Carbenicillin as Inhibitor of β-Lactamase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

CARBENICILLIN was produced as a new, semi-synthetic penicillin with antibacterial activity against Pseudomonas aeruginosa and some other microorganisms¹, but this compound was known to be destroyed by staphylococcal penicillinase². Newsom et al.³ described the substrate profile of a constitutive β-lactamase from one strain of Pseudomonas aeruginosa and reported the hydrolysis of carbenicillin at a rate higher than benzylpenicillin. When compared with the inducible enzyme described by Sabath et al.⁴, it differed both in the substrate profile and the ability to hydrolyse carbenicillin. Lack of activity of the inducible enzyme on carbenicillin was also reported by Garber and Friedman⁵ when studying eight strains of Pseudomonas aeruginosa. Sykes and Richmond⁶ were able to identify three types of β-lactamases among fifty-six strains of Pseudomonas aeruginosa according to induci-bility, substrate profile and activity on carbenicillin. Type I (Sabath et al.⁴) was inducible, highly active on cephaloridine and showed no activity on carbenicillin. Types II (Sykes and Richmond⁷) and III (Newsom et al.³) were constitutive and inactivated carbenicillin at different rates. Only the constitutive enzymes conferred resistance towards carbenicillin. We have investigated the activity on carbenicillin of β-lactamases from strains of Pseudomonas aeruginosa isolated from clinical specimens. Activity on benzylpenicillin and cephaloridine was also studied.     

Isolation and Characterization of Antigenic Components of a New Heptavalent <Emphasis Type="Italic">Pseudomonas</Emphasis> Vaccine

ONE of the most serious opportunistic bacterial pathogens is Pseudomonas aeruginosa¹ and the inability of the common antimicrobial agents to combat such infections suggested the investigation of specific immunological prophylactic and therapeutic approaches. Therefore Fisher et al. began to develop a new serotype schema as a prerequisite for an immunizing preparation which would protect humans against the most prevalent strains of Pseudomonas. This communication describes the isolation, purification and preliminary characterization of seven new lipopolysaccharide antigens which were obtained from strains representative of each of the seven protective serotypes identified by Fisher et al.².     

On the Mechanism of Action of <Emphasis Type="Italic">lac</Emphasis> Repressor

Chen et al. have proved conclusively that lac repressor and RNA polymerase bind independently to wild type lac DNA in vitro. To explain the lacp ^s mutation, which causes competitive binding between repressor and polymerase, they suggest that a new promoter site has been created near the lac operator.     

Inhibition of Bacterial DNA Replication by 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo)-uracil

THE semi-conservative replication of DNA of Gram-positive bacteria is specifically inhibited by 6-(p-hydroxyphenyIazo)-uracil (HPUra; obtained from ICI) in an apparently novel mechanism^1–4. We have attempted to characterize the HPUra-sensitive site in replication using in vitro preparations of drug-sensitive bacteria. In particulate and soluble preparations of sensitive bacteria, however, HPUra at high concentration does not significantly inhibit polymerization of deoxyribonucleotides^2,4. Since these systems may not accurately represent the process of DNA replication as it occurs in vivo, we have examined the effect of HPUra on a more suitable, toluene-treated preparation of Bacillus subtilis described by Matsushita et al.⁵. In this preparation, DNA replication is ATP-dependent, utilizes deoxyribonucleotides to give biologically active DNA, semi-conservatively and sequentially in the proper gene order. HPUra can inhibit DNA replication by this system. We describe here the characteristics of HPUra inhibition and the conditions necessary for it to occur.     

Probiotic Strawberry Yogurts: Microbiological,Chemical and Sensory Properties

This study was performed to determine the viability of Lactobacillus acidophilus and Bifidobacterium bifidum in yogurt made with strawberry marmalade (SM) and to examine the quality properties of probiotic yogurt. Acidity, pH, bacterial counts and sensory analysis of the yogurt samples were investigated on days 1, 3, 5, 7, 10 and 14 during storage at 4 °C. The survival rate of L. acidophilus was greater than that of B. bifidum. The viability of L. acidophilus decreased during the storage period, but B. bifidum numbers remained stable during the storage period. The highest L. acidophilus count (7.20 log cfu/g) was found in L. acidophilus + B. bifidum SM yogurt on day 1. The highest B. bifidum count (6.13 log cfu/g) was detected in yogurt containing L. acidophilus + B. bifidum SM yogurt on day 7. Yeast and mould counts of all yogurts increased during the storage period. Coliform bacteria and Staphylococcus aureus were not detected in the yogurt samples. The highest overall acceptance sensory score was observed in yogurts containing L. acidophilus. Considering the sensory and probiotic characteristics of all yogurt samples, this study suggested that strawberry yogurt with a suitable 5–7 day storage period can be produced with single L. acidophilus addition or single B. bifidum addition.     

