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.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Repair deficiency in Escherichia coli UV-sensitive mutator strain uvr502

The effect of ultraviolet irradiation (UV) has been studied in $\text{Escherichia}\text{coli}$ mutator UV-sensitive mutant $\text{uvr502}$, its $\text{uvrA6}$ derivative and wild-type strain. The $\text{uvr502}$ mutant is about 5 times more UV-sensitive than the $\text{uvr}$⁺ isogenic strain, but 3 times less sensitive than the $\text{uvrA6}$ single mutant. Cells of the $\text{uvr502}$ mutant are unable to rejoin the fragments of parental DNA formed after UV as a result of incision. The double mutant $\text{uvrA6}\text{uvr502}$ as well as the single $\text{uvrA6}$ mutant irradiated with UV is unable to introduce breaks into parental DNA. The extent of postreplication repair is essentially normal in the $\text{uvr502}$ cells. There is no significant difference between the $\text{uvr}{}^{\mathrm{+}}$ and $\text{uvr502}$ cells in the rate and extent of UV-induced DNA degradation.     

Cycloheximide inhibition of insulin control of liver glycogen synthase b into a conversion.

Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase $\text{b}\text{into}\text{a}$ conversion without affecting the level of synthase $\text{b}$. The effect of cycloheximide, believed to elevate cAMP in liver of normal rats, is independent of cAMP levels of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase $\text{b}$ to $\text{a}$ conversion by cycloheximide does not appear to be the result of a cycloheximide-induced cAMP-dependent phosphorylation of synthase $\text{a}$ to $\text{b}$ and suggests that insulin control of synthase $\text{b}$ and $\text{a}$ interconversions is dependent upon cycloheximide-sensitive protein synthesis.     

Transformability of DNA polymerase-deficient mutants of Bacillus subtilis

The effect of a deficiency in DNA polymerase on recombination in $\text{Bacillus}$$\text{subtilis}$ has been studied. It is concluded that the major DNA polymerase of $\text{B.}$$\text{subtilis}$ is not required for recombination, and that the recombination deficiency of a previously described DNA polymerase-deficient mutant is actually due to a $\text{rec}$ mutation. Genetic crosses imply that this recombination deficiency is not $\text{recA}$ or $\text{recB}$.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

EPR study of the effect of formate on cytochrome C oxidase

The addition of formate to oxidized cytochrome $\text{c}$ oxidase (ferrocytochrome $\text{c}$: oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high spin heme signal at g = 6 and a splitting of g = 3 signal to g = 2.98 and 3.07. When formate-cytochrome $\text{c}$ oxidase is reduced, the g = 2.98 signal decreases significantly. The spectrophotometric studies showed that formate is a specific ligand to cytochrome $\text{a}$₃. Data suggest that binding of formate to oxidized cytochrome $\text{c}$ oxidase produces a ligand-$\text{a}$₃ interaction leading to the splitting of g = 3 signal hitherto considered as due to cytochrome $\text{a}$. Thus both cytochrome $\text{a}$ and $\text{a}$₃ contribute to the resonance of g = 3 signal of cytochrome $\text{c}$ oxidase.     

The accumulation of glycinamide ribotide by ade3 and ade8 mutants of Saccharomyces cerevisiae

The purine intermediate GAR is present in cell free extracts of $\text{ade3}$ and $\text{ade8}$ mutants of yeast. It is also detectable following acid hydrolysis of extracts of $\text{ade6}$ and $\text{ade7}$ which accumulate FGAR and FGAM respectively. GAR accumulation is repressed by growing cells in high levels of adenine. Neither $\text{ade4}$ nor $\text{ade5}$ accumulate GAR and both prevent accumulation of GAR in $\text{ade3}$ and $\text{ade8}$ and FGAR in $\text{ade6}$. Since $\text{ade3}$ is known to be defective in folate metabolism these results indicate that $\text{ade8}$ is blocked in the conversion of GAR→FGAR.     

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.     

Trichomonasfetus—Induced abortion

$\text{History}$ and $\text{Clinical}$$\text{Signs}$: Herds infected with $\text{Trichomonas}$$\text{fetus}$ have histories of infertility, occasional abortions, and pyometra.$\text{Gross}$$\text{Lesions}$: There are no specific gross lesions in the fetus. The fetuses are usually aborted in the first half of gestation and may or may not be accompanied by the placenta.$\text{Microscopic}$$\text{Lesions}$: There are no specific microscopic lesions.$\text{Cultural}$$\text{Procedures}$: It is ordinarily not necessary to culture $\text{T.}$$\text{fetus}$ in order to demonstrate its presence in placental fluids and/or abomasal contents.$\text{Serologic}$$\text{Procedures}$: There are no suitable seroligic procedures for diagnosing Trichomoniasis.$\text{Special}$$\text{Procedures}$: Wet mounts of abomasal contents and/or placental fluids are examined microscopically for $\text{T.}$$\text{fetus}$.$\text{Preferred}$$\text{Diagnostic}$$\text{Procedures}$: Demonstrate the presence of $\text{T.}$$\text{fetus}$ by microscopic examination of wet mounts of placental fluids and/or abomasal contents.     

