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.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

Absorption of Escherichiacoli endotoxin from the small intestines of mature non-pregnant gilts

The small intestines of 31 mature crossbred gilts were infused with $\text{Escherichia}$$\text{coli}$ endotoxin through permanent jejunostomies. A total of 86 infusions were done. The endotoxin was absorbed after 4 infusions. Evidence of absorption included variations in body temperatures, decline in white blood cell counts, detection of the endotoxin in circulating blood plasma samples by a limulus amebocyte lysate gelation test, and abnormal clinical signs. $\text{E.}$$\text{coli}$ endotoxin placed in the small intestines of normal, healthy, mature gilts does not readily cause illness.     

Origin of the labile sulfide in the iron-sulfur proteins of Escherichiacoli

A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of $\text{Escherichia}\text{coli}$ cells. Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells. The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with [sulfane-³⁴S]thiocystine, a compound which can differentially label $\text{in}\text{vivo}$ the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown $\text{E.}\text{coli}$     

Chromosomal incompatibility in Escherichia,coli K-12: A new explanation for the observed instability of minichromosome inheritance

The minichromosome pWS6 was unstable in $\text{Escherichia}$,$\text{coli}$ K-12 but became stable upon transfer to $\text{Salmonella}$,$\text{typhimurium}$. The instability of pWS6 was restored when pWS6 was brought back to $\text{E.}$,$\text{coli}$, an observation consistent with the proposed phenomena of chromosomal incompatibility.     

Cupric ion resistance as a new genetic marker of a temperature sensitive R plasmid, Rtsl in Escherichia coli.

A temperature sensitive kanamycin (Km) resistant R plasmid, Rtsl, was found to confer cupric ion (Cu²⁺) resistance on its hosts in $\text{Escherichia}\text{coli}$. At conjugal transfer, two kinds of segregants were obtained from Rtsl, i.e. Cu²⁺ resistant, Km sensitive and Km resistant, Cu²⁺ sensitive plasmids. Protein T existed in $\text{E.}\text{coli}$ cells harboring Rtsl or the Cu^rKm^s-plasmid. The inhibitory effect on the host cell growth at 43°C was observed with Rtsl⁺ or the Km^rCu^s-plasmid⁺ cells. A relationship between these Rtsl derivatives and Rtsl in $\text{Proteus}\text{mirabilis}$ which has been studied was discussed.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

Iron enhances the bactericidal action of streptonigrin

The lethal action of streptonigrin on strains of $\text{Escherichia}\text{coli}$ is greatly enhanced by citrate (10^?2 M). Desferrioxamine (2×10^?4 M), when added with streptonigrin and citrate, eliminates the citrate enhancement. These observations point to a role for iron in the bactericidal mechanism of streptonigrin. Extracellular citrate is known to promote the acquisition of iron by $\text{E.}\text{coli}$ by delivering it as a ferric citrate complex to a specific transport apparatus on the cell envelope. Therefore, it may promote action of streptonigrin by increasing the intracellular concentration of available iron. Desferrioxamine, which forms a much stronger complex with ferric ion than does citrate, would be expected to suppress the ferric citrate effect, and this was observed.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.     

Anesthetics alter outer membrane architecture and temperature range of growth of Escherichia coli K12

The addition of local anesthetics procaine and 2-phenylethanol during cell growth and membrane isolation lowered the phase transition temperature of purified outer membranes of $\text{Escherichia}\text{coli}$. Furthermore, when added to growth media, these anesthetics lowered to an equal extent the maximum temperature of growth without affecting growth at low temperatures. The phase transition of the cytoplasmic membrane was not affected by the presence of the drugs. These data substantiate the hypothesis that the temperature range over which the cell can maintain the outer membrane in a mixed (gel + liquid crystalline) lipid state determines the temperature range over which growth can occur.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.     

Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichiacoli

$\text{O}$⁶-Methyl[8-³H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m⁶dG), is demethylated by cell-free extracts of $\text{Escherichia}\text{coli}\text{B}\text{r}$ adapted by exposure to $\text{N}$-methyl-$\text{N}$′-nitro-$\text{N}$-nitrosoguanidine, as shown by the appearance of ³H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted $\text{E}\text{.}\text{coli}$. These results provide direct evidence that a previously described inducible repair activity in $\text{E}\text{.}\text{coli}$ acts by demethylating $\text{O}$⁶-methylguanine at the DNA level.     

Kinetics of CO and O 2 complexes of rabbit liver microsomal cytochrome P 450

An endonuclease has been isolated and purified from $\text{Escherichia}$$\text{coli}$ which degrades RNA hydrogen bonded to DNA and no other polynucleotide substrates, including double stranded RNA, single stranded RNA, double stranded DNA or single stranded DNA.     

Ligand displacement reactions of oxyhemocyanin: comparison of reactivities of arthropods and molluscs.

The ligand displacement reactions of oxyhemocyanin have been compared over a series of arthropods and molluscs. The arthropods (with the exception of $\text{Limulus}$) are found to be more reactive than the molluscs, (k_cancer = .04 hr^?1, k_busycon = .002 hr^?1, k_limulus ? 10^?4hr^?1 N₃^? reactions). Correlation of the spectral properties of the oxy sites require these to be extremely similar with small differences being associated with shifts in the dd transition energies. The met produced by ligand displacement contains variable amounts of EPR detectable, group 2 exogenous ligand damaged sites (arthropods 25–35%, molluscs 3–9%, $\text{Limulus}$ <1%). A parallel (arthropod > mollusc ～ $\text{Limulus}$ ～ 0) group 2 ligand damaged site is also reported for the half met derivatives.     

Interaction of CDP-diglyceride spin-label with Escherichia coli B membrane fractions and its relationship with phospholipid synthesis

Interaction of CDP-diglyceride with $\text{Escherichia}\text{coli}$ B membrane was studied by ESR spectroscopy. The results showed that the micelles of CDP-diglyceride molecules associate with membrane surface in the presence of Mg²⁺, whereas, when Mg²⁺ was omitted from the system, CDP-diglyceride molecules diffuse rapidly into membrane bilayer. The latter condition was shown to be more preferable for phosphatidylglycerol biosynthesis.     

A role for ribonuclease III in synthesis of bacteriophage T4 transfer RNAs.

Synthesis of T4 tRNA^Gln depends on normal levels of $\text{Escherichia}\text{coli}$ ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNA^Gln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNA^Gln points to an essential cleavage by the enzyme of a precursor RNA containing tRNA^Gln.     

Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

Thermosensitive cell growth and permeability coded by an Rts1 plasmid and their suppression upon integration into the chromosome in Escherichia coli.

A multiphenotypically thermosensitive plasmid, R_ts1, was found to confer upon the host cell of $\text{Escherichia}\text{coli}$ K12 an increased permeability through the cell surface to Actinomycin D and rifampicin as well as a detrimental host cell growth at 42°C only when it existed autonomously but not in an integrated state.