Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

Pharmacokinetics of triclabendazole in rabbits

1.Pharmacokinetic profiles of triclabendazole (TCBZ) following intravenous (i.v.) and oral administration of the drug in rabbits were carried out.2. In normal rabbits, TCBZ was metabolized rapidly to its sulphoxide (TCBZ-SO) and sulphone (TCBZ-SO₂) derivatives following administration, with undetectable concentrations of unchanged TCBZ in the plasma of the treated animals at any time (detection limit, 10 ng/ml).3. The disposition kinetics of this drug in rabbits can be described by a two-compartment open model.4. Mean peak concentrations in plasma of TCBZ-SO and TCBZ-SO₂ of 12.41 μg/ml and 9.5 μg/ml occurred 7.5 and 9.5 hr after oral administration, respectively.5. Both metabolites were eliminated slowly from plasma with elimination half-lives of 16.86 hr for the sulphoxide and 13 hr for the sulphone.6. The area under the plasma concentration versus time curve (AUC) was 240 mg hr/l for the sulphoxide, higher than that found for the sulphone, 185 g hr/l.     

THE EFFECT OF HYDROCORTISONE ON HeLa CELL GROWTH

HeLa Chessen cells have a doubling time of 18 hr when grown in MEM containing 10% calf serum and antibiotics. When hydrocortisone (1.7 μg/ml) is added to exponentially distributed cells in log growth in this medium, a new pattern of growth begins to emerge after 10–12 hr. This pattern is characterized by a transitional state lasting for about 6 hr, and then a new doubling time of about 35 hr is maintained thereafter. Hydrocortisone removes about 5% of the cells from the proliferative pool and extends the generation time of proliferating cells to about 30 hr. The extension of the generation cycle appears to occur almost entirely in late G₁. Cells grown as clones (average 6 cells/clone) prior to the addition of hydrocortisone, undergo these changes with doses as low as 0.00017 μg/ml of medium. When the average clone size is 1.5 cells per clone, the drug concentration must be 0.017 μg/ml or higher to initiate this response. The HeLa S₃ strain continues to grow with an 18-hr doubling time in the presence of hydrocortisone after a temporary delay in growth occurring between the 12th and 16th hour.     

ENDOGENOUS LEVELS OF INDOLE-3-ACETIC ACID IN SYNCHRONOUS CULTURES OF CHLORELLA PYRENOIDOSA12

Endogenous levels of indole-3-acetic acid were mesaured in synchronous cultures of Chlorella pyrenoidosa (TX-7-11-05). The cultures were synchronized by alternating light:dark periods of 15:9 hr at a temperature of 40 ± 1 C. After 2 synchronous cycles the cultures were exposed to a low light treatment of 350 ± 100 ft-c. The time to incipient cell division under these conditions was 6 hr and 15 min. Samples were taken at 3 sampling periods during the low light treatment period:low light 0 hr (LL0); low light 3 hr (LL3); and low light 6:15 hr (LL6:15). The algal extracts were analyzed by a fluorometric procedure which measured the indole-α-pyrone product formed by the action of the trifluoracetic acid-acetic anhydride reagent with IAA. The IAA levels increased gradually from the autospore stage (5.19 μg × 10^?4/mg dry wt) to the adolescent stage (7.13 μg × 10^?4/mg dry wt) and more rapidly when approaching the ripened adult stage (14.55 μg × 10^?4/mg dry wt). The mean percentage increase from autospore to adolescent was 36.9%, and from adolescent to ripened adult 104.6%. The total percentage increase from autospore to adult was 180.3%. Levels of IAA were 2 times higher just prior to division than in the autospore stage.     

Infusions of chemicals into the brain and the development of sustained elevations of blood pressure in the rat

Osmotic minipumps were used to infuse carbachol chloride (1.23 μg/hr), echothiophate iodide (0.5 μg/hr), histamine dihydrochloride (10 μg/hr), prostaglandin E₂ (1.0 μg/hr) and thyrotropin-releasing hormone (0.5 and 5.0 μg/hr) solutions into the cerebral ventricles of unanesthetized rats and blood pressure was measured by the tail-cuff method. Histamine dihydrochloride, prostaglandin E₂ and thyrotropin-releasing hormone produced an initial rise in blood pressure, but were not effective in producing sustained elevations in blood pressure. Carbachol infusions elevated blood pressure throughout the 7-day infusion period when results were compared to saline-infused animals. Infusions of echothiophate iodide, an anticholinesterase agent, produced an initial rise in blood pressure but these pressor effects were not sustained. In animals infused with echothiophate for 7 days, the pressor response to a challenge dose of echothiophate was diminished.     

