The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

On the defect of a dCMP hydroxymethylase mutant of bacteriophage T4 showing enzyme activity in extracts

Infection by $\text{L}\text{13}$, a temperature-sensitive mutant of gene 42 of phage T4, the structural gene for dCMP hydroxymethylase, previously was shown not to form T4 DNA at nonpermissive temperatures. Yet the enzyme activity was found in extracts. Since inactivation of the enzyme was not reversible, we have examined acid-soluble extracts of cells infected at nonpermissive temperature by $\text{tsL}\text{13}$ for 5-hydroxymethyldCMP in order to determine whether the enzyme functioned $\text{in vivo}$. A double mutant of $\text{tsL13}$ and $\text{amB}\text{24}$ (5-hydroxymethyldCMP kinase) did not form the nucleotide at nonpermissive temperature, but the control, $\text{amB}\text{24}$, formed large quantities. From these results and previous temperature-shift studies it is suggested that the enzyme is normally activated to function $\text{in vivo}$ between 5 and 8 minutes after infection.     

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro

Preovulatory follicles isolated from immature rats, treated $\text{in vivo}$ with pregnant mare's serum gonadotropin, were incubated $\text{in vitro}$ and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

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.     

Early to late switch in bacteriophage T7 development: functional decay of T7 early messenger RNA

Infection of ultraviolet light-irradiated Escherichia coli with T7 phage in the presence of chloramphenicol results in synthesis of T7 early messenger RNA but not late mRNA. T7 early mRNA accumulates in terms of acid-insoluble, T7 DNA-hybridizable RNA. However, messenger activity of the same RNA decays rapidly with a half-life of about 6.5 minutes at 30 °C when tested for the ability to direct in vitro protein synthesis. This functional decay of T7 early mRNA is attributable to a loss of structural integrity of the RNA. Polyacrylamide-agarose gel electrophoresis shows that T7 early mRNAs are cleaved, generating smaller-size RNAs. Kinetics of the appearance of T7-specific RNA polymerase, one of the early gene products, during normal T7 infection show that the capacity of the cells to produce the enzyme decays very rapidly when early mRNA synthesis is terminated either by rifampicin or by a natural mechanism programmed by T7. Preferential synthesis of late proteins in the presence of chemically stable early mRNA late in T7 infection may be explained by the observed functional decay of early mRNA.     

Methylation of transfer RNA during transformation of human lymphocytes by phytohemagglutinin

Methylation of transfer RNA during phytohemagglutinin induced transformation of human lymphocytes was studied by labeling the tRNA $\text{in}\text{vivo}$ with (methyl-H³)-methionine and measuring the distribution of tritium in the methylated nucleotides. An alteration in the pattern of methylation occurred within hours after PHA-stimulation and this change was maintained through several cell generations. There was a 50 to 94% increase in the relative amount of methylated N²-methyl-guanine (1 to 3 hr) and a 40 to 59% decrease in the relative amount of 1-methyladenine (1 to 12 hr). Treatment of the stimulated cells with Actinomycin D prevented the subsequent methylation not tRNA. However, inhibition of protein synthesis by puromycin did not effect the early changes observed in the methylation of tRNA.     

Induction of angiotensin converting enzyme in human monocytes in culture.

Angiotensin converting enzyme (E.C.3.4.15.1, peptidyl dipeptidase) in circulating human monocytes rose from undetectable or minimal levels $\text{in}\text{vivo}$ to as high as 35.5 nmol/min·mgprotein (>300-fold increase) after 6 or 7 days in culture. Enzyme induction was enhanced by autologous serum and exposure for two days to 0.45 μM dexamethasone. Potent inhibition of enzyme induction by 370 μg/ml of actinomycin D and 1 μM cycloheximide suggested that new messenger RNA and enzyme biosynthesis are involved in the induction. Human monocyte and lung enzyme were similar with respect to EDTA inhibition, CoCl₂ activation and inhibition by an antienzyme antiserum. Human lymphocytes had minimal or undetectable enzyme which was not induced after 4 days in culture.     