4-ethenylidene steroids as mechanism-based inactivators of 3β-hydroxysteroid dehydrogenases

The synthesis of 4-ethenylidene-5α-androstane-3β, 17β-diol ($\text{5}$) and of 4-ethenylidene-5α-androstane-3,17-dione ($\text{4}$) is described. Compound $\text{5}$ is a competitive inhibitor of solubilized bovine microsomal adrenal Δ⁵-3β-hydroxysteroid dehydrogenase, with Ki =2.7μM, and is converted by the enzyme to the corresponding 3-ketone. Compound $\text{4}$ shown to irreversibly inactivate the enzyme in a time-dependent manner ($\text{t}\text{1}\text{2}\text{=31}\text{min}\text{; 55μ}\text{M}\text{;}\text{pH}\text{=7.0}$). The substrate, dehydroepiandrosterone, protects against inactivation by compound $\text{4}$. In contrast, compound $\text{5}$ is not oxidized at the 3-position by the 3β-(and 17β)-hydroxysteroid dehydrogenase from $\text{P. testosteroni}$, but is oxidized at the 17-position. Nevertheless, the 4-ethenylidene-3,17-diketone ($\text{4}$) causes irreversible time-dependent inactivation ($\text{t}\text{1}\text{2}\text{=28}\text{min}\text{; 64μ}\text{M}\text{;}\text{pH}\text{=7.0}$) when incubated directly with this bacterial enzyme, acting as an affinity label.     

The effects of narcotic analgesics and narcotic antagonists on ganglionic transmission in the rat.

The effects of narcotic analgesics, narcotic-antagonist analgesics and narcotic antagonists on ganglionic transmission in the superior cervical ganglia of the rat were studied $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$ administration of morphine, meperidine, methadone, pentazocine or naltrexone blocked ganglionic transmission. Levorphanol, cyclazocine, nalorphine and naloxone had no effect on ganglionic transmission in this procedure. $\text{In}$$\text{vitro}$ studies confirmed the $\text{in}$$\text{vivo}$ results with the exception of levorphanol, cyclazocine and nalorphine, which were also found to block ganglionic transmission $\text{in}$$\text{vitro}$. In both preparations, naloxone did not antagonize the effect of morphine, suggesting that the effects of morphine and the other opiates were nonspecific. Similar potency of $\text{d}$- and $\text{l}$-isomers of pentazocine and cyclazocine support this conclusion. The observation that naltrexone blocked ganglionic transmission, but the other pure narcotic antagonist, naloxone, was inactive is somewhat unique to this test procedure and possibly significant.     

The biosynthesis of phosphorylated tyrosine hydroxylase by organ cultures of rat adrenal medulla and superior cervical ganglia: a correction.

Determination of the complete amino acid sequence of the rubredoxin isolated from the sulfate reducing bacterium $\text{Desulfovibrio}$$\text{gigas}$ showed that the molecule consists of a single polypeptide chain of 52 residues. The sequence of the first 42 residues was determined using an automatic Protein Sequencer. Peptides derived from tryptic hydrolysis and from specific cleavage at tryptophan residue were used to construct the total sequence. Compared with the sequence of $\text{Desulfovibrio}$$\text{vulgaris}$ rubredoxin, 37 positions are identical, and with the sequences of $\text{Clostridium}$$\text{pasteurianum}$, $\text{Peptostreptococcus}$$\text{elsdenii}$, $\text{Micrococcus}$$\text{aerogenes}$ and $\text{D.}$$\text{vulgaris}$ rubredoxins, 20 matching residues occur. A crystallographic study of the $\text{D.}$$\text{gigas}$ rubredoxin is in progress.     

Stereoselectivity in the metabolism of drobuline (d1-1-(isopropylamino) -4,4-diphenyl-2-butanol) a new anti-arrhythmic agent

The metabolism of drobuline has been examined in the dog, rabbit, rat, guinea pig and hamster. In the dog, unlike the other species, glucuronide conjugation is the major route of metabolism. The structure of the conjugate has been established as an O-glucuronide by isolation using HPLC following by field desorption mass spectral analysis. When the separate $\text{d}$- and $\text{l}$-isomers of drobuline were administered to a series of dogs the $\text{l}$-isomer reached plasma levels approximately three time higher than those of the $\text{d}$-isomer. Deuterium labeled drobuline was synthesized and resolved by multiple crystallizations of the malate salts. Racemic mixtures containing $\text{d}$₆-$\text{d}$ and $\text{h}$₆-$\text{l}$ drobuline and $\text{d}$₆-$\text{l}$ and $\text{h}$₆-$\text{d}$ drobuline were prepared and analyzed by GC-MS as the pentafluoropropionate derivatives. When either racemic mixture was administered to dogs (10 mg/kg, p.o.) the plasma levels of the $\text{l}$-isomer were found to be approximately three times those of the $\text{d}$-isomer. Using these deuterium labeled mixtures the disposition of the two isomers has been examined in the isolated perfused dog liver, in hepatocytes and isolated microsomes. The results indicate that the difference in plasma levels of the $\text{d}$- and $\text{l}$-isomers is not dependent upon stereospecific absorption or excretion but rather it is caused by metabolism of the $\text{d}$-isomer at a faster rate than that of the $\text{l}$-isomer.     