Amino-acid Sequence of λ Phage Endolysin

WE wish to present a preliminary report of the amino-acid sequence of λ endolysin. This protein is a lytic enzyme¹ and its structural gene, R, maps toward the right end of λ DNA². Conditional mutants as well as frame-shift mutants (R. Thomas, personal communication) have been isolated and analysed³. Hogness et al.⁴ developed a technique to assay the gene activity of the fragmented λ DNA, which suggested that it might be possible to isolate a small segment of DNA containing the endolysin gene. Purification, immunological properties and end group analysis of λ endolysin were studied by Black and Hogness^5–7.     

Repair of Thermally Induced DNA Breakage in <Emphasis Type="Italic">Escherichia coli</Emphasis>

BACTERIA grown at above optimal temperatures progressively lose viability^1,2. The degree of sensitivity is a genetic trait, correlated with sensitivity to ionizing radiation in E. coli and is characterized by single-strand DNA breaks³. The loss of viability is partly reversible by keeping the cells in buffer at a lower temperature⁴. Rosenberg et al.⁵ found a numerical correlation between the thermodynamic parameters of protein denaturation and observed death rates of various organisms and suggested that protein denaturation was a likely cause of cell death. We show here that strand breakage and the repair of this breakage parallel the observed changes in viability during thermal inactivation in E. coli.s     

<Emphasis Type="Italic">Pseudoscopelus sagamianus</Emphasis> Tanaka, 1908 (Chiasmodontidae): Designation of the neotype,morphological characteristics of the North Pacific population,and species variation within its whole area

North Pacific Pseudoscopelus sagamianus (from the Kuroshio zone) are characterized morphologically in detail. The neotype is established. The North Pacific population and the group of populations from the Atlantic and Indian oceans are compared in detail, significant differences are found between them in coloration of the orobranchial cavity and in the pattern of photophores of series trf and prcf. In the author’s opinion, these differences are of the subspecies level. Potential areas of subspecies are discussed, and the area of the whole species is verified. A possibility of validity of the name P. pierbartus Spitz et al., 2007, for the group of populations from the Atlantic and Indian oceans is considered. Additions and changes to methods of investigations of Pseudoscopelus suggested by Melo et al. (2007) are critically discussed.     

Effect of 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenyl)-azouracil on <Emphasis Type="Italic">B. subtilis</Emphasis> DNA Polymerases

DNA replication in Bacillus subtilis^1,2 and other Gram-positive organisms³ is specifically inhibited by 6-(p-hydroxyphenyl)-azouracil (HPUra). The site of action of this compound has not so far been identified, but important progress was made by Brown et al.⁴, who studied the effect of HPUra on DNA synthesis in B. subtilis cells made permeable to externally supplied deoxynucleoside triphosphates by treatment with toluene. In this in vitro system, HPUra had no inhibitory effect when added alone, but in the presence of NADPH or dithiothreitol (DTT) the drug was reduced to a colourless form which specifically inhibited DNA synthesis.     

Chemical shift assignments of Ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

RpsA, also known as ribosomal protein S1, is an essential protein required for translation initiation of mRNAs when their Shine-Dalgarno sequence is degenerated (Sorensen et al. 1998). In addition, RpsA of Mycobacterium tuberculosis (M. tb) is involved in trans-translation, which is an effective system mediated by tmRNA-SmpB to release stalled ribosomes from mRNA in the presence of rare codons (Keiler 2008). Shi et al. found that POA binds to RpsA of Mtb and disrupts the formation of RpsA–tmRNA complex (Shi et al. 2011) and mutations at the C-terminus of RpsA confer PZA resistance. The previous work reported the pyrazinoic acid-binding domain of RpsA (Yang et al. Mol Microbiol 95:791–803, 2015). However, the HSQC spectra of the isolated S1 domain does not overlap with that of MtRpsA280-438, suggesting that substantial interactions occur between the flexible C-terminus and the S1 domain in MtRpsA .To further study the PZA resistance and how substantial interactions influence/affect protein structure, using heteronuclear NMR spectroscopy, we have completed backbone and side-chain ¹H, ¹⁵N, ¹³C chemical shift assignments of MtRpsA280-438 which contains S1 domain and the flexible C-terminus. These NMR resonance assignments provide the framework for detailed characterization of the solution-state protein structure determination, dynamic studies of this domain, as well as NMR-based drug discovery efforts.     