The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

Non-specific uterine infection and bovine fertility. I. Infection patterns and endometritis during the first seven weeks post-partum

From a total of 93 dairy cows which were sampled repetitively throughout the non-gravid period, 14 cows developed persistent purulent endometritis, failed to complete uterine involution and were chronically infertile. The remaining 79 cows had completed uterine involution prior to 50 days post-partum. Forty-seven of these cows subsequently proved to be fertile and 32 cows were infertile. Fertility was not influenced by the variable uterine flora or by the endometritis which was found in all the cows within the first two weeks post-partum. Cows which had uterine infection with $\text{Corynebacterium pyogenes}$ subsequent to Day 21 developed severe endometritis and were almost invariably infertile to the first service. Fertility to subsequent services was not necessarily impaired in those cows which eliminated $\text{C. pyogenes}$ sufficiently early to allow complete uterine involution prior to Day 50. Cows with abnormal oestrous cycles were not particularly susceptible to uterine infection.In all the cows the composition of the uterine flora fluctuated constantly throughout the first 7 weeks pp. as a result of spontaneous contamination, clearance and recontamination. Contamination of the uterus by other non-specific bacteria during the 3 to 4 week period which is required for resolution of severe endometritis induced by $\text{C. pyogenes}$ could lead to a false correlation between the endometritis and the bacterial flora as determined by a single sample. It is concluded that the information which is derived from $\text{in vivo}$ sampling of the uterus in clinically normal cows during the first 7 weeks pp. is of little value in predicting subsequent fertility.     

Preparation and biological activity of taxol acetates

Synthesis and biological activity of 2′-acetyltaxol and 7-acetyltaxol are reported. Activity is measured $\text{in}\text{vivo}$ by cytotoxicity toward the macrophage-like cell line J774.2, and $\text{in}\text{vitro}$ by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2′ results in loss of $\text{in}\text{vitro}$ activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on $\text{in}\text{vitro}$ microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for $\text{in}\text{vitro}$ activity and this position is available for structural modifications.     

Deamination of 5-methoxytryptamine, serotonin and phenylethylamine by rat MAO in vitro and in vivo.

Pretreatment of rats with clorgyline, a selective inhibitor of MAO-A, significantly inhibited the $\text{in vivo}$ deamination of intraventricularly administered serotonin (5-HT) and 5-methoxytryptamine (5-MT), but not phenylethylamine (PEA). Pretreatment with d, l-deprenyl, a selective inhibitor of MAO-B, significantly inhibited the $\text{in vivo}$ deamination of all three substrates. Brain and liver homogenates from rats pretreated with clorgyline showed a decreased ability to deaminate ($\text{in vitro}$) 5-MT and 5-HT, but not PEA. Homogenates from animals pretreated with d,l-deprenyl showed a decreased capacity to deaminate PEA, but not 5-MT or 5-HT. Clorgyline, when added to brain and liver homogenates, selectively blocked the deamination of 5-MT and 5-HT, but not PEA, whereas, d,l-deprenyl blocked the deamination of PEA without affecting that of 5-MT or 5-HT. In addition, 5-MT was found to be 100 X more potent than PEA at inhibiting the $\text{in vitro}$ deamination of 5-HT. These findings suggest that 5-MT and 5-HT are favored substrates for MAO-A $\text{in vitro}$ and $\text{in vivo}$. However, $\text{in vivo}$, significant amounts of 5-MT and 5-HT can also be deaminated by MAO-B.     

Some observations on Corynebacterium pyogenes infection of the bovine uterus

Throughout the non-gravid period, bacteriological samples and endometrial biopsy specimens were taken repetitively from the uteri of 93 cows in 9 dairy herds. The genital organs of 7 of the 14 cows which developed chronic purulent endometritis or pyometra were examined at slaughter 8 to 9 months after parturition. $\text{C.}$$\text{pyogenes}$ was recovered at least once from 61 (65.6 $\text{per}$$\text{cent.}$) of the cows. The highest rate of infection was found during the second week post-partum. Intrauterine infection with $\text{C.}$$\text{pyogenes}$ invariably induced endometritis. The severity of the endometrial reaction was determined by the duration of the infection but the lesions never progressed to the “gland site mass” lesions and extensive stromal sclerosis which have been described as the “significant lesions” in endometritis.The duration of the infection also determined the effect of $\text{C.}$$\text{pyogenes}$ on fertility. Transient infection during the puerperium did not affect fertility; transient infection at a later period reduced fertility to one service, rarely to a second or third service; persistent infection induced chronic purulent endometritis or pyometra accompanied initially by functional anoestrum and, after some months, by organic anoestrum. The factors which determined the duration of the intrauterine infection with $\text{C.}$$\text{pyogenes}$ were not identified.     

