Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Effect of colchicine on virus-induced fusion of human erythrocytes.

Colchicine was found to stimulate the virus-induced fusion of human erythrocytes. Colchicine also stimulated the rate of hemolysis, but had no effect on its final extent, suggesting that the enhanced rate of envelope fusion, $\text{i}$. $\text{e}$. virus to cell, caused by colchicine resulted in the stimulation of cell to cell fusion. The fact that effective doses of colchicine were at millimolar concentrations, together with the absence of microtubules in human erythrocytes, indicates that the target of colchicine action is not this subcellular tubular system. Instead, the peripheral membrane protein, spectrin, may be a likely candidate for the site of colchicine action.     

Stimulation of insulin secretion from mouse pancreatic islets, maintained in tissue culture, by the pituitary neurointermediate lobe of the genetically obese mouse (ob/ob)

The rapid stimulation of insulin release by a perifusate from the pituitary neurointermediate lobe of the $\text{ob}\text{ob}$ mouse has been demonstrated by perifusing collagenase prepared mouse islets maintained for 48 hours in tissue culture. The maximal stimulation occurred in about 2 minutes and insulin secretion remained slightly above basal levels for 10 minutes. Freshly prepared collagenase islets showed no response to the pituitary factor and after 24 or 72 hours in culture there was a significant but reduced response compared to the 48 hour cultured islets.     

Quantitative turbidimetric assay for potency evaluation of colchicine-like drugs.

A modified Shelanski method has been applied to quantitatively compare cochicine-like drugs with regard to their inhibition of the rate of rat brain tubulin polymerisation $\text{in vitro}$. The potency of six known microtubule-disrupting drugs was in increasing order: griseofulvin < colchicine < podophyllotoxin < rotenone < vinblastine < vincristine. Two other drugs which have been claimed to interfere with microtubules, melatonin and isopropyl-n-phenylcarbamate were inactive. Methyl benzimidazol-2-yl-carbamate showed only a marginal effect. The usefulness of the method in screening colchicine-like drugs is discussed.     

Contrasting effects of maytansine and vinblastine on the alkylation of tubulin sulfhydryls

The ansa macrolide maytansine is a competitive inhibitor of vinblastine for binding to tubulin. Both drugs are potent inhibitors of microtubule assembly in vitro but maytansine, unlike vinblastine, is unable to induce tubulin aggregation or to stabilize colchicine binding. In this study, the effects of maytansine and vinblastine on the accessibility of tubulin's sulfhydryl groups were compared. It was found that 10 μm vinblastine inhibited the reaction of bovine brain tubulin with [¹⁴C]iodoacetamide by 45%. In contrast, maytansine, even up to 100 μm, had no effect on the reaction. However, when the two drugs were tested in combination, maytansine was a potent inhibitor of vinblastine's effect, consistent with the two drugs competing for the same or overlapping sites, but suggesting that the nature of the binding was different. In contrast, maytansine did not affect the suppression of alkylation induced by colchicine and podophylotoxin, consistent with these drugs binding to different sites. Maytansine and vinblastine were each able to increase the formation of $\text{β}{}^1$ by the bifunctional reagent, N,N′-ethylenebis-(iodoacetamide); $\text{β}{}^1$ is the designation for an electrophoretically faster migrating form of β-tubulin which apparently contains an intrachain crosslink. Thus, in at least the portion of the tubulin molecule involved in $\text{β}{}^1$ formation, the two drugs have similar effects. Since maytansine does not appear to suppress any competing alkylation reactions, it is possible that the enhancement of $\text{β}{}^1$ formation represents a genuine conformational effect. Since the sulfhydryl groups of tubulin may be involved in regulating microtubule assembly, it is likely that maytansine and vinblastine differ in the manner in which they inhibit microtubule assembly.     

Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

Colchicine-binding properties of the total cytoplasmic pool of tubulin from rat liver were evaluated in tubulin-stabilizing (TS) supernates. Microtubules were separated from free tubulin using a microtubule-stabilizing solution (MTS) and ultracentrifugation. [³H]Colchicine-binding properties of microtubule-derived tubulin were investigated in supernates prepared after resuspension of MTS pellets in TS. In TS buffer at 37 °C the colchicine-binding activity of the total cytoplasmic pool of tubulin decayed with $\text{T}\text{1}\text{2}$ of 3.39 h. Resuspended pellet tubulin decayed much more rapidly under the same conditions with a $\text{T}\text{1}\text{2}$ of 0.72 h. This rapid time decay of microtubule-derived tubulin was found to be at least partially attributable to prior microtubule-stabilizing solution exposure. Since tartrate has been reported to increase the rate of colchicine binding to tubulin, sodium tartrate (150 mm) was added to our colchicine-binding system. This addition increased the detectable [³H]colchicine binding by 10% in the total cytoplasmic preparation and by 85% in the resuspended pellet preparation. Addition of tartrate (150 mm) also resulted in a 105% increase in the $\text{T}\text{1}\text{2}$ for total cytoplasmic tubulin and a 412% increase for microtubule derived tubulin. Total cytoplasmic supernates of liver bound [³H]colchicine linearly over a wide range of tissue concentrations. However, resuspended microtubule-stabilizing solution pellet supernates in tubulin-stabilizing solution showed some increase in colchicine binding per tissue weight in the more dilute samples. Our data which demonstrate differences in colchicine-binding properties for total cytoplasmic and microtubule-derived pools of tubulin suggest that present assays for hepatic tubulin polymerization which assume identical binding properties should be interpreted with caution.     

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine. 30 seconds later uteri were frozen $\text{in}$$\text{situ}$ and assayed for cyclic AMP and glycogen phosphorylase. The cyclic AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50% of that in non-estrogen-treated controls. Catecholamine-induced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls. At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls. Estrogen regulates uterine β-adrenergic sensitivity but the time courses of estrogen effects on the cyclic AMP and glycogen phosphorylase response changes are different. Catecholamine-induced uterine cyclic AMP formation is biphasic: suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours. Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24–48 hours of estrogen. It is concluded that estrogen has independent effects on the β-adrenergic-glycogen phosphorylase activation pathway at two different points; one prior to cyclic AMP formation and another after cyclic AMP formation.     

An attempt to isolate Brucellaabortus from uterine flushings of brucellosis-reactor donor cattle

Two experiments were conducted to test for the recovery of brucella organisms from uterine flushings and harvested embryos of sero-positive embryo donor females. In Experiment I, 16 sero-positive cows were superovulated with FSH treatments and artificially inseminated at 12, 24 and 36 hours following the onset of estrus with brucella-free semen. At 48 hours after the onset of estrus, one half the potential donor females were administered an intrauterine inoculation of 3.3 to 4.6 × 10⁴$\text{Brucella}$$\text{abortus}$ (strain 2308) organisms while the remainder received a control inoculation. In Experiment II, the same 16 cows were similarly administered superovulatory treatments and inseminated following estrus. The uterine inoculation was increased to 1.5 to 2.5 × 10⁸ organisms administered 48 hours following estrus. Samples of recovered flushing medium and homogenized embryo residues were placed into a validated $\text{in}$$\text{vitro}$ culture system to detect the presence of brucella bacteria. Uterine flushings and embryos recovered from 31 females exhibiting estrus following FSH treatments were free from either field strain or the inoculated $\text{B.}$$\text{abortus}$ (strain 2308) contamination. The flushings obtained from a single female, which did not respond with estrus following FSH treatment but was inoculated at appointment, did contain $\text{B.}$$\text{abortus}$ which was identified as the inoculated strain 2308 and not field strain organisms. These results indicate that brucella contamination of flushing media and harvested embryos will not likely be incurred when collecting embryos from sero-positive donor females. These findings offer further encouragement for the use of embryo transplantation as a method to produce brucella-free offspring from infected cows.     

Differences in effects of norharman with various classes of chemical mutagens and amounts of S-9.

The mutagenicities of aniline, $\text{o}$-toluidine and yellow OB were demonstrated only in the presence of the β-carboline compound, norharman. The effect of norharman increased linearly with increase in the amount of S-9. The mutagenicity of 4-dimethylaminoazobenzene was greatly enhanced by the presence of norharman, and again dose-dependency on the amount of S-9 was observed. In the presence of a large amount of S-9, norharman caused several fold enhancement of the mutagenicities of $\text{N}$-2-fluorenylacetamide, benzo(a)-pyrene, and 1,4-dimethyl-3-amino-5$\text{H}$-pyrido(4,3$\text{b}$) indole, isolated from a tryptophan pyrolysate. However, norharman suppressed the mutagenicities of these compounds in the presence of a small amount of S-9. The mutagenicity of kaempferol, a flavonoid, was inhibited by norharman with either a large or small amount of S-9.     