Effects of depurination on the fidelity of DNA synthesis.

The effect of depurination of polynucleotide templates on the fidelity of DNA synthesis in vitro has been determined. The fidelity of DNA synthesis with Escherichia coli DNA polymerase I, avian myeloblastosis virus DNA polymerase and human placenta DNA polymerase-β is decreased as a result of depurination of the poly[d(A-T)], poly[d(G-C)]and poly[d(A)]templates. The error rate with poly[d(A-T)]increased from $\text{1}\text{17,500}$ to $\text{1}\text{2100}$ using E. coli Pol I, and from $\text{1}\text{4100}$ to $\text{1}\text{1500}$ using the myeloblastosis virus DNA polymerase. Depurination of poly[d(A)]increased the error rate from $\text{1}\text{21,000}$ to $\text{1}\text{6500}$ using E. coli Pol I, and from $\text{1}\text{19,300}$ to $\text{1}\text{6100}$ using the DNA polymerase-β from human placenta. Depurination of poly[d(G-C)]resulted in an increase in the error rate with E. coli Pol I from $\text{1}\text{9200}$ to $\text{1}\text{2200}$, and with the virus DNA polymerase from $\text{1}\text{2400}$ to $\text{1}\text{1300}$. This misincorporation is shown to be directly proportional to the extent of depurination. Deletion experiments and alkaline sucrose gradient analyses suggest that the incorporation of complementary and non-complementary nucleotides is dependent on polymerization, and occurs in the same newly synthesized product. Kinetic studies and nearest-neighbor analyses indicate that the incorporation of non-complementary nucleotides occurs randomly as single-base substitutions. The nearest-neighbor studies also suggest that any of the four deoxynucleotides can be incorporated opposite apurinic sites. The number of each nucleotide incorporated relative to the number of apurinic sites was determined to be $\text{1}\text{490}$ for dGTP, $\text{1}\text{115}$ for dCTP, $\text{1}\text{2·5}$ for dATP and $\text{1}\text{1·7}$ for dTTP with both the poly[d(A-T)] and poly[d(A)] templates. The frequencies of misincorporation relative to the number of apurinic sites with the poly[d(G-C)]template were $\text{1}\text{230}$ for dATP, $\text{1}\text{120}$ for dTTP, $\text{1}\text{2·4}$ for dGTP and $\text{1}\text{1·8}$ for dCTP. Hydrolysis at the apurinic sites by alkali treatment reversed the effects of depurination on fidelity. The error rates with the depurinated templates were reduced to within 2% of those obtained prior to depurination, providing additional evidence that the misincorporation after depurination results from apurinic sites on the template. These results suggest a possible relationship between depurination of DNA and errors in DNA replication and/or repair.     

The influence of dna A and dna C mutations on the initiation of plasmid DNA replication

The dependence of the replication of several plasmids on the chromosome-determined initiation products, $\text{dna}$ A and $\text{dna}$ C, has been studied. The initiation of the replication of $\text{Col}$ E₁ DNA requires the chromosomal $\text{dna}$ A product. In contrast two de-repressed transfer factors ($\text{R 1 drd 16}$ and $\text{Hly}$₁₅₂) seem to determine a corresponding plasmid-specific factor. The $\text{dna}$ C-product is necessary for the ordered initation of all plasmids studied. The addition of low concentrations of chloramphenicol leads to a relaxed replication of $\text{Col}$ E₁ DNA at the restrictive temperature in $\text{dna}$ A-mutants, but not in $\text{dna}$ C-mutants.     

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.     

Enzymatic release of nitric oxide from L-alanosine,an antineoplastic antibiotic

L-Alanosine is an antineoplastic drug which is the 3-isonitramino analog of $\text{L}$-aspartic acid. The drug is known to be metabolized to the corresponding 2-oxo acid. Unlike the parent amino acid, the 2-oxo acid is unstable under mild conditions. When the 2-oxo acid is generated $\text{in}\text{vitro}$ by the aerobic action of $\text{L}$-amino acid oxidase on $\text{L}$-alanosine, the reaction mixture contains products capable of diazotizing sulfanilamide and of reducing ferricytochrome $\text{c}$ to ferrocytochrome $\text{c}$. It is thus likely that, as expected from model reactions, the unstable 2-oxo acid derived from $\text{L}$-alanosine decomposes into nitric oxide and other reactive free-radical species. Enzymatically promoted production of highly cytotoxic nitric oxide may pertain to the biological activity of the antibiotic. The reaction should prove extrapolable to the design of other enzyme-activated cytotoxic agents.