Regulation of argF mRNA synthesis,performed in vitro

The specific synthesis of $\text{arg}$F mRNA directed by the $\text{arg}$F gene carried on the specialized transducing bacteriophage $\text{λh80C}{}_1\text{857darg}$F, performed $\text{in vitro}$, is described with the use of an S180 extract from a strain carrying $\text{arg}$R^?. Synthesis of $\text{arg}$F mRNA is biphasic at approximately 7 minutes. The regulation of $\text{arg}$F mRNA synthesis by the specific arginine holorepressor present in an S180 extract prepared from a strain carrying the $\text{arg}$R⁺ allele is described.     

Antigen-Induced leukotriene relaese from rat lung in vitro

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

Prostaglandins and in vitro ovarian progestin biosynthesis

Prostaglandin F_2α (PGF_2α) did not alter the $\text{in vitro}$ biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF_2α did not affect the incorporation of acetate-1-¹⁴C into progestins. PGF_1α, 15-keto PGF_2α, and PGE₁ did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE₂ inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF_2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF_2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF_2α (10 μg/ml) did not stimulate the $\text{in vitro}$ biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.     

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed in vitro and in vivo

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed $\text{in vitro}$ were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found $\text{in vitro}$ (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.     

Monitoring the stereospecificity of morphine action in vivo and in vitro through brain membrane lipid fluidity

Differential scanning calorimetry of crude brain mitochondrial lipids obtained from control and morphine treated rats was carried out and the lipid phase transition measured. Morphine treatment resulted in a significant decrease in the temperature range and enthalpy of the phase transition. This effect was found to be dose dependent and reversible both $\text{in vivo}$ and $\text{in vitro}$ by naloxone. Studies with levorphanol and dextrorphan demonstrated stereospecificity. Furthermore, the ether precipitable fraction of total lipid extracts was shown to mediate the opiate response.     

Prostaglandin-induced enhancement of the in vitro anamnestic response

The ability of various prostaglandins (PGs) to affect the $\text{in vitro}$ anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the $\text{in vitro}$ anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

LH-releasing activity of potent LH-RH analogs in vitro

The LH-releasing activity of eight superactive analogs of LH-RH was measured in pituitary cells in primary culture. Introduction of the C-terminal ethylamide modification into [D-Ala⁶]- and [D-Leu⁶]-LH-RH (two peptides already 3 times more active than LH-RH) increases their activities 10-fold. [D-Phe⁶]- and [D-Trp⁶]-LH-RH are 90 and 100 times more active than LH-RH, respectively. The ethylamide derivatives of these two compounds are however approximately six times less active than the parent peptides.     

Angiotensin stimulates cortisol biosynthesis in human adrenal cells in vitro

Adrenal glands obtained from patients undergoing therapeutic adrenalectomy were used to study the effects of angiotensin on human adrenal steroidogenesis. It was observed that angiotensin stimulated cortisol biosynthesis. Although this has been demonstrated to occur in canine and bovine adrenals, angiotensin-induced cortisol biosynthesis has not been established in man. The possibility that angiotensin merely stimulated glomerulosa cells to secrete precursor steroids which accumulated in the medium and then diffused into fasciculata cells to provide substrate for cortisol biosynthesis was excluded by demonstrating that 3β-hydroxy-5-pregnen-20-one (pregnenolone) and progesterone (the only pertinent precursors) did not accumulate in angiotensinstimulated cell suspensions. In addition, angiotensin stimulated cortisol biosynthesis in a fasciculata cell suspension in which angiotensin did not stimulate aldosterone production. Therefore, in human adrenal cell suspensions angiotensin appeared to act directly to stimulate cortisol synthesis by fasciculata cells. In normal subjects pre-treated with dexamethasone, angiotensin infusions failed to stimulate an increase in plasma cortisol. The physiological importance of angiotensin as a regulator of cortisol secretion remains, therefore, to be established.     