The measurement of human endometrial prostaglandin production a comparison of two in vitro methods

Two $\text{in vitro}$ methods for measuring human endometrial prostaglandin production were compared. Endometrial samples from eight patients were incubated over eight hours by a perifusion and a superfusion technique. The collected fractions were assayed by radioimmunoassay for PGE₂ and PGF_2α.There was no significant difference between the perifusion and superfusion methods for the pattern and amount of PGE₂ and PGF₂ production with time. Significantly higher production levels of PGE₂ and PGF_2α were found in secretory phase endometria than in proliferative phase endometria. Histological examination of the tissue specimens by light and electron microscopy showed that both methods caused gross tissue damage after eight hours experimentation. The superfusion method produced more morphological damage than the perifusion method. However, no tissue damage could be detected after one hour of incubation with either method.Over an eight hour period neither the perifusion nor the superfusion technique appears to be a good indicator of $\text{in vivo}$ endometrial prostaglandin production. Either reflect the $\text{in vitro}$ situation.     

Oral ingestion of insulin liposomes: Effects of the administration route

We prepared an insulin liposome suspension by hot dispersion (50 °C) of a lipid mixture comprising dipalmitoyl phosphatidylcholine (DPPC) and cholesterol (7:2 molar ratio) in an 80 UI/ml acid bovine insulin solution, followed by two minutes of cold sonification (4 °C). Free insulin was removed by ultracentrifugation and the washed insulin liposomes were resuspended in a 1% aqueous saline solution (pH 3). Administration of these liposomes in the buccal cavity of normal rats caused clear hypoglycemia (?37% of the initial glycemia after one hour and ?44% after $\text{2}\text{1}\text{2}$ hours), but the solution was inactive when introduced by a strictly intragastric route. Hypoglycemic effects were also obtained when a mixture containing a liposome suspension devoid of insulin and 10 UI/100 g b.w. of free insulin was given by the buccal route (?56% of initial glycemia one hour later and ?55% after $\text{2}\text{1}\text{2}$ hours). These results show that the route of liposomal insulin administration strongly influences its biological effects.     

Transfer of cultured bovine embryos

A total of 126 bovine embryos were surgically collected from 16 superovulated donor heifers 5 days after estrus and randomly selected for either immediate transfer to synchronized recipients or $\text{in}$$\text{vitro}$ culture at 37°C for 24 hours and subsequent transfer. Twenty-four of 56 (42.8%) embryos maintained for 24 hours in Ham's F10 medium supplemented with 10% heat treated fetal calf serum (HTFCS) and transferred to 32 recipients produced live calves. Survival of 70 noncultured embryos transferred to 35 recipients was 55.7% (39 calves). The percentages of recipients that were diagnosed pregnant at 42 days with cultured and control embryos were 59.4% ($\text{19}\text{32}$) and 74.3% ($\text{26}\text{35}$), respectively. No statistical difference was observed between the $\text{in}$$\text{vitro}$ cultured and control embryos for viability following transfer to recipient females.In a second study, Day 7 embryos maintained in Ham's F10 medium supplemented with 10% HTFC serum for various culture periods were tested for viability following nonsurgical transfer to recipient females. A total of $\text{1}\text{5}$, $\text{1}\text{3}$ and $\text{0}\text{4}$ embryos cultured for 24, 48 and 72 hours, respectively, resulted in pregnant recipients following transfer.     

