The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.     

Observations on 6-mercaptopurine activity in Saccharomyces cerevisiae

Abstract Hydrogen fluoride treatment of [¹⁴C-glycerol]lipoteichoic acid synthesized by growing Streptococcus faecium ATCC9790 in the presence of 1,3[¹⁴C]glycerol produced five radioactive, water-soluble products which were identified by chromatographic and analytical techniques to be tetraglucosyl glycerol, triglucosyl glycerol, diglucosyl glycerol, monoglucosyl glycerol and unsubstituted glycerol. The percent composition of each varied modestly from culture to culture and ranged between 7 and 8% for the tetra-, 20.5 and 31.2% for the tri-, 11.3 to 23.5% for the di-, 20.9 to 26.8% for the mono-, and 23.1 to 34.8% for the unsubstituted glycerol. The same glucosylated glycerol compounds could be obtained in an in vitro reaction in which a 30 000 × g particulate enzyme catalyzed the incorporation of [³H]glucose from UDP [³H]glucose into lipoteichoic acid.     

Analysis of Dysgenesis-Induced Lethal Mutations on the X Chromosome of a Q Strain of DROSOPHILA MELANOGASTER

The Q strain known as v6 was tested for its ability to induce X-linked lethal mutations in male and female hybrids from crosses with M strains in the P-M system of hybrid dysgenesis. All measurements of the mutation rate were made on the X chromosome derived from the v6 strain. The lethal rate for young hybrid males from the cross M female X v6 male was 1.11% per chromosome. For older males, it was only 0.44%, suggesting that there is less mutational or more repair activity in the germ cells of the older males or that mutant cells are selectively eliminated as the hybrid males age. The lethal rate for hybrid females from comparable crosses was approximately the same for both ages that were tested. However, it was substantially less than the rate for the hybrid males--only 0.26% per chromosome. Genetically identical hybrid females from reciprocal crosses also showed a low mutation rate, 0.13% per chromosome. Again, there was no difference between young and old flies. Mapping experiments established that most of the lethal mutations that were recovered from the male and female hybrids were located in two regions on the X chromosome, one between bands 14B13 and 15A9 , the other between bands 19A1 and 20A , which encompasses the maroonlike locus. More refined mapping of the lethals in the maroonlike region demonstrated that the vast majority of these affected a single gene located in band 19C4 . Cytological analysis of the lethal chromosomes revealed that several carried rearrangements, including inversions, duplications and deficiencies. Chromosome breakage occurred primarily in bands 14D1 -3 and 18F- 20A , and most of the breaks in the latter segment were located in 19C . However, rearrangements involving 19C and mutations of the gene in 19C4 were mutually exclusive events. In situ hybridization of a P element probe to the chromosomes of v6 demonstrated that P elements reside at a minimum of five sites on the X chromosome. These P element sites correspond to the mutational and breakage hot spots on that chromosome. The combined genetic and cytological data imply that most of the X-linked lethal mutations that occur in M X v6 hybrids are due to local P element action. Consideration of these and other data suggest that v6 is a weak P strain in the P-M system of hybrid dysgenesis and that other Q strains might also be regarded in this way.     

Arrangement of pili in colonies of Neisseria gonorrhoeae.

The morphology and arrangement of pili in the P++ colony phenotype of Neisseria gonorrhoeae were examined by a variety of electron microscopic techniques. The apparent structure and organization of gonococcal pili varied depending upon the method of specimen preparation. Pili as thin, individual, unbranched structures were demonstrated by negative staining and in sections of epoxy-embedded specimens. Pili forming thick structures which branch, subdivide, and rejoin to form an irregular lattice were demonstrated in specimens processed by the critical-point drying method and by rapid freezing and low temperature sublimination. We propose that in gonococcal colonies of the P++ phenotype, pili exist as individual threadlike structures only on the bacterial surfaces; as the pili leave the bacterial surfaces, they form thick bundles which branch, subdivide, and rejoin to form a supporting framework interconnecting the colony members. This arrangement of pili is usually disrupted by the commonly used method of negative staining and cannot be clearly detected within epoxy-embedded specimens. These data are summarized in a model depicting the organization of pili in the P++ colony phenotype of N. gonorrhoeae.     

Photoaffinity labeling of mammalian alpha 1-adrenergic receptors. Identification of the ligand binding subunit with a high affinity radioiodinated probe

We have synthesized and characterized a novel high affinity radioiodinated alpha 1-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido - 3 - [125I]iodophenyl) pentanoyl] - 1 - piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (KD = 130 pM) in a reversible and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an alpha 1-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical alpha 1-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at Mr = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the Mr = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the alpha 1-adrenergic receptor.     