Anticoccidial drugs: Effects on infectivity and survival intracellularly of Eimeria tenella sporozoites

The effects of various anticoccidial drugs on extracellular and intracellular sporozoites were studied in cell culture and in chickens. Treatment of freshly excysted, extracellular sporozoites of Eimeria tenella for 18 hr with monensin, decoquinate, or robenidine at 100 ppm had no effect on oocyst production 7–10 days after the sporozoites were rinsed free of drugs and fed to chickens. Treatment of cultures of E. tenella in chick kidney cell monolayers with monensin (0.001 μg/ml), decoquinate (0.01 μg/ml), zoalene (20.0 μg/ml), or robenidine (0.01 μg/ml) had no effect on intracellular sporozoites at 4 hr following introduction of sporozoites and drugs into the culture. A significant reduction of intracellular parasites occurred at 24 hr in the cultures treated with monensin or zoalene. Remaining intracellular sporozoites in monensin-treated cultures were morphologically abnormal or degenerate, while sporozoites in other cultures appeared normal. The number and condition of sporozoites in the nontreated cultures were unchanged at 24 hr postinoculation. These results indicate that sporozoites undergo changes subsequent to penetration of host cells that render them susceptible to drug action.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Prepartum onset of maternal behavior in hamsters and the effects of estrogen and progesterone.

The onset of maternal behavior in pregnant hamsters was measured by presenting foster pups at 0900 and 2100 hr on Day 15 and at 0300, 0500, and 0700 hr on Day 16 and then at hourly intervals until parturition began. The occurrence of parturition was determined at each maternal test and at 0.5 hr intervals beginning at 0700 hr on Day 16. Nulliparous and primiparous animals became maternal at approximately the same time on Day 16, 2 and 6 hr prepartum, respectively, demonstrating that parturition is not essential for maternal behavior. The second experiment showed that nulliparous females injected with either 1 μg or 10 μgm estradiol-17β (E₂), 0.1 mgm progesterone (P), 10 μgm E₂ plus 0.1 mgm P, or oil at 1200 hr on Day 15 became maternal at the same time of day (0800–1000 hr) while parturition was delayed 8 hr in females receiving P. The results suggest a dissociation between the regulation of parturition and maternal care and are compared to previous research into the hormonal basis of maternal behavior in rats.     

Chemical N-desulfation of heparin negates its ability to capacitate bovine spermatozoa

Heparin specifically and saturably binds to bovine spermatozoa and stimulates capacitation as assessed by the ability of spermatozoa to undergo a zona pellucida-induced acrosome reaction (AR) in vitro. However, the structural features of heparin important for capacitation are poorly understood The purpose of this study was to determine the importance of the sulfatc content of heparin for its potency to bind to bull spermatozoa and promote agglutination and capacitation. The pyridine salt of heparin was Nndesulfated, which reduced its mean sulfate content from 19.7% to 11.6%. The N-desulfated heparin was then resulfated by incubation with trimcthylamine sulfur trioxide for 6,12, or 24 hr, raising sulfate to original concentrations. Heparin but not N-desulfated beparin competed with [³H]-heparin to bind to spermatozoa. Heparin at 11.6 μg/ml reduced [³H]-heparin binding by half when competing with a saturating concentration of the radidabeled compound (12 μg/ml). N-desulfated heparin did not displace [³H]-heparin. Heparin, resulfated 6 hr or 12 hr, was equal to native heparin in binding potency. Heparin at 50,100, or 250 μg/ml caused more than 40% of the cells to head-to-head agglutinate in aggregates of 8 or more. N-desulfated heparin did not cause agglutination. After spermatozoa were incubated with 0, 5,10, 50, 100, or 250 μg/ml of heparin for 4 hr, 100 μg/ml of lysophosphatidylcholine (LPC)-induccd AR within 20 min in 21.3, 37.7, 27.8, 45.3, 54.2, or 42.5% of the cells, respectively. Sperm exposed to the same concentrations of N-dcsulfated heparin exhibited AR of 17.7,27.3,24.3,22.5,27.7, or 33.8%, respectively, following exposure to LPG Resulfated heparin did not agglutinate or capacitate spermatozoa. In conclusion, N-desulfation of heparin abolished heparin's ability to bind to, agglutinate, and capacitate bovine spermatozoa. Resulfation of N-desulfated heparin restored binding activity but not agglutination or capacitation activity.     

Uptake of glucose and orthophosphate by the american oyster.