The exchange and maximal net flux of glucose across the human erythrocyte. II. The effect of two sulphydryl enzyme inhibitors,chlormerodrin and p-chloromercuribenzene sulfonic acid

The exchange and maximal net fluxes of [¹⁴C]glucose across the membrane of the human red cell were measured. The effects of $\text{p-}\text{chloromercuribenzene}$ sulfonic acid and chlormerodrin on these two parameters of glucose transport were determined. At low concentrations $\text{p-}\text{chloromercuribenzene}$ sulfonic acid (a non-penetrating organic mercurial) was found to readily inhibit the exchange flux but not the net efflux. At extremely high concentrations of $\text{p-}\text{chloromercuribenzne}$ sulfonic acid the maximal net efflux showed some degree of inhibition. Chlormerodrin (a penetrating organic mercurial) inhibited both the exchange and net fluxes in the same manner. The addition of insulin in certain instances reduced the degree of inhibition caused by the organic mercurials. Insulin had no effect on the amount of either $\text{p-}\text{chloromercuribenzene}$ sulfonic acid or chlormerodrin which bound to the red cell. From the results obtained, it is suggested that there exist glucose-reactive sites on both the outer and inner surfaces of the membrane. The results also suggest a carrier system possessing different sites or molecular arrangements for glucose egrees and for glucose entry.     

Selective enhancement of bleomycin cytotoxicity by local anesthetics

The cytotoxic effect of the antitumor antibiotic bleomycin toward cultured mouse FM3A cells was greatly enhanced by exposure of the cells to local anesthetics either before or together with treatment with bleomycin. Such local anesthetics include dibucaine, tetracaine, butacaine, lidocaine and procaine. Dibucaine-induced cell sensitization to bleomycin cytotoxicity produced a decrease in cell survival that became dependent on dose and time of bleomycin treatment. This effect of local anesthetics seems to be selective to bleomycin, since dibucaine and lidocaine do not enhance the cytotoxic effect of other antitumor agents including adriamycin, mitomycin C and $\text{cis}$-diamminedichloroplatinum(II).     

Calcium requirement in compound 48/80 induced histamine release from rat mast cells in vitro

The effect of magnesium and EDTA on compound $\text{48}\text{80}$-induced histamine release and adenosine triphosphate (ATP) content of mast cells has been studied. Inhibition of histamine release after preincubation of the cells with or without EDTA in the absence of calcium and the reversal by calcium indicate that calcium is required for compound $\text{48}\text{80}$-induced histamine release. The presence of magnesium potentiate the inhibition caused by the lack of calcium. The inhibition of histamine release is not related to changes in cellular ATP content. The observations with EDTA suggest that calcium may be provided for the release process from intracellular sources.     

Comparative effects of ftorafur and 5-fluorouracil on DNA synthesis in rat small intestine.

The effect of equimolar doses of ftorafur (100 mg/kg) and 5-fluorouracil (65 mg/kg) on the $\text{in}$$\text{vivo}$ incorporation of deoxyuridine and thymidine into the DNA of rat small intestine was studied. 5-fluorouracil produced a greater than 90% inhibition of deoxyuridine incorporation within one hour after injection. This degree of inhibition was sustained for at least 12 hours. Deoxyuridine incorporation was inhibited by 30 to 65% during the initial six hours after the injection of ftorafur. By 12 hours the rate of incorporation had returned to 66% of the control value. Neither drug inhibited thymidine incorporation into DNA. A study of the metabolic disposition of radioactively labeled ftorafur and 5-fluorouracil showed that the latter drug was more rapidly and completely converted to fluorouracil-containing nucleotides in the small intestine. The possible relationship between these findings and the reported differences in the toxicity of the two drugs is discussed.     

Activation and detoxification of bromobenzene in extrahepatic tissues

Bromobenzene causes hepatic and extrahepatic toxicity in rats. Toxicity is related to the presence of covalently bound material in these tissues. A major bromobenzene metabolite, p-bromophenol, has been shown to give rise to covalently bound material in liver, lung and kidney $\text{in vivo}$, but is not toxic. p-Bromophenol is formed from bromobenzene in liver, lung and kidney microsomes and is subsequently metabolized to 4-bromocatechol and covalently bound material. Bromobenzene-3, 4-oxide generated $\text{in situ}$ by liver microsomes, is detoxified by kidney, liver and lung cytosol. The results suggest that the kidney toxicity caused by bromobenzene is probably not mediated by either bromobenzene-3, 4-oxide or the reactive metabolites of p-bromophenol. In contrast, bromobenzene-3, 4-oxide may play a role in the lung toxicity observed after bromobenzene administration. However, the covalently bound material found in extrahepatic tissues may be derived from both bromobenzene-3, 4-oxide or the reactive metabolites of p-bromophenol, which may be formed directly by these tissues or transported there from the liver.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.