Comparative foraging behavior of Neotropical robber flies (Diptera: Asilidae)

Summary Robber fly species within a Panamanian rain forest comprised distinct shade-seeking (SS) and light-seeking (LS) groups. Thoracic temperatures of LS species averaged 9.2°C greater than ambient, whereas those of SS species averaged only 1.3°C above ambient. Among SS species, attack rate decreased with increasing body mass, whereas relocation rate and attack and relocation distances increased with increasing body mass. Attack and relocation distances of LS species were similar to those of SS species of similar size. Large (>100 mg) LS species, however, had much higher attack and relocation rates than large SS species. The potential costs and benefits of basking are briefly discussed.     

Perception of solid shape from shading

Observers judged the slants and tilts of numerous regions within shaded images of ellipsoid surfaces that varied in shape, orientation, surface reflectance, and direction of illumination. The perceived three-dimensional structure of each surface was calculated from these judgments. Much of the error in observers' responses resulted from a tendency to perceive surfaces whose axes were aligned with the display screen. The presence of specular highlights or cast shadows, in contrast, had no effect on performance. The results of the experiment indicate that several assumption of certain formal models for perception of shape from shading are not psychologically valid. The most notable of these assumptions are that the visual system initially assumes that all surfaces have Lambertian reflectance and that illuminant direction must be known before shape detection can proceed. These assumptions are often accompanied by a third assumption that surface orientation is detected locally, and global shape determined by smoothing over local surface orientation estimates. The present experiment indicates that an alternative approach offered by Koenderink and van Doorn may be more psychologically accurate, as it avoids all three assumptions.Supported in part by the Air Force Office of Scientific Research (AFOSR 82-0148). The research reported in this paper was performed while the first author was a graduate student at the University of Connecticut at Storrs     

Antimicrobial activity and physico-chemical properties of some alkyldimethylbenzylammonium chlorides

A series of alkyldimethylbenzylammonium chlorides have been synthesized with n-alkyl chain lengths of C1 leads to C18. Octanol/water partition coefficients were determined and the antimicrobial activity assessed as the minimum growth inhibitory concentrations towards twelve strains of micro-organisms, representative of Gram-negative and Gram-positive bacteria, yeasts and fungi. The data were subjected to a numerical analysis. Antimicrobial activity of the compounds was found to be a parabolic function of their lipophilicity and maximized with n-alkyl chain lengths of between C12 and C16. The data fit to quadratic functions estimated for low (C1-C7) and high (C8-C16) alkyl chain length compounds was better than for a single quadratic describing the activity of the complete series (C1-C18). These maximized at log P values corresponding to alkyl-chain lengths of approximately C7 and C14 respectively, and were suggestive of low and high affinity binding sites upon the cell surface. The data analysis allowed the chain lengths of compounds with optimal activity towards the various groups of organisms to be determined. Generally yeasts and fungi were most sensitive towards C12, Gram-positive bacteria towards C14, and the Gram-negative bacteria towards C16. Gram-negative cells were the most resistant towards all the compounds and Gram-positive cells the least.     

Expression of terminal deoxynucleotidyl transferase in human thymus during ontogeny and development

Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in human thymus during ontogeny and development. In five fetal thymus samples, the enzyme activity was barely detectable. At birth, the terminal transferase activity remained low. Maximum expression of the enzyme activity occurred between 10 and 40 mo of age. Analysis of six other enzyme activities, adenosine kinase, deoxyadenosine kinase, AMP deaminase, dAMP deaminase, 5' nucleotidase, and adenosine deaminase confirmed the normal status of the thymic tissue. A careful analysis of thymic architecture revealed that involution did not occur as a result of the disease process that necessitated cardiac surgery. By immunofluorescence, the TdT antigen was localized exclusively in the nucleus of cortical thymocytes. Protein immunoblotting studies indicated that human thymic terminal transferase exists as a single high m.w. species in individuals under 30 mo of age. Thereafter, a variant m.w. species is detectable. The increase in expression of this enzyme coincides with the increase observed in serum immunoglobulin levels during maturation and precedes the maximum development of the human thymus.     

Identification of penicillin-binding protein 5a of Bacillus megaterium KM as a DD-carboxypeptidase.

Measurement of the stabilities of DD-carboxypeptidase activity and the penicillin-binding activity of proteins 5 and 5a in membranes isolated from vegetative cells and stage-V forespores suggests that the unique sporulation-specific protein 5a may be a penicillin-sensitive DD-carboxypeptidase.