A Novel function of cytochrome C (555, Chlorobium thiosulfatophilum) in oxidation of thiosulfate

Thiosulfate-cytochrome c-551 reductase derived from $\text{Chlorobium}\text{thiosulfatophilum}$ has been highly purified. The enzyme reduces cytochrome $\text{c}\text{-551 of}\text{C}\text{.}\text{thiosulfatophilum}$ in the presence of thiosulfate while cytochrome $\text{c}$-555 of the organism is not reduced by the enzyme. Cytochrome $\text{c}$-555 reacts with the enzyme at an appreciable rate only in the presence of cytochrome $\text{c}$-551. However, the reduction rate of cytochrome $\text{c}$-551 by the enzyme is greatly enhanced on addition of a catalytic amount of cytochrome $\text{c}$-555. Therefore, cytochrome $\text{c}$-555 seems to function as an effector on thiosulfate-cytochrome $\text{c}$-551 reductase as well as it acts as the electron donor to the light-excited chlorobium chlorophylls.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Evidence for water as the product for oxygen reduction by cytochrome cd.

Evidence is presented that water is the final product of electron donation to molecular oxygen by cytochrome $\text{cd}$ from $\text{Paracoccus}\text{denitrificans}$ when ferrocytochrome $\text{c}$ acts as donor to $\text{cd}$. Negative evidence for the accumulation of superoxide and peroxide was obtained by rate effect experiments in the presence of superoxide dismutase, catalase, and peroxidase. Positive evidence for water was obtained by showing a 4 to 1 stoichiometric balance for rates of electron acceptance from ferrocytochrome $\text{c}$ to rates of donation to molecular oxygen.     

Myelin basic protein: a substance that releases immunoreactive growth hormone in vitro.

A protein has been isolated from ovine hypothalamus on the basis of its ability to stimulate release of growth hormone by $\text{in}$$\text{vitro}$ cultures of dispersed pituitary cells. This protein has been identified as being myelin basic protein. With no similar biological activity $\text{in}$$\text{vivo}$, myelin basic protein is thus to be recognized as a potentially interfering substance in any search for the physiological growth hormone releasing factor using $\text{in}$$\text{vitro}$ assay systems.     

Chromatographic detection of the acetohydroxy acid synthase isoenzymes of Escherichia coli K-12.

Two valine-sensitive acetohydroxy acid synthase activities were separable from $\text{Escherichia}\text{coli}$ K-12 cells by virtue of their different affinities for DEAE-cellulose eluted with a KC1 gradient. These activities appeared to be independent from a valine-resistant cryptic component expressed only in $\text{ilvO}$ regulatory mutants. The properties of the first and second activity were coincident to those of extracts of $\text{ilvB}$ and $\text{ilvHI}$ mutants, respectively. These data prove that the $\text{ilvB}$ and $\text{ilvHI}$ gene products exist in the cell as physically distinct acetohydroxy acid synthase isoenzymes.     

Isolation of coccidioides immitis from bat guano and preliminary findings on laboratory infectivity of bats with Coccidioides immitis

$\text{Coccidioides}$$\text{immits}$ has been isolated from the guano of bats obtained beneath bat roosting places deep within deserted mine tunnels. Certain species of bats have been demonstrated to be susceptible to experimental infection by $\text{C.}$$\text{immitis}$. There is, however, a difference in the laboratory susceptibility between the species studied ($\text{Antrozous}$$\text{pallidus}$ and $\text{Macrotus}$$\text{californicus}$). $\text{Antrozous}$$\text{pallidus}$ requires 8 times more arthrospores (400 as compared to 50) than does $\text{Macrotus}$$\text{californicus}$ to produce a defineable infection. Our cultures for $\text{C.}$$\text{immitis}$ in the tissue of $\text{Antrozous}$ produced colonies of the bacterium $\text{Serratia}$$\text{marcescens}$. Because these tissues were handled under sterile conditions the presence of this red pigment (prodigiosin) producing bacterium was thought not the result of contamination. Prodigiosin suggests a possible mechanism for the apparent greatly reduced susceptibility to $\text{C}$. $\text{immitis}$ found in the laboratory population of $\text{Antrozous}$. Skin and serological tests on bats were negative as were cultures of guano from experimentally infected bats and mice. It is speculated that infected dying bats that fall to the floor beneath their roosting sites may contaminate the guano. We of course realize that other animals (we have found $\text{Neotoma}$ and $\text{Bassariscus}$ rarely in these tunnels) may have tracked the fungus into these deep tunnel sites.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

Occurrence of cis-7-tetradecenoic acid in the envelope phospholipids of Escherichia coli K12

We have isolated a tetradecenoic acid from $\text{E}\text{.}\text{coli}$ and have identified this new acid as $\text{cis}$-7-tetradecenoic by its ¹³C nuclear magnetic resonance spectrum. This identification was confirmed by conventional structural studies. The acid is a component of the phospholipids of $\text{E}\text{.}\text{coli}$ and comprises about 15% of the total phospholipid unsaturated fatty acid.     