Transfer of Immunity to Tumour Isografts by the Systemic Administration of Xenogeneic “Immune” RNA

SPECIFIC immunoreactivity can be conferred on lymphoid cells by incubation with RNA-rich extracts prepared from lymphoid tissues exposed to specific antigens in vivo¹ and in vitro^2,3. We have shown transfer of immunity to tumour specific antigens in vivo⁴ and in vitro⁵ by incubation of syngeneic spleen cells in vitro with RNA extracted from the lymphoid tissues of xenogeneic or syngeneic animals immunized with the tumour to be treated. Administration of these spleen cells to normal animals decreased the development and growth of isografts of the same tumour.     

Effect of BCG on Friend Disease Virus in Mice

NONSPECIFIC stimulation of the reticulo-endothelial system and resistance against tumorigenesis are produced by injection of attenuated Mycobacterium bovis (BCG). Biozzi et al.¹ demonstrated resistance to Ehrlich's ascites tumour in BCG-immune mice and Halpern et al.² showed that such immunization effected control of T-8 tumours in rats. Further, at least partial control of other tumours^3–6 has been effected by this procedure in mice and hamsters.     

Equisetin as potential quorum sensing inhibitor of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Objective

To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa.

Results

QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes.

Conclusions

Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

     

Reclassification of <Emphasis Type="Italic">Halomonas caseinilytica</Emphasis> Wu et al. 2008 as a later synonym of <Emphasis Type="Italic">Halomonas sinaiensis</Emphasis>—Comments on the proposal by Hwang et al., Antonie van Leeuwenhoek 109:1345–1352, 2016

Hwang et al. (Antonie van Leeuwenhoek 109:1345–1352, 2016) proposed the reclassification of Halomonas caseinilytica (Wu et al. 2008) as a later synonym of Halomonas sinaiensis, based on the publication of the latter name in 2007 by Romano et al. However, the name H. sinaiensis was validly published only in 2011. Therefore the proposal by Hwang et al. is not appropriate; instead, the name H. sinaiensis can be proposed as a later synonym of H. casinilytica.     

Regulatory Properties of Intergeneric Hybrids of Aspartate Transcarbamylase

THE regulatory enzyme aspartate transcarbamylase (ATCase) from Escherichia coli contains two non-identical protein sub-units, one the catalytic subunit which provides the active sites of the enzyme and the other the regulatory subunit which provides the binding sites for nucleotide inhibitors and activators^1,2. The catalytic subunit is a trimer of “C” polypeptide chains, associated by three heterologous c: c domains of bonding (terminology given by Monod et al.³ and Cohlberg et al.⁴). The regulatory subunit is a dimer of “R” chains, associated by an isologous r: r domain. Two catalytic and three regulatory subunits interact specifically across six r: c domains of inter-subunit bonding to complete the quaternary structure of the ATCase molecule.     

Effect of Rifampicin on the Development of Tumours induced by Adenovirus in Male Hamsters

INITIAL in vitro studies established that rifampicin, one of a group of rifamycin SV derivatives^1,2, prohibits bacterial growth and phage replication by binding to a polypeptide component of the microbial DNA-dependent RNA polymerase^3–7. The trachoma agent and related psittacosis-lymphogranuloma agents are also inhibited in vitro and in embryonated eggs by this drug⁸. Further studies have shown that rifampicin is active against a number of bacteria in vivo, both after parenteral and oral administration^1,9,10. It also inhibits malaria in mice¹¹ and trachoma in monkeys^12,13 and is of special value in the treatment of human tuberculosis^14–16. The low toxicity of the rifamycins in mammals¹⁷ has been attributed to the observed relative insensitivity of mammalian RNA polymerase to the rifamycins in vitro^3,18.     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

A review of the <Emphasis Type="Italic">Chrysolina lineella</Emphasis> species-group of the subgenus <Emphasis Type="Italic">Anopachys</Emphasis> Motschulsky, 1860, genus <Emphasis Type="Italic">Chrysolina</Emphasis> Motschulsky, 1860 (Coleoptera,Chrysomelidae, Chrysomelinae)

The Chrysolina lineella species-group of the subgenus Anopachys Motschulsky, 1860 is revised. Chrysolina watanabei Takizawa, 1970 is revalidated from the synonymy with Ch. lineigera (Jacobson, 1901). Ch. sundukovi Mikhailov, 2006 is a new junior synonym of Ch. watanabei. Special attention is paid to the infraspecific variability of the male and female genitalia. The structure of the aedeagal flagellum and the spermatheca are used as diagnostic characters. A key to species is included. Ch. watanabei is for the first time recorded from the Asian continent (Primorskii Territory of Russia and Northeastern China).     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.