Stimulation of leydig cell testoterone secretion in vitro and in vivo in hypophysectomized rats by an agonist of luteinizing hormone releasing hormone

Injection of a luteinizing hormone-releasing hormone (LHRH) agonist into 55-day-old male rats which had been hypophysectomized 3 days earlier resulted in a 10- to 30-fold increase in the levels of testosterone in serum and testicular interstitial fluid (IF) in the 4h following injection. The levels achieved were within or above the normal range for intact untreated rats of this age. In similar animals, injection of LHRH agonist also enhanced the serum testosterone response to injected hCG at $\text{1}\text{1}\text{2}\text{h}$, but not at later times after injection, and by 24h reduced IF levels of testosterone suggested that LHRH agonist had begun to inhibit stimulation by hCG. $\text{In vitro}$, dispersed Leydig cells from untreated hypophysectomized rats showed a 2-fold increase in testosterone responsiveness to LHRH agonist when compared to cells from intact rats, and this change was associated with an 80% increase in the number of Leydig cell LHRH-receptors.     

Amphetamine-induced release of dopamine from the substantia nigra in vitro

Incubation of chopped tissue from the substantia nigra of the rat brain with d-amphetamine resulted in a significant release of [³H]dopamine into the incubation medium. This effect was observed with both exogenous [³H]dopamine previously taken up by the tissue and [³H]dopamine endogenously synthesized from L-[3,5-³H]tyrosine. The observed release was greater in magnitude when the apparent conversion of released dopamine to 3-methoxytyramine was taken into account. The relevance of the present results to the previously postulated self-inhibition by dopaminergic neurons of the substantia nigra pars compacta is discussed. The present data also provide support for the concept that catechol-O-methyltransferase (E.C.2.1.1.6.) is located primarily extraneuronally in brain.     

Decreased in vivo protein and phospholipid methylation after in vivo elevation of brain S-adenosyl-homocysteine

The administration of adenosine together with homocysteine resulted in a dose-related elevation of cerebral S-adenosyl-L-homocysteine without concomitant perturbation of S-adenosyl-L-methionine levels. The adenosine + homocysteine treatment also decreased the incorporation of labile and stable methyl groups into brain proteins. Brain [³H]-phosphatidyl N,N-dimethylethanolamine and [³H]-phosphatidylcholine were also significantly decreased while [³H]-phosphatidyl-N-monomethylethanolamine remained unchanged. The data indicate that elevated brain S-adenosylhomocysteine can markedly and selectively inhibit the $\text{in vivo}$ methylation of brain proteins and phospholipids.     

Prostaglandin F production in vitro by granulosa cells from rabbit pre-ovulatory follicles

The concentration of PGF in rabbit Graafian follicles increases at ovulation but the cell type responsible for PGF secretion has not been identified. We have found that a pure population of granulosa cells isolated from pre-ovulatory follicles of estrous rabbits secrete prostaglandin F in tissue culture (total secretion, 446 ng/10 days; 0.09 pg/cell/day). LH/FSH did not influence the rate of PGF secretion, but there was a 50% inhibition after dibutyryl cAMP treatment, and complete inhibition by indomethacin. These results indicate that granulosa cells could secrete the prostaglandin which accumulates in the follicle at ovulation, and that PGF secretion may be modified by the addition of cAMP to the medium.     

N-hydroxymethylpentamethylmelamine,a major in vitro metabolite of hexamethylmelamine

N-Hydroxymethylpentamethylmelamine (HMPMM) was identified by HPLC and by GLC-MS after derivatization, as a metabolite of the anticancer drug hexamethylmelamine (HMM) in incubation mixtures with fortified mouse liver 9000 × g and microsomal preparations. HMPMM formation was dependent on the presence of NADPH and oxygen. N-demethylated metabolites were also found. HMPMM displays appreciable chemical stability and 29% was recovered after 60 min incubation in buffer. HMPMM constituted more than 50% of total HMM metabolites in 30 min incubations. The known chemical reactivity of carbinolamines means that HMPMM could be involved in the pharmacological or toxic effects of HMM.     