The uptake, by oysters, of glucose is approximately 10–15 ng/hr/g wet weight, when glucose concentration in the saline was at 50 μg/ml. The removal of inorganic orthophosphate was approximately at a rate of 0.36 ng/hr/g wet weight. Radioactive studies indicate that these substrates are metabolized by oysters. The technique developed can be used to study oyster metabolism in the laboratory.     

Effect of 17 beta-estradiol and testosterone on guanosine 3', 5'-cyclic monophosphate in the rat adrenal cortex

The levels of guanosine 3′, 5′-cyclic monophosphate (cGMP) were measured in the rat adrenal cortex after administration of a single dose of either 17β-estradiol or testosterone. Young immature rats received 10 μg 17β-estradiol (females) or 100 μg testosterone (males). After testosterone administration, cGMP levels progressively rose to about 150 per cent of the control values after 4–6 hrs, and remained elevated until at least 9 hr. Administration of 17β-estradiol resulted in a similar increase in cGMP, which began at 2 hr and persisted until 9 hr, reaching levels of about 180 per cent of the controls. Our data are further evidence of general effect of steroid hormones on cGMP in their target tissues.     

Total carbohydrate determination in dimethyl sulfoxide and guanidine hydrochloride as solvents

An automated total carbohydrate determination method based on phenol-sulfuric acid is presented for use with dimethyl sulfoxide or 4 M guanidine hydrochloride as solvents. The analysis system may be adjusted to yield linear optical density versus concentration plots up to concentrations of 100 μg carbohydrate/ml at concentration gradients of 10 μg/ml between samples. Sampling rates of 20 samples/hr and 30 samples/hr are used for 4 M guanidine hydrochloride and dimethyl sulfoxide, respectively.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Toxic effects of mercury on the hard clam,Meretrix lusoria,in various salinities

1. The 96-hr lc₅₀ values for juvenile hard clams, Meretrix lusoria, were 328, 392 and 194 μg/l Hg in 10, 20 and 30 ppt salinities at 25 ± 1°C, respectively; for adult hard clams 341 and 140 μg/l Hg in 20 and 30 ppt salinities, respectively.2. Acclimatizing the adult clams to low salinity of 10 ppt lessened the toxicity of mercury. However, juvenile animals appeared to be more sensitive to mercury poisoning after 96 hr exposure in 10 ppt salinity.3. All embryos exposed to 40 μg/l Hg and above died within 30 hr. In the control, 44% of hatched embryos had developed into D-stage larvae, while those exposed to 20 μg/l Hg were still in the trochophore stage. Most of the retarded larvae developed into abnormal forms within 30 hr at 28°C in 15 ppt salinity.4. In order to maintain water quality and protect natural resources, the recommended safe level of mercury is 0.046 (0.039–0.053) μg/l Hg, based on the estimated 30-hr EC₅₀ for the clam embryos, with an application factor of 0.01.     

The reversible inhibition of steroid-induced sexual behavior by intracranial cycloheximide

Cycloheximide(Cyclo), an inhibitor of protein synthesis by a direct action on protein synthesis at the ribosomal level, was used to reversibly inhibit estrogen-induced sexual receptivity. Cyclo (100 μg per rat) was infused into the preoptic area(POA) of ovariectomized rats at varying times before, simultaneously with, and after 3 μg of subcutaneous estradiol benzoate (EB). All animals received 0.5 mg progesterone (P) 36 hr after EB, and were tested for sexual receptivity 4–6 hr after P. The females were placed with stud males and a lordosis quotient was computed for each female (lordosis quotient = number of lordosis responses/20 mounts by the male × 100). Females receiving Cyclo 6 hr before, simultaneously with, or 12 hr after EB showed significantly lower levels of sexual receptivity when compared to females receiving Cyclo 36 hr before and 18 and 24 hr after EB. When those animals that showed low levels of sexual behavior after Cyclo infusion were reprimed with EB and P 7 days later and presented with a male they showed high levels of sexual receptivity. Thus, the effect of Cyclo was reversible. Only Cyclo infusions into the POA (bilateral) and third ventricle were effective in suppressing sexual behavior. Caudate nucleus, lateral ventricle, and unilateral POA infusions were without effect.The data presented are in agreement with earlier work that utilized actinomycin D to inhibit steroid-induced sexual behavior. Cyclo was found to be less toxic than actinomycin D. All of the available evidence is consistent with the hypothesis that estrogen stimulates RNA and/or protein synthesis in its facilitation of sexual behavior in the female rat.     

