Complete amino acid sequence of the coat protein of the Pseudomonas, aeruginosa RNA bacteriophage PP7

As part of a project intending to assess the evolutionary kinship between the RNA coliphages and RNA bacteriophages of other bacterial genera, we have sequenced the coat protein of $\text{Pseudomonas}\text{,}\text{aeruginosa}$ RNA phage PP7. Like the coat proteins of coliphages MS2 and Qβ and of the broad host range RNA phage PRR1, PP7 coat protein (127 residues) is highly hydrophobic, and contains a cluster of basic residues between positions 40 to 60. Minimal mutation distance values were calculated for comparison of PP7 coat protein with each MS2, Qβ and PRR1 coat proteins. Application of the Moore-Goodman criterion to those values, shows that these four RNA bacteriophage coat proteins very likely descent from a common ancestor.     

Lymphocyte function in experimental African trypanosomiasis. IV. Immunosuppression and suppressor cells in the athymic nu/nu mouse

The role of T cells in the development and expression of antigen-nonspecific immunosuppression in experimental African trypanosomiasis was addressed. Nude ($\text{nu}\text{nu}$) C57BL/ 6 NIH mice and their thymus-bearing ($\text{nu}\text{+}$) littermates were infected with Trypanosoma rhodesiense and examined for suppression of splenic B-cell responses in vitro to the mitogen LPS. All animals developed splenic unresponsiveness to LPS. Further, both nu/nu and nu/ + infected mice displayed suppressor cell activity in their spleen cell populations upon transfer to normal uninfected mouse spleen cell cultures. On the basis of these findings we suggest that both the generalized immunosuppression and the development of suppressor cell activity in the spleens of mice infected with T. rhodesiense are T-independent processes.     

Limulus anti-LPS factor: An anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system

Exposure of $\text{Limulus}$ amebocytes to bacterial endotoxins (lipopolysaccharides, LPS) results in the activation of the coagulation system, which consists of several protein components. During the separation of these components, a potent anticoagulant, named tentatively anti-LPS factor, which inhibits the endotoxin-mediated coagulation reaction, was found in both amebocytes from the hemolymphs of $\text{Tachypleus}\text{tridentatus}$ and $\text{Limulus}\text{polyphemus}$. The principle purified partially from $\text{Tachypleus}$ amebocyte lysate had a molecular weight less than 10,000, as judged with the ordinary gelfiltration experiment. It inhibited specifically the activation of factor B, which has recently been characterized to be a coagulation factor highly sensitive to LPS, but it did not inhibit the activities of the active factor B and the active clotting enzyme separated from the lysate. The inhibitory activity of anti-LPS factor disappeared almost completely by the treatments with pronase-P and subtilisin, suggesting its polypeptide-like substance, but it resisted to a boiling treatment. A possible site of the anticoagulant action on the $\text{Limulus}$ coagulation system was discussed.     

Dancylcadaverine eliminates calmodulin stimulation of phosphodiesterase

Dansylcadaverine, which structurally resembles the calmodulin antagonists W-7 and W-5, prevented the calmodulin dependent stimulation of 3′:5′-cyclic nucleotide phophodiesterase $\text{in}$$\text{vitro}$. Dansylcadaverine and trifluoperazine sensitized cells to $\text{Pseudomonas}$$\text{aeruginosa}$ exotoxin A in apparently the same way, exept that 40 times higher concentrations of dansylcadaverine than of trifluoperazine was required.     

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions.

Nitrite reductase (cytochrome $\text{cd}$) from $\text{T. denitrificans}$ has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from $\text{P. aeruginosa}$. EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both $\text{c}$- and $\text{d}$-hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a ¹⁴N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant.To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.     

Adsorption of bacteriophages specific for Pseudomonas aeruginosa R factors RP1 and R1822

Short, thick pili were found by electron microscopy on bacteria carrying the P group drug resistance plasmids RP1 and R1822. The R1822-specific phage PRR1 was seen to adsorb to the bases of the pili. Three RP1-specific phages, one filamentous (Pf3), and two with very thick capsids (PR3, PR4), were seen to attach all around the surface of $\text{P. aeruginosa}$ cells, and were thought to be somatic, since pilus phages appear to be strictly polar on this species. PR3 and PR4 also lysed a strain of $\text{E. coli}$ containing an N group plasmid, suggesting a relationship between the N and P group plasmids.     