Extremely rapid degradation of [3H] methionine-enkephalin by various rat tissues in vivo and in vitro

Thirty sec after the intrajugular injection of [³H] methionine-enkephalin (met-enkephalin) in the rat, the radioactivity was already distributed in an apparent volume of 53 ml and the metabolic clearance rate calculated from the characteristics of the plasma disappearance curve was 10 ml/min. As shown by partition chromatography plasma extracts obtained 15 sec after injection of [³H] met-enkephalin, only 5% of the total radioactivity migrated as the intact pentapeptide, while no detectable intact pentapeptide remained 2 min after injection, thus indicating a half-life of [³H] met-enkephalin of the order of 2 to 4 sec. Incubation of rat cerebral tissue with [³H] met-enkephalin indicates that the first step in the breakdown of met-enkephalin in both plasma and brain tissue is cleavage of the Tyr-Gly amide bond. These data offer an explanation for the low activity of met-enkephalin after intraventricular or intravenous administration.     

Differential translation of turnip yellow mosaic virus mRNAs in vitro

Total TYMV RNA was incubated in a reticulocyte lysate, and the initiation peptides of the main proteins synthesized $\text{in vitro}$ (195 K, 150 K and 20 K daltons) analyzed after tryptic digestion. The 195 K and the 150 K dalton proteins present analogous patterns, different from the one obtained with the 20 K dalton protein (coat protein), suggesting that only one initiation site exists on the genomic RNA for the synthesis of the two high molecular proteins. The results of competition experiments between genomic and coat protein mRNA indicate that the ribosomes have a much greater affinity for the coat protein mRNA. This may represent a regulatory mechanism for the preferential amplification of coat protein synthesis in the infected cells.     

Effect of metoclopramide on rat prolactin secretion in vivo

Metoclopramide, a potent antagonist to apomorphine, was used to the rats $\text{in vivo}$ to determine its effect on the release of prolactin. A single i.p. injection of metoclopramide at 10 or 100 μg/100 g b.w. under urethane anesthesia increased serum prolactin levels by 1.6 or 7.2 fold, respectively, compared with basa levels. This prolactin increase was completely abolished by 2-bromo-α-ergocryptine (CB-154).These data suggest that metoclopramide stimulates prolactin secretion in rat and this secretion is abolished by dopaminergic stimulant.     

Phagocytosis of microspheres containing an anticancer agent by tumor cells in vitro

The uptake of suspended human albumin microspheres containing 6-mercaptopurine-8-¹⁴C by several tumor cell lines has been followed in cell culture. In a preliminary study, HeLa, KB, and human glioblastoma cells have all been shown to phagocytize the drug-containing spheres. We suggest that if studies now in progress confirm the ability of the ingested anticancer agent to survive lysosomal digestion of the albumin spheres, phagocytosis of such spheres may prove to be a method of delivering drug to malignant cells while preventing these agents from producing toxic effects in nonphagocytic tissues.     

Effect of ovarian hormone pretreatment on gallbladder motility in vitro

Adult male guinea pigs were pretreated with estrogen, progesterone, or estrogen plus progesterone and the $\text{in vitro}$ contractile response of the gallbladder to cholinergic stimulation examined. The data were compared with results obtained from control animals. Progesterone pretreatment was associated with a significant decrease in the maximal contractile response of the tissues and with a significant increase in the dose of acetylcholine needed to produce a threshold response. Estrogen pretreatment significantly decreased the threshold dose requirements but had no effect on maximum tension development. In addition, estrogen pretreatment antagonized the inhibitory effect of progesterone pretreatment. The data support the hypothesis that the ovarian steroid hormones can affect gastrointestinal smooth muscle. Furthermore, the hormones appear to exert independent and opposite effects on gallbladder motility. Additional studies will be required to determine the physiological significance of these observations.     

Inhibition of natural killer cell activity in vitro by alcohols

The effect of the addition of small quantities of alcohols to cocultures of natural killer effector cells from $\text{C57BL}\text{6}$ mice and BDIX rats with YAC-1 tumor cell targets has been studied. The order of inhibition of NK cell-mediated killing is 1-butanol > 1-propanol > 2-propanol > ethanol > methanol. The inhibition of killing due to the addition of alcohol correlates with decreases of effector-target cell binding. Therefore caution should be exercised in interpreting results of cellular experiments in which these alcohols have been used to solubilize inhibitors.