Pediocin-like antimicrobial peptides (class IIa bacteriocins) and their immunity proteins: biosynthesis, structure, and mode of action.

Pediocin-like antimicrobial peptides (AMPs) form a group of lactic acid bacteria produced, cationic membrane-permeabilizing peptides with 37 to 48 residues. Upon exposure to membrane-mimicking entities, their hydrophilic, cationic, and highly conserved N-terminal region forms a three-stranded antiparallel beta-sheet supported by a conserved disulfide bridge. This N-terminal beta-sheet region is followed by a central amphiphilic alpha-helix and this in most (if not all) of these peptides is followed by a rather extended C-terminal tail that folds back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal half. There is a flexible hinge between the beta-sheet N-terminal region and the hairpin C-terminal region and one thus obtains two domains that may move relative to each other. The cationic N-terminal beta-sheet domain mediates binding of the pediocin-like AMPs to the target-cell surface through electrostatic interactions, while the more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the hydrophobic part of the target-cell membrane, thereby mediating leakage through the membrane. The hinge provides the structural flexibility that enables the C-terminal hairpin domain to dip into the hydrophobic part of the membrane. Despite extensive sequence similarities, these AMPs differ markedly in their target-cell specificity, and results obtained with hybrid AMPs indicate that the membrane-penetrating hairpin-like C-terminal domain is the major specificity determinant.Bacteria that produce pediocin-like AMPs also produce a 11-kDa cognate immunity protein that protects the producer. The immunity proteins are well-structured, 4-helix bundle cytosolic proteins. They show a high degree of specificity in that they largely recognize and confer immunity only to their cognate AMP and in some cases to a few AMPs that are closely related to their cognate AMP. The C-terminal half of the immunity proteins contains a domain that is involved in specific recognition of the C-terminal membrane-penetrating specificity-determining hairpin domain of the cognate AMP.     

Differential expression of the epidermal K1 and K10 keratin genes during mouse embryo development.

Induction of genes coding for the K1 and K10 keratins during mouse development was studied by measuring the accumulation of their respective mRNAs in day 10 to 17 embryos using an RNase protection assay. Although these two keratins are coexpressed in the suprabasal layers of the epidermis, it was found that while K1 mRNA was detectable as soon as day 10, K10 mRNA was not detectable before day 12. The expression of these genes at this stage of development was not expected since they are specifically associated with keratinization, a process that does not begin before day 17 of gestation. Histological examination of the epidermis of day 10 to 17 embryos suggests that both genes are induced in cells committed to epidermal differentiation, after stratification has started but before the onset of keratinization. It was also found that the two mRNAs increased in abundance steadily and significantly until day 16 and that, in spite of the expectation that filaments should contain equivalent amounts of each subunit, K1 mRNA remained more abundant than K10 mRNA at all times including in adult epidermis. These observations indicate that the two genes are regulated independently during development.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic Subunit and in a Regulatory factor of the glutamy-transfer ribonucleic acid synthetase.

The glutamyl-transfer ribonucleic acid synthetase (GluRS) of a partial revertants (ts plus or minus) of the thermosensitive (ts) mutant strain JP1449 (LOcus gltx) and of a ts mutant strain EM111-ts1 with a lesion in or near the locus gltx have been studied to find the relation between these two genetic loci known to influence the GluRS activity in vitro and the presence of a catalytic subunit and of a regulatory subunit in the GluRS purified from Escherichia coli K-12. The ts character of strain JP1449-18ts plus or minus is co-transduced with the marker dsdA at the same frequency as is the ts character of strain JP1449. Its purified GluRS is very thermolabile and its Km for glutamate is higher than that of a wild-type GluRS. These results indicate that the locus gltX is in the structural gene for the catalytic subunit of this enzyme. The location of the mutation causing the partial ts reversion in strain JP1449-18ts plus or minus is discussed. The GluRS purified from the ts mutant strain EM111-ts1 has the same stability as the wild-type enzyme, but its Km forglutamate increases with the temperature, suggesting that the locus gltE codes for a regulatory factor, possibly for the polypeptide chain that is co-purified with the catalytic subunit.     

Coronary hypertonia and angina

Coronary hypertonia was observed in a patient with unstable angina. It was possible on one occasion to reproduce the clinical picture, electrocardiographic changes, lactate production and coronary hypertonia by means of atrial pacing. He had a normal left coronary arteriogram when the diffuse spasm was relieved by nitroglycerin. Therefore hypertonia (or spasm) of the left coronary artery was believed to be the cause of his variant angina with subendocardial ischemia.     

Analysis of the androgenic activity of synthetic "progestins" currently used for the treatment of prostate cancer

In order to assess the androgenic activity of synthetic "progestins" currently used as "antiandrogens" for the treatment of prostate cancer in men, the effect of a series of these compounds has been measured following 14 days of treatment of adult castrated rats on specific and sensitive parameters of androgenic activity, namely ventral prostate weight and prostatic ornithine decarboxylase (ODC) activity. Medroxyprogesterone acetate (MPA) is almost equipotent with 5 alpha-dihydrotestosterone (DHT), a 49% increase in prostatic weight being observed at the low dose of 0.15 mg, twice daily (P less than 0.01). Megestrol acetate (Megace), chlormadinone acetate (CMA) and spironolactone were less potent but caused a 36-59% increase in prostatic weight at the highest dose used, namely 10 mg. At the 5 mg dose, cyproterone acetate (CPA) caused a 75% increase in prostatic weight. The androgenic activity of the compounds is even more clearly illustrated by their marked stimulatory effect on prostatic ornithine decarboxylase (ODC) activity. MPA, at the low dose of 0.15 mg, caused a 20-fold increase (relative to the effect of placebo) in the activity of the enzyme while the same dose of DHT caused a 15-fold stimulation of enzymatic activity. At the 10 mg dose, megestrol acetate, CMA and spironolactone caused 13.1, 11.8 and 8.6-fold stimulations of ODC activity, respectively. Flutamide, on the other hand, had no stimulatory effect on either ventral prostate weight or prostatic ODC activity. In agreement with glucocorticoid activity, MPA, megestrol acetate and CMA caused a marked inhibition (45-64%) of adrenal weight. The present data show that MPA is a highly potent androgen while megestrol acetate, CMA, CPA and spironolactone have lower but significant androgenic activity on all the parameters measured. It should be added that MPA, megestrol acetate and CMA are completely devoid of antiandrogenic activity while spironolactone shows weak antiandrogen action and CPA is a mixed agonist-antagonist. Flutamide, the compound used as reference, is the only compound devoid of any androgenic action and is thus acting as a pure antiandrogen on both ventral prostate weight and prostatic ODC activity. The present data have major implications for the choice of drug to be used for the treatment of androgen-sensitive diseases, especially prostate cancer. As shown by the present data, the synthetic "progestins" so-far available all possess variable levels of androgenic activity and are thus not recommended for the treatment of prostate cancer.     

Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.

Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids. These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences. From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci. Two, possibly three, of these loci are physically linked. A. caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components. Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants. When wild-type A. caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold. In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h. While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate. Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation. Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism. During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2. In A. caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate. Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate. Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.