoing reaction. The present study uses this estimate of this fraction of reactive material to permit the evaluation of overall rate constants and equilibrium constants. When the estimate of 65% of reactive material derived without making nonideality corrections is applied to the kinetics data, very satisfactory agreement is obtained between the equilibrium constant acquired from equilibrium data and the equilibrium constants derived from the kinetics data.     

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.     

Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.     

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.