Concerning the specificity of heme oxygenase: the enzymatic oxidation of synthetic hemins.

Hemin XIII $\text{4}\text{～}$, hemin III $\text{5}\text{～}$, and iron $\text{1,4-di(β-hydroxyethyl)porphyrin}\text{6}\text{～}$ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX $\text{1}\text{～}$. The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-¹⁴C]$\text{6}\text{～}$ and [α-¹⁴C]$\text{4}\text{～}$ as substrates. The chemical oxidation of $\text{4}\text{～}$, $\text{5}\text{～}$, and $\text{6}\text{～}$ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Cytochrome oxidase from an extreme thermophile. Thermus thermophilus HB8

The cytochrome oxidase (EC 1.9.3.1) of $\text{Thermus}\text{thermophilus}$ HB8 was isolated from the membrane fraction, and was highly purified. The oxidase contained heme $\text{a}$ and heme $\text{c}$ as the prosthetic groups. The purified preparation showed a single band in polyacrylamide gel electrophoresis, and three major polypeptides with apparent molecular weights of 52,000, 37,000 and 29,000 were observed in the presence of sodium dodecyl sulfate. The enzyme reacted rapidly with $\text{T}\text{.}\text{thermophilus}$ cytochrome c-552. The oxidation of $\text{T}\text{.}\text{thermophilus}$ cytochrome $\text{c}$-555,549 by the enzyme was very slow, and was stimulated by the addition of cytochrome $\text{c}$-552. The enzyme was highly stable to heat.     

Template specificities of the RNA dependent DNA polymerase of different murine C-type virus particles.

The crude RNA dependent DNA polymerase of seven different C-type viruses (AMV, Kirsten-MSV produced by NRK or $\text{NIH}\text{3T3}$ cells, Moloney-MuLV, Kirsten-MuLV, the murine myeloma associated virus (MuMAV) from FLOPC-1 and MOPC-21) was analyzed for their ability to utilize four different synthetic $\text{RNA}\text{DNA}$ hybrids or three different $\text{DNA}\text{DNA}$ duplexes as templates. The polymerases from AMV and murine sarcoma or leukemia viruses were distinctly different in their template stimulated activities and the two MuMAV polymerases were different from all of the other enzymes. MuMAV RDDPs were not stimulated by any of the synthetic $\text{RNA}\text{DNA}$ hybrid templates to the same level as the enzymes of the other C-type viruses and their ability to distinguish between templates was also different.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Multiple forms of starch branching enzyme of maize: Evidence for independent genetic control

Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, $\text{sugary}$ and $\text{amylose-extender}$, showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from $\text{sugary}$ kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by $\text{sugary}$ branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of $\text{amylose-extender}$ kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of $\text{amylose-extender}$ maize, but this activity was regenerated by the addition of any branching enzyme.     

Role of protein binding and pharmacokinetics in the lack of correlation between in vitro inhibition of PG-synthetase and in vivo antiinflammatory activity of a homologous series of nonsteroidal antiinflammatory compounds.

The antiinflammatory activity of a homologous series of α-alkyl substituted [4-(1-oxo-2-iso-indolinyl)-phenyl]-acetic acid has been assayed by some $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ tests.These compounds were shown to be particularly active in inhibiting prostaglandin biosynthesis from bovine seminal vesicles, and their potency was seen to increase as the size of the substituents in the side chain increased.The antiinflammatory activity $\text{in}$$\text{vivo}$ is not correlated with $\text{in}$$\text{vitro}$ inhibition of PG-synthetase. Discussion of the data takes into account the plasma protein binding and pharmacokinetics of these compounds.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Two sex pheromone components of the tobacco budworm moth, Heliothis virescens

Two compounds were isolated from female $\text{Heliothis}$$\text{virescens}$ (Lepidoptera: Noctuidae) extracts and identified as $\text{cis}$-9-tetradecenal and $\text{cis}$-ll-hexadecenal. Together they elicit intense male $\text{H}$. $\text{virescnes}$ response in laboratory tests and have attracted males in the field. Although $\text{cis}$-ll-hexadecenal is an $\text{H}$. $\text{zea}$ sex pheromone, no evidence was obtained for $\text{cis}$-9-tetradecenal in $\text{H}$. $\text{zea}$.