Induction of sexual receptivity in the female lizard, Anolis carolinensis: Effects of estrogen and the antiestrogen CI-628

Daily behavioral testing revealed that there is a latency period of at least 48 hr from the administration of a single injection of estradiol benzoate (EB) to the first significant increase in female sexual receptivity in the ovariectomized female lizard, Anolis carolinensis. This latency period did not vary with dosage of EB used in these experiments (i.e., 0.8, 1.4, and 4.0 μg) nor with method of injection (subcutaneous vs intraperitoneal for dose of 1.4 μg EB). Following a single EB injection, female sexual receptivity increased after the 48-hr latency period, reached an observed peak from Day 3 to Day 6, and thereafter declined to pretreatment levels by Day 19. Although both 1.4 and 4.0 μg of EB produced higher levels of female sexual receptivity than did treatment with 0.8 μg of EB, results obtained with 4.0 μg EB did not differ from those obtained with 1.4 μg EB. Administration of the nonsteroidal antiestrogen CI-628 (80 μg) at either 4 or 24 hr following a single subcutaneous injection of 1.4 μg EB significantly reduced subsequent female sexual receptivity. These results suggest that there is a critical length of time during which estrogen must act on the brain and support the concept of an estrogen “maintenance” effect during this priming period.     

The effects of actinomycin D,cycloheximide and puromycin on the 20-hydroxyecdysone induced autophagocytosis in larval fat body cells of Pieris brassicae

The effects of 20-hydroxyecdysone (5 μg/g body weight), cycloheximide (5 μg/g body weight), puromycin (30 μg/g body weight) and actinomycin D (30 μg/g body weight) were investigated on the larval fat body cells of 48–70-hours-old last instar larvae of Pieris brassicae.20-Hydroxyecdysone was found to induce the formation of autophagic vacuoles 3 hr after its administration, but this was, however, prevented by simultaneous cycloheximide treatment in a parallel experiment. On the contrary, puromycin proved to induce autophagocytosis. These diverse effects of the two translational inhibitors on hormone-induced autophagocytosis may be explained by differences in their modes of action.Actinomycin D, when administered 21 hr prior to the hormone, exerted a preventive effect on induced autophagocytosis, but was ineffective when applied 3 hr before the injection of 20-hydroxyecdysone.It was concluded that the presence of RNA synthesized several hours prior to the hormone treatment was a prerequisite for induction of autophagocytosis by 20-hydroxyecdysone.     

Duration of estrogen stimulation and progesterone inhibition of maternal behavior in pregnancy-terminated rats

The duration of the effectiveness of estradiol benzoate (EB) on the latency to the onset of maternal behavior was measured in 16-day pregnant rats that were hysterectomized-ovariectomized (HO). Eight groups of HO animals were treated with either a single SC injection of 5 μg/kg of EB or oil at surgery and were initially presented with foster pups at either 24, 48, 72, or 96 hr postoperatively. Compared to their respective controls, EB-treated animals showed singificantly shorter latencies when testing began at 48 and 72 hr but not 24 or 96 hr. In the second experiment, 16-day HO rats were treated with 5 μg/kg of EB at surgery and either oil or 0.5 mg of progesterone at 0, 24, or 44 hr postoperatively. Additional groups received either progesterone or oil at surgery (instead of EB) and a second injection of oil 44 hr later. Testing began 48 hr following surgery for all groups, and the results showed that only the groups injected with EB alone or EB plus progesterone at 44 hr displayed short-latency maternal behavior. It was concluded that a significant reduction in the latency to the onset of maternal behavior can be obtained between 24 and 72 hr after EB treatment and that progesterone when injected concurrently or 24 hr later can inhibit the effectiveness of EB.     

Leishmania donovani: Oral efficacy and toxicity of formycin B in the infected hamster

Formycin B, a structural analog of inosine, was evaluated as an orally administrable antileishmanial agent. Against Leishmania donovani in hamsters, it achieved an 85–92% reduction in numbers of parasites in livers of infected animals after oral administration at 13 mg/kg/day for 4 days. Its efficacy by oral administration was approximately four to eight times that by intramuscular administration and four times that of the positive control drug Glucantime by intramuscular administration. The levels of formycin B in serum after the final oral administration of 26 mg/kg/day were 1.4 μg/ml at 1 hr and 0.3 μg/ml at 2 hr. The concentration in liver was greater (9.0 μg/ml at 1 hr) and declined more slowly. With this latter dosage or with 104 mg/kg/day there was no acute toxicity of formycin B to bone marrow or formed elements of the blood. The only statistically significant toxicity to the liver was a doubling of serum total bilirubin levels. Comparison of the in vivo efficacy of formycin B against L. donovani to the mild acute toxicity of the drug suggests that formycin B has potential as an oral agent against visceral leishmaniasis.