Carbon monoxide and hydroxymercuribenzoate sensitivity of a fatty acid (omega-2) hydroxylase from Bacillus megaterium.

DNA-free minicells of $\text{Escherichia coli}$ will not allow growth of phage T-7, nor is RNA synthesis stimulated by phage infection. Thus, these miniature cells seem not to contain $\text{in vivo}$ RNA polymerase activity. However, DNA-dependent RNA polymerase activity can be unmasked in extracts with poly(dA-T) and Mn²⁺. This activity may represent a cytoplasmic pool of inactive RNA polymerase in normal cells.     

In vitro production of immune interferon by spleen cells of mice immunized with herpes simplex virus.

In Vitro production of Immune Interferon (IF) in response to Herpes Simplex Virus (HSV) antigen by sensitized spleen cells from C57B1/6 (B6) mice could be detected as early as 3 and for at least 20 days after ip infection of HSV. Maximal levels of IF were produced after 10 hr of culture, but there was no decay of activity when supernatants were sampled during the subsequent 3 days. The IF produced shared certain known properties of immune IF and was not neutralized by an antiserum against viral-induced (type I) IF. DBA/2 (D2) mice which are considerably more sensitive in vivo to HSV infection than B6 mice produced significantly lower amounts of immune IF in the in vitro test system regardless whether high or low doses of virus were injected. The same pattern of results was observed when resistant B6D2F1 hybrid mice were compared with $\text{A}\text{J}$ and Balb/c mice which are about as sensible to ip infection with HSV as DBA/2 mice in our laboratory. These results demonstrate a remarkable defect of in vitro cellular immunity in mice susceptible to a virus infection when compared with resistant mice. Conceivably, a similar defect may be of in vivo relevance.     

Effects of pyrazole on rat liver tryptophan oxygenase

The effects of pyrazole administration on rat liver tryptophan oxygenase have been studied both under basal conditions and after induction by cortisol or activation by tryptophan.Pyrazole administration is followed by a decrease of the basal holoenzyme and total enzyme activities. It induces furthermore a considerable inhibition of the cortisol mediated tryptophan oxygenase induction. These effects are not mediated by a modification of a tryptophan oxygenase effector, as shown by mixed homogenate experiments. The tryptophan enhancement of total tryptophan oxygenase activity is not affected by pyrazole administration contrary to the holoenzyme activity. Pyrazole added $\text{in vitro}$ inhibits liver tryptophan oxygenase activity only when used at concentrations which are considerably higher that those occuring $\text{in vivo}$ after pyrazole administration.     

Comparison of invivo and invitro uterine contractility in the ewe

Uterine contractions were observed $\text{in}$$\text{vivo}$ by laparotomy and exposure of the uterus. Ten hours after the beginning of estrus, an average of 39 contractions per 10 min originated in the posterior ends of the uterine horns and moved toward the oviducts, while an average of, 13 contractions originated in the anterior ends of the horns and moved toward the cervix. Two days later (58 hr after the beginning of estrus), the contractions had changed in origin and direction; only 6 contractions originated in the posterior ends of the horns and moved anteriorly, while 39 originated in the anterior ends and moved posteriorly.Experiments were done to determine whether the change in origin of contractions $\text{in}$$\text{vivo}$ was reflected in the contractility of strips of myometrium in a tissue bath. The number and amplitude of contractions were recorded from strips of circular and of longitudinal myometrium taken from the posterior and anterior ends of the uterine horns at 10 and 58 hr after the beginning of estrus. The myometrial strips contracted approximately 3 to 4 times per minute regardless of the time after the beginning of estrus, the end of the uterine horn from which the tissue was taken, or whether the contracting muscle was circular or longitudinal. Thus, the physiological mechanisms that controlled the number and origin of uterine contractions $\text{in}$$\text{vivo}$ did not maintain that control over myometrial tissue $\text{in}$$\text{vitro}$.