Effect of prostaglandin E2 on vascular responses of the rabbit kidney to nerve stimulation and noradrenaline, in vitro and in situ

The effect of prostaglandin E₂ on vascular responses of the rabbit kidney to renal nerve stimulation and noradrenaline was examined $\text{in}$$\text{vitro}$ and $\text{in}$$\text{situ}$ as a test of the hypthesis that prostaglandins of the E series may be involved in the regulation of adrenergic neuroeffector transmission. Intraarterial administration of prostaglandin E₂ to the $\text{in}$$\text{vitro}$ kidney caused marked inhibition of vascular responses to nerve stimulation whereas the responses to noradrenaline were not significantly altered. In the $\text{in}$$\text{situ}$ preparation, vascular responses to both nerve stimulation and noradrenaline were inhibited by prostaglandin E₂ infusion, although its effect on responses to nerve stimulation was approximately twice that observed on responses to noradrenaline.It is concluded that prostaglandin E₂ acts primarily at a prejunctional level of adrenergic neuroeffector transmission in the kidney, although a postjunctional effect has also been observed.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

Trichinella spiralis: genetic basis for differential expression of phase-specific intestinal immunity in inbred mice

Responsiveness of mouse strains after phase-specific immunization with Trichinella spiralis is compared. Two strains ($\text{NFR}\text{N, NFS/N}$) showed strong overall responsiveness. The response type could be characterized in phase-specific terms as: strongly anti-adult, weakly to moderately anti-preadult, and strongly antifecundity. By comparison, congenic mice of the C₅₇B1 $\text{10}\text{Sn}$ background (B10·A, B10·D₂, B10·S, B10·Q) displayed poor total responses that could be characterized as: weakly anti-adult, very weakly anti-preadult, weakly anti-fecundity after preadult immunization, and mixed (weak and strong) after adult immunization. The $\text{C}{}_3\text{H}\text{HeJ}$ mouse appeared to be intermediate between the B10·BR and the $\text{NFR}\text{N}$ strains in overall responsiveness. Genetic determinants of anti-preadult or anti-adult responses of $\text{NFR}\text{N}$ strain mice were dominant over their B10 congenic counterparts as shown in F₁, crosses of $\text{NFR}\text{N}$ × B1O·BR mice. Since the $\text{NFR}\text{N}$ (predominantly H-2^q) and the $\text{NFS}\text{N}$ (H-2^S) are both strong responders, while the B10·Q(H-2^q) and B10·S (H-2^S) are weak, it is suggested that the major genes controlling anti-preadult and anti-adult responses are not linked to the major histocompatibility complex. However, variations in anti-adult immunity and anti-fecundity in the B10 congenic mice (B10·Q and B10·S are the strongest responders) suggest that minor genes linked to the MHC exert some control over these responses. Some evidence was obtained for gene complementation as the F₁ cross of $\text{NFR}\text{N}$ and $\text{NFS}\text{N}$ mice responded more vigorously than the parental lines. We conclude that multiple genes determine anti-T. spiralis intestinal responses in mice. The major genes are unlinked to the major histocompatibility complex whereas several minor genes are linked.     

The steroidogenic activity of biotinylcorticotropin-avidin complexes.

The steroidogenic activity of complexes of [biocytin²⁵]-corticotropin_1–25 amide (biotinylcorticotropin) with avidin (1:1), (4:1) and (1:10) was compared to that of biotinylcorticotropin using isolated rat adrenal cells. Parallel log-dose responses and maximal stimulation were observed with all these materials. The 1:1 complex is approximately 25% as active as biotinylcorticotropin (ED₅₀22.5 and 5.6 $\text{nM}$ respectively). The 4:1 complex is more active than the 1:1 complex (ED₅₀9.0 $\text{nM}$). The presence of an excess of avidin (1:10 complex) does not interfere with the ability of biotinylcorticotropin to stimulate steroidogenesis (ED₅₀18.0 $\text{nM}$). It is concluded that biotinylcorticotropin attached to avidin binds specifically to receptors on the rat adrenal cell and elicits its biological response. These results indicate that biotinylcorticotropin can be noninvasively labeled with ¹²⁵I-avidin.     

Kinetics of chlorotetracycline uptake in Staphylococcus aureus by a fluorescence technique

The antibiotic chlorotetracycline (CTC) is used as a fluorescent chelate probe to investigate the kinetics of its uptake into $\text{Staphylococcus aureus}$. CTC binds to divalent cations in an aqueous solution with enhanced fluorescence. This fluorescence is polarity dependent, being higher in apolar solutions. Upon addition of CTC to dispersions of $\text{S. aureus}$, a time dependent fluorescence enhancement is detected demonstrating that the CTC-divalent cation complex migrates into the apolar regions of the membrane. This uptake, which follows saturation kinetics, is energy dependent. A K_m of 162 μ$\text{M}$ was obtained for CTC concentration ranges of 0.2–100 μgm/ml.     