Ribonuclease F,a putative processing endoribonuclease from Escherichia coli

A new endoribonuclease activity, RNase F, was partially purified from $\text{Escherichia coli}$ cells. This activity can cleave a precursor RNA molecule (of Species 1), isolated from T4 infected cells, in a specific site. This activity is different from the other three know processing endoribonucleases of $\text{E. coli}$ RNase III, RNase E and RNase P.     

On the primary structure of the Escherichia coli R4 cell wall lipopolysaccharide core.

From $\text{Escherichia coli}$ R4, and from some deeper rough mutants of it, the cell wall lipopolysaccharides were isolated and subjected to mild acid hydrolysis. By methylation/g.l.c./m.s. and other analyses of the core oligosaccharides thus obtained, the primary structure of the $\text{E. coli}$ R4 core (hexose and heptose region) was elucidated:

     

Cytotoxicity of pyocin S2 to tumor and normal cells and its interaction with cell surfaces

Cytotoxicity and adsorption of pyocin S2 produced by Pseudomonas aeruginosa M47 (PAO 3047) to virally transformed mammalian cells, human malignant cells and normal cells in the same species were studied. Pyocin S2 inhibited the growth of not only tumor cells (XC, TSV-5, mKS-A TU-7, HeLa-S3 and AS-II cells) but also normal cells (BALB/3T3 and BHK 21 cells). The inhibitory effects on the cells increased with an increase of pyocin S2 activity. On the other hand, there were some tumor cells (155-4 T2 and HGC-27 cells) and normal cells (normal rat kidney and human embryo lung cells) which were resistant to pyocin S2. The pyosin S2 activity was neutralized by the cell membrane preparations from pyosin S2-sensitive cells, but not by those from pyocin-resistant cells. This neutralization ability was inhibited by high concentrations of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine and}\text{N-}\text{acetyl}$ neuraminic acid and completely destroyed by periodate and neuraminidase. The inhibition by the saccharides was concentration dependent. These results suggest that the toxicity of pyocin S2 to several mammalian cells is due to the presence of the binding site for pyocin S2 in the cell membrane and further, that the carbohydrate moiety, especially of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine}$ and sialic acid, may play an important role as an initial binding site for pyocin S2.     

Bacteriophage-induced lytic enzyme which hydrolyzes L-alanine-D-glutamic acid peptide bond in peptidoglycan

The mode of action of bacteriophage-induced lytic enzyme “LE95” was investigated. The LE95 hydrolyzed peptide portion in peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The exposed amino terminal amino acid was identified as glutamic acid by analysis of terminal amino acid by dinitrophenylation. This result suggested the LE95 hydrolyzed the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The enzyme did not hydrolyze various peptides prepared from bacterial cell wall. This experimental result suggested that the glycan chain of peptidoglycan would be essential for the enzymic activity.     

Gene m3 mediated efflux of intracellular leucine from bacteriophage P22 infected Salmonella typhimurium

Infection of $\text{Salmonella}\text{typhimurium}$ with the $\text{m}{}_3$ mutant of bacteriophage P22 leads to a rapid and severe efflux of intracellular leucine. The superinfection exclusion ($\text{sie}$) genes of P22 interfere with the function of $\text{m}{}_3$ gene, the product(s) of which is speculated to be an internal protein of phage P22.     

31P NMR studies of metabolism in Acanthamoebacastellanii: Polyphosphate release from encysted cells

${}^{31}\text{P NMR}$ studies of $\text{Acanthamoeba}\text{castellanii}$ have shown that encysting cells release polyphosphate into the encystment medium. Mature cysts contain low levels of polyphosphate, as do vegetative cells. Young cysts (7 days) show detectable levels of nucleotide diphosphates and triphosphate similar to those observed in vegetative cells. Mature cysts (90 days) show only excreted polyphosphate as well as a component which has a chemical shift of a phosphodiester. The inorganic phosphate peak in the cyst shows that the cyst milieu is liquid-like and that the intracellular environment maintains a pH between 6 and 7.5 in the presence of extracellular values from 4 to 9.     

Effect of ribosomal loading on the structural stability of bacteriophage T7 early messenger RNAs.