Application of selected ion monitoring technique for quantitative determination of picomole levels of pyrrolidine in the brain

A specific and sensitive method for analysis of brain pyrrolidine, a volatile amine with potent synaptotropic actions on the peripheral and central nervous systems, was devised. The method involves the isolation of volatile amines by steam distillation and the qualification and quantification of pyrrolidine by gas chromatography/mass spectrometry ($\text{gc}\text{ms}$) including a selected ion monitoring technique with deuterium-labeled pyrrolidine as an internal standard. The lower limit of quantification for the method was 2 pmol, and the mean concentration of pyrrolidine in the rat whole brain was determined to be 95 pmol/g of tissue.     

Kinetics of hydrolysis to tetraols and binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol derivatives to DNA. Conformation of adducts

When the major reactive metabolite of benzo(a)pyrene, $\text{trans}$ -7,8-dihydroxy - $\text{anti}$-9,10-epoxy -7,8,9,10-tetrahydrobenzo(a)pyrene ($\text{anti}$-BPDE) is incubated with DNA in aqueous solution at 25°C, both covalent binding and hydrolysis of $\text{anti}$-BPDE to its tetraols occur. Using fluorescence and absorption spectroscopy it is shown that hydrolysis of $\text{anti}$-BPDE is markedly accelerated by DNA. In the presence of 5A₂₆₀ units of DNA per ml in cacodylate buffer solution, at an initial concentration of DNA phosphate/$\text{anti}$-BPDE ratio of 100, both the extent of covalent binding to DNA ( < 7% of the total $\text{anti}$-BPDE initially present) and hydrolysis of $\text{anti}$-BPDE reach their maximum levels within less than five minutes after mixing. Absorption and electric linear dichroism spectra indicate that the tetraols bind non-covalently to DNA by an intercalation mechanism, whereas the covalent product displays the characteristics of an externally bound complex.     

Differential block by 1-hyoscyamine of the salivary and vascular responses of the dog mandibular gland to prostaglandin F2alpha.

Prostaglandin F_2α (PGF_2α) (3–300 pmol) administered to the dog mandibular gland $\text{via}$ the glandular artery produced salivation and an increase in blood flow rate in a dose-related manner. The salivary responses to PGF_2α and to electrical stimulation of the chorda-lingual nerve were abolished by intra-arterial infusion of $\text{1}$-hyoscymine (30 nmol/min), whereas the vascular responses to both were not affected. The salivary and vasodilator responses to PGF_2α were not affected by intra-arterial infusion of hexamethonium (0.6–2 μmol/min) which abolished those to stimulation of the chordalingual nerve. These results support the prevous conclusion that PGF_2α produces the two responses by exciting the parasympathetic ganglion or postganglionic neurons in the dog mandibular gland.     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Mechanism of inhibition of bacterial luciferase by anaesthetics.

The effects of volatile anaesthetics on bacterial luciferase were studied $\text{in vitro}$. It was shown that the concentration of anaesthetic required to inhibit the reaction velocity by 50% was similar to that required to reduce light output by 50% $\text{in vivo}$ and this concentration was also in the clinical range for each agent. A kinetic response suggestive of competitive inhibition is occuring at the aldehyde binding site on the luciferase and it is postulated that this is related to the very hydrophobic nature of this site.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Glycyl-tRNA synthetase: Evidence for two enzyme forms and sigmoidal saturation kinetics

Two forms of glycyl-tRNA synthetase from $\text{E}\text{.}\text{coli}$ have been resolved by DEAE-Sephadex chromatography. The two forms display different kinetic responses to increasing tRNA concentration. One gives a sigmoidal response and a Hill coefficient of 2.2 ± 0.2 while the other shows Michaelis-Menten kinetics. It is suggested that these data may indicate a role for glycyl-tRNA synthetase in the regulation of protein biosynthesis. During DEAE-Sephadex chromatography and ammonium sulfate fractionation the addition of substrates (glycine, ATP, and MgCl₂) has proven to be of significant value in resolving the two enzyme forms from each other and from contaminating proteins.     

The dextro and levorotatory isomers of N-phenylisopropyladenosine: stereospecific effects on cyclic AMP-formation and evoked synaptic responses in brain slices.

The stereoisomers of N⁶-phenylisopropyladenosine elicit accumulations of cyclic AMP in brain slices via interaction with adenosine-receptors. The response in guinea pig cerebral cortical slices and in rat hippocampal slices is blocked by theophylline and potentiated by biogenic amines. A chelator, EGTA, potentiates the response to phenylisopropyladenosine in guinea pig cerebral cortical slices. The $\text{1}$-isomer (EC₅₀ 25 μM) is four- to five-fold more potent than the $\text{d}$-isomer in eliciting accumulations of cyclic AMP in brain slices. In a rat coronal hippocampal slice $\text{in vitro}$, $\text{1}$-phenylisopropyladenosine (IC₅₀ ～ 0.7 μM) reduces the amplitude of evoked synaptic responses generated $\text{via}$ a monosynaptic pathway to the CA1 pyramidal neurons. The $\text{d}$-isomer is nearly one hundred-fold less potent. Thus, the adenosine-receptors involved in the electrophysical response appear much more stereoselective for the $\text{1}$-isomer of phenylisopropyladenosine than the adenosine-receptors involved in cyclic AMP-generation in brain slices.     

Induction of erythroid differentiation in Friend murine erythroleukemic cells by inorganic selenium compounds.

Inorganic selenium compounds are shown to be inducers of hemoglobin synthesis in malignant murine erythroleukemia (MEL) cells. SeO₂ can induce hemoglobin synthesis at $\text{1}\text{20}$ the concentration of butyric acid and $\text{1}\text{5000}$ the concentration of dimethylsulfoxide (DMSO), two potent inducers of erythroid differentiation in MEL cells. SeO₂ and H₂SeO₃ showed an equivalent capacity to stimulate hemoglobin synthesis in three different MEL cell lines. The incorporation of ³H-glycine into hemoglobin was demonstrated in lysates of SeO₂-induced MEL cells.     

The characterization of energized and partially de-energized (respiration-independent) β-galactoside transport into escherichia coli

Unidirectional fluxes of [¹⁴C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN^?) conditions are analyzed kinetically.When the cells are energized, the value for $\text{V}$ influx is $\text{0.45 ± 0.01}\text{mM}$ internal concentration increment/s and $\text{K}{}_{\mathrm{t}}$ is $\text{0.26 ± 0.03}\text{mM}$. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is $\text{2 · 10}{}^3$.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of $\text{8.8 ± 0.2 · 10}{}^{\mathrm{?4}}\text{s}{}^{\mathrm{?1}}$.The transport in the presence of CN^? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O₂).The effects of CN^? are to lower the $\text{V}$ for influx and to change the efflux from a non-saturable to a saturable process with a value for $\text{K}{}_{\mathrm{t}}$ (60 mM) intermediate between that for energized efflux ($\text{> 250}\text{mM}$) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.     

Treadmilling of actin at physiological salt concentrations: An analysis of the critical concentrations of actin filaments

Treadmilling of actin was investigated at physiological salt concentrations (100 mm-KCl, 0.5 to 2.0 mm-MgCl₂, 200 μm-ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid or 50 μm-CaCl₂ at 37 °C. The concentration at which monomers bind to the lengthening end of filaments with the same rate as subunits are released (low critical concentration c₁ was determined by mixing unmodified actin filaments with various concentrations of monomeric actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Above a monomeric actin concentration of about 0.12 μm, incorporation of actin molecules into filaments was detected, whereas below this concentration no incorporation was found (c₁ = 0.12 μm). Combination of various concentrations of labeled monomers with labeled filaments permitted determination of the net critical concentration ($\text{c}{}_1$) at which filaments lengthen at one end with the same rate as they shorten at the other end ($\text{c}{}_1\text{= 0.16}\text{μm}$). A lower limit of the high critical concentration at the shortening end (c_h) was estimated by measuring the release of subunits from labeled filaments in the presence of various concentrations of unlabeled monomers (c_h >0.5 μm). The differences in the three critical concentrations demonstrate that under physiological conditions actin filaments lengthen at one end by, on the average, one subunit during the time that four association reactions take place at the two ends (efficiency parameters $\text{s =}\text{1}\text{4}$). The small difference between the low and the net critical concentration suggests that the rates of both association and dissociation are considerably greater at the lengthening end than at the shortening end of actin filaments.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Effects of l-cysteine and O-acetyl-l-serine in the synthesis and mutagenicity of azide metabolite

The ability of L-cysteine to inhibit azide-metabolite synthesis and mutagenecity is investigated in Salmonella typhimurium TA1530 and cys E₆ strains. L-cysteine specifically inhibits the synthesis of the mutagenic azide metabolite as other compounds containing SH group did not affect the production of this metabolite. Azide mutagenicity is completely inhibited by L-cysteine at a concentration (5 μmoles/plate) where the metabolite mutagenicity was not affected. $\text{O-}\text{Acetyl-}\text{L}\text{-serine}$ can reverse the L-cysteine mediated inhibition of the metabolite synthesis and thus mutagenicity in the same strains. These results suggest that $\text{O-}\text{acetyl-}\text{L}\text{-serine}$ may be required to synthesize the azide metabolite or its precursor.     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.