The $\text{in vivo}$ structural stabilities of the T7 early mRANs were measured and found to vary according to whether chloramphenicol or puromycin were added before or after infection with phage T7. These antibiotics had little effect upon messenger stability when they were added prior to infection. When chloramphenicol (but not puromycin) was added after completion of T7 early mRNA synthesis, the structural stability of the messages was enhanced. Messages which are inefficiently translated $\text{in vivo}$ due to altered 5′-termini were not stabilized by the late addition of chloramphenicol. We interpret these results to mean that ribosomal protection of the T7 early mRNAs is responsible for the increase in messenger structural stability in the presence of chloramphenicol.     

Analysis of chromatin repeat units in logarithmically and stationary growing cells of Paramecium aurelia and Tetrahymena pyriformis.

We have used micrococcal nuclease as a probe of the repeating structure of chromatin isolated from the macronuclei of logarithmically and stationary grown $\text{Paramecium aurelia}$ and $\text{Tetrahymena pyriformis}$. For both these lower eukaryotes, the monomer size is shown to vary depending on the stage in the growth cycle. $\text{P. aurelia}$ exhibits a monomer size of 153±7 bp and 178±6 bp and $\text{T. pyriformis}$ 207±10 bp and 230±10 bp in logarithmic and stationary cells, respectively. Both exhibit a nucleosome size of 140 bp. We discuss the possible association of these changes with histone content and nuclear activity changes, and also a possible reason for the divergence from the size pattern of monomer repeats seen in lower eukaryotes by $\text{T. pyriformis}$.     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy.

Ribosomal proteins $\text{L}\text{7}\text{L}\text{12}$ have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the $\text{L}\text{7}\text{L}\text{12}$ stalk. The $\text{L}\text{7}\text{L}\text{12}$ stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the $\text{L}\text{7}\text{L}\text{12}$ stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibody binding to the stalk was shown to be solely dependent upon the presence of $\text{L}\text{7}\text{L}\text{12}$ by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of $\text{L}\text{7}\text{L}\text{12}$ were unable to bind anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ restored the ability of $\text{L}\text{7}\text{L}\text{12}$-depleted cores to react with anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies. Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies pre-absorbed with $\text{L}\text{7}\text{L}\text{12}$ did not react with 50 S subunits.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies used in these experiments reacted only with the $\text{L}\text{7}\text{L}\text{12}$ stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, $\text{L}\text{7}\text{L}\text{12}$-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the $\text{L}\text{7}\text{L}\text{12}$ stalk from more than 95% of the subunits, and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ into depleted cores restores the $\text{L}\text{7}\text{L}\text{12}$ stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-$\text{L}\text{7}\text{L}\text{12}$ immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that $\text{L}\text{7}\text{L}\text{12}$ are located only on the $\text{L}\text{7}\text{L}\text{12}$ stalk.     

Inhibition of exonuclease V after infection of E. coli by bacteriophage T7

Exonuclease V ($\text{recBC}$ DNase) is inactivated in $\text{E. coli}$ between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.     

Immunoassay for honey bee cytochrome c in single animals with cytochrome c-coated bacteriophages: A sensitive tool for the study of caste formation in the honey bee, Apis Mellifera

The development of a sensitive viroimmunoassay for honey bee cytochrome $\text{c}$ and its usage for early detection of caste differentiation is described. Pure honey bee cytochrome $\text{c}$ was isolated from workers and used to produce antibodies in rabbits. Bacteriophage T4 was chemically modified by covalent attachment of honey bee cytochrome $\text{c}$ using tolylene-2,4-diisocyanate as a cross-linking agent. The immunospecific inactivation of this bacteriophage-cytochrome c conjugate by anti-cytochrome $\text{c}$ antibodies can be inhibited by free cytochrome $\text{c}$. In quantitative determinations, 50% inhibition is reproducibly achieved at a concentration of 6 ng/ml (5 pmol/ml) and as little as 0.3 ng/ml (0.25 pmol/ml) could be detected by this system. Cytochrome $\text{c}$ concentrations were measured in individual animals and substantial differences between corresponding larval stages of worker and queen bees are reported.     

Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins

Core particles of 50S ribosomes depleted of $\text{L7}\text{L12}$ proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating $\text{L7}\text{L12}$ proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of $\text{L7}\text{L12}$ proteins has less ordered structure which, on removal of $\text{L7}\text{L12}$ proteins, becomes more organized. Apparently, binding of $\text{L7}\text{L12}$ proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA.