Plasma catecholamines: effects of footshock level and hormonal modulators of memory storage

Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in male Sprague-Dawley rats before and at several times after training injections of agents known to enhance or to impair later retention performance for a one-trial inhibitory (passive) avoidance task. Two days before testing, each animal was surgically prepared with a chronic tail artery catheter that allows for repeated blood sampling in unhandled rats. Exposure to a single, intense training footshock (3.0 mA, 2.0 sec duration) resulted in an immediate but transient increase in plasma levels of EPI and to a lesser extent NE. Plasma levels of both catecholamines did not differ between unshocked controls and animals that received a weak training footshock (0.6 mA, 0.5 sec duration). An injection of EPI at a dose that enhances retention performance (0.1 mg/kg, sc) resulted in increments in plasma EPI levels of 0.8-1.9 ng/ml from 5 to 40 min after injection. An injection of EPI (0.5 mg/kg, sc) at a dose that produces retrograde amnesia resulted in increments in plasma EPI ranging from 3.7 to 4.5 ng/ml during the 40 min after injection. Plasma NE levels were not significantly altered following an EPI injection. A single injection of adrenocorticotropin (ACTH, 0.3 or 3.0 IU per rat) did not alter the plasma catecholamine responses to training with a weak footshock. Similarly, the synthetic ACTH analog, Organon 2766 (125 or 250 mg/Kg) did not affect plasma catecholamines in untrained (unshocked) rats.These results demonstrate that significant increments in plasma levels of NE and EPI occur shortly after inhibitory avoidance training. Furthermore, an injection of EPI that enhances retention of an inhibitory avoidance task mimics the magnitude, though not the temporal characteristics, of the endogenous adrenal medullary response to a training footshock. Other hormonal treatments (ACTH and Organon 2766) which enhance memory storage do not affect plasma levels of NE and EPI.     

The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.     

Modifications of glomerular basement membrane cross-links in experimental diabetic rats

Glomerular basement membranes were isolated from normal and streptozotocin-diabetic rats. after elimination of non collagenous components with 8M urea the extent of ketoamine-linked hexoses and cross-links in the preparation was determined using NaB³H₄ reduction and radiochromatography. The observed increase in the level of N-glycosylation appeared to be closely related to a lowered amount of intermolecular cross-links in diabetic rats as compared to normal animals. Such a glycosylation may affect the stability and physicochemical properties of G.B.M. and thereby contribute to the vascular sequelae of diabetes mellitus.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Role of food intake in estradiol-induced body weight changes in female rats

In two experiments, we examined the relationship between estradiol-induced undereating and body weight loss in ovariectomized (OVX) rats. In the first experiment, both estradiol benzoate (EB) and the nonsteroidal anti-estrogen, MER-25, produced body weight losses that could not be duplicated simply by pair-feeding. In the second experiment, we compared the effects of EB treatments in obese OVX rats and in OVX rats in which the post-OVX obesity was prevented by food restriction. When fed ad libitum, both groups of oil-treated OVX rats exhibited substantial body weight gains that were not accompanied by overeating. In lean OVX rats, EB treatments caused a transient hypophagia but did not reduce body weight. These results suggest three conclusions. (1) Changes in food intake are neither necessary nor sufficient to cause some of the body weight changes induced by ovarian hormones. (2) Estradiol can depress food intake in female rats without altering the regulated body weight. (3) More attention should be paid to metabolic factors when studying gonadal influences on body weight.     

Binding of radioiodinated human beta-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).     

Multiple immunoreplica Technique: screening for specific proteins with a series of different antibodies using one polyacrylamide gel

A simple method for immunological identification of proteins resolved electrophoretically is presented. Proteins from one polyacrylamide gel can be subjected to a series of electrophoretic transfers to nitrocellulose paper (partial “western-blots”), providing several replicas of the gel. Each replica can be reacted with a series of different antisera (at least three), where the preceding antibody is removed by treatment with pH 2.2. The antigen-antibody complexes are visualized using ¹²⁵I-Protein A. Reactivity and antigenic specificity of proteins immobilized on nitrocellulose paper is not affected by repeated incubations and low pH treatments. Identical size of the replicas and superimposable profiles of proteins detected by antibodies allow a precise localization of particular polypeptides in the original gel.     

Differences in the protein binding of several drugs and bilirubin in serum and heparinized plasma of rats.

The purpose of this investigation was to compare the protein binding of salicylic acid, phenytoin, warfarin and bilirubin in serum and heparinized plasma of rats. Protein binding was determined by equilibrium dialysis (drugs) or by a reaction rate method (bilirubin), using serum and plasma obtained from the same animals. The three drugs were significantly less protein bound in heparinized plasma than in serum; this difference was particularly pronounced in the case of warfarin. Addition of heparin to serum also resulted in a decrease in the protein binding of the drugs but to a lesser extent than in plasma. The protein binding of bilirubin was more extensive in plasma than in serum, irrespective of the anticoagulant used (heparin, sodium citrate, or disodium ethylenediamine-tetraacetate). It may be desirable to perform all binding studies with serum rather than plasma.     

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Metabolism of cyclophosphamide by purified cytochrome P-450 from microsomes of phenobarbital-treated rats

Incubation of [³H]-sidechain-labeled and [¹⁴C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent.     

Supernormal insulin: [D-PheB24]-insulin with increased affinity for insulin receptors

[D-Phe^B24]- and [D-Phe^B25]-human insulin were semisynthesized from porcine insulin by enzyme assisted coupling method. Receptor binding ability of [D-Phe^B24]- and [D-Phe^B25]-insulin was 180% and 4%, respectively, of that of human insulin. Increased affinity of [D-Phe^B24]-insulin was ascribed to markedly decreased dissociation rate in binding to human cultured lymphocytes. Negative cooperative effect of [D-Phe^B24]insulin was also increased to twice of that of human insulin. Biological activity of these analogues was assessed by 2-deoxy-glucose uptake studies in isolated adipocytes and the ability of [D-Phe^B24]- and [D-Phe^B25]-insulin was 140% and 4%, respectively, of that of human insulin. These findings suggest that B25 L-Phe is more crucial for receptor binding and that [D-Phe^B24]-insulin is the first semisynthetic insulin to show increased affinity for insulin receptors.     

Hormone induced lordosis and its relation to masculine sexual activity in male rats

Intact and castrated male rats were injected with a single dose of estrogen (75 μg), followed by progesterone (1 mg) or by oil. Intact males showed higher lordosis quotients as compared to castrated males. Progesterone facilitated lordosis behavior in castrated as well as in intact males. An estrogen-progesterone treated group of sexually inactive male rats and of sexually exhausted males showed lower levels of lordosis as compared to the intact sexually active males.     

Onset of the receptive and proceptive components of feminine sexual behavior in rats following the intravenous administration of progesterone

The present study was carried out in order to assess the time course of action of progesterone (P) in the facilitation of complete feminine sexual behavior. Female rats (estrogen primed via 5% E₂ Silastic capsules) were given 200 μg of P either intravenously (iv) or subcutaneously (sc), and tested for estrous behavior at $\text{1}\text{4}$, $\text{1}\text{2}$, 1, 2, and 4 hr after treatment. Among iv-treated animals, significant amounts of lordosis behavior were seen as early as $\text{1}\text{2}$ hr, and a dramatic rise in solicitation behavior was observed at 2 hr. Although sc-treated animals displayed significant amounts of lordosis and solicitation behavior at 2 hr, the behavior was not maximal until 4 hr. Intravenous administration of 400 μg P was equipotent to 200 μg P, whereas 50 μg of iv P was relatively ineffective. A dual mechanism hypothesis pertaining to progesterone's actions in the facilitation of both the receptive and preceptive components of feminine sexual behavior in rats is discussed.     

Tryptophan metabolism and its interaction with gluconeogenesis in mammals: Studies with the guinea pig,mongolian gerbil,and sheep

The metabolism of l-tryptophan by liver cells from guinea pigs, gerbils, and sheep was studied. The rate of tryptophan oxidation was less in all three species examined than in the rat. In all three species, a much higher proportion of tryptophan carbon was metabolized through the citric acid cycle than in the rat. The accumulation of quinolinate was very low in guinea pig and sheep, and this correlated with the lack of inhibition of gluconeogenesis by tryptophan in these species. Tryptophan is a weak inhibitor of gluconeogenesis in the gerbil, and this again is consistent with a limited capacity for quinolinate formation. There was no correlation between the extent of tryptophan inhibition of gluconeogenesis and the intracellular distribution of phosphoenolpyruvate carboxykinase. Administration of tryptophan to guinea pigs in vivo had no effect on glucose turnover or on phosphoenolpyruvate carboxykinase activity.     

Metabolic clearance rate and half-time disappearance rate of human N-terminal and adrenocorticotropin of pro-opiomelanocortin in the rat: a comparative study

In metabolic clearance rate (MCR) and plasma half-time disappearance rate (t 1/2) of human N-terminal (1-76) and adrenocorticotropin(hACTH 1-39) of pro-opiomelanocortin were compared after intravenous bolus injection of both peptides simultaneously into rat. The level of immunoreactive (IR) hNT and IR-ACTH in plasma and urine samples were measured by specific and homologous radioimmunoassays (RIAs). The MCR and hNT and hACTH were 3.01 +/- 0.20 ml/min (M +/- S.D., N = 4) and 2.04 +/- 0.06 ml/min, respectively (p less than 0.05), The curve for the disappearance rate of IR-hNT was triphasic (rapid t 1/2 = 0.96 +/- 0.39 min, intermediate t 1/2 = 6.7 +/- 2.25 min, and slow t 1/2 = 74 +/- 15.8 min), while that of IR-ACTH was biphasic (rapid t 1/2 = 3.3 +/- 0.68 min, and slow t 1/2 = 41.5 +/- 3.03 min) as analyzed by the non-linear least-squares methods. Statistically significant difference (p less than 0.01) was found between IR-hNT and IR-hACTH in the rapid t 1/2 and in the slow t 1/2. Subsequent analysis of pooled plasma sample (30 min post-injection) by molecular sieve chromatography on Sephadex G-50 superfine column revealed that the majority of IR-hNT (90-95%) and IR-ACTH (60-70%) are co-chromatographed with [125I]iodo hNT and [125I]iodo ACTH respectively. Similarly, gel filtration of pooled urine sample (120 min post-injection) on Sephadex G-50 superfine revealed that 80-90% of IR-hNT and less than 50% of IR-ACTH co-eluted with [125I]iodo hNT and [125I]iodo ACTH, respectively. Smaller molecular forms of IR-hNT and IR-ACTH were definitely apparent in the urine sample. In conclusion, hNT has a larger MCR and a longer half-time disappearance rate (t 1/2) than IR-hACTH in rat plasma and it appears that hNT is more resistant to degradation by plasma and by kidney than hACTH.     

The effects of progestins,mineralocorticoids, glucocorticoids,and steroid solubility on the induction of sexual receptivity in rats

In the first experiment, progesterone and its 5α-reduced metabolite, 5α-dihydroprogesterone, dissolved in two different vehicles were compared for their effectiveness in facilitating lordosis behavior in ovariectomized estrogen-primed rats. When dissolved in oil vehicle, 5α-dihydroprogesterone was less effective than progesterone. However, when dissolved in Tween 80 solution, the two progestins were equally effective. In the second experiment, adrenal corticoids dissolved in Tween 80 solution were tested for their relative ability to facilitate sexual receptivity. Progesterone, desoxycorticosterone, and desoxycorticosterone acetate were equally effective in facilitating sexual receptivity. Aldosterone, corticosterone, and corticosterone acetate were no more effective than the vehicle in facilitating sexual receptivity.     

Evidence for participation of sperm proteinases in fertilization of the solitary ascidian,Halocynthia roretzi: Effects of protease inhibitors

Effects of 15 proteinase inhibitors and an inhibitor against aminopeptidases on fertilization of the solitary ascidian, Halocynthia roretzi were studied in search of lysins. Fertilization of intact eggs was blocked by three trypsin inhibitors, leupeptin, antipain, and soybean trypsin inhibitor, and by two chymotrypsin inhibitors, chymostatin and potato proteinase inhibitor I. On the other hand, the fertilization of naked eggs was not blocked at all by leupeptin and was only partially blocked by chymostatin at the concentrations sufficient for blocking that of intact eggs. This indicates that spermatozoa utilize trypsin-like and chymotrypsin-like proteinases probably as lysins for penetrating through the chorion. The chymotrypsin-like activity appears to be also required for some step besides sperm penetration through the egg investments.     

Progesterone-induced meiosis in Xenopus laevis oocytes: a role for cAMP at the "maturation-promoting factor" level.

Cholera toxin inhibition of progesterone-induced meiosis of Xenopus laevis oocytes in vitro has been correlated with increased cAMP levels. Inhibition of germinal vesicle breakdown (Gvbd) and cAMP increase occurred after a lag period of 2 hr, when cholera toxin was injected, or 4--5 hr, when applied externally. The ability of the maturation-promoting factor (Mpf) to provoke Gvbd when injected into recipient oocytes was found to be dependent upon whether the oocytes had been exposed to cholera toxin alone or to toxin and progesterone. With the former, cAMP levels were elevated and Mpf activity was abolished, whereas with the latter, the increase in cAMP was less pronounced and Mpf activity was observed. Injection of cAMP or its 8-thio derivatives shortly before the appearance of progesterone-induced Mpf abolished Gvbd. If injected earlier or later, no inhibition was observed. In contrast, cholera toxin inhibited maturation even when added several hours before progesterone, suggesting a sustained accumulation of cAMP. No Gvbd occurred when 8-thio-methyl-cAMP was injected together with Mpf. These data suggest that cAMP is involved in the control of the formation/amplification and/or activity of Mpf-a result which may be of general significance in cell division mechanisms.     

Male copulatory behavior and the induction of ovulation in female voles: a quest for species specificity.

Two experiments were conducted to investigate species specificity in the neuroendocrine responsiveness of female prairie voles to the copulatory patterns of males. In Experiment 1, prairie vole males mated for one ejaculatory series were not significantly more effective in inducing ovulation in prairie vole females than montane voles mated with prairie vole females for one series, two series, or to satiety. Mating with conspecific males did result in significantly more implanted embryos than did heterospecific matings. In Experiment 2, it was found that, when the amount of vaginal stimulation was both low and equated across groups, prairie vole males were significantly more effective in triggering ovulation in female prairie voles than were either meadow voles or montane voles. Although there appears to be some species specificity to the “vaginal codes” of these congeneric species, its biological significance is unclear.     

Messenger RNA for myosin polypeptides: Isolation from single myogenic cell cultures

Messenger RNA which stimulates the synthesis of myosin heavy chain in a reticulocyte lysate has been isolated from single myogenic cell cultures. Specific myosin polypeptides have been identified by immunoprecipitation with an antibody made to purified adult chicken skeletal muscle myosin. This mRNA binds to oligo(dT)-cellulose, and an active fraction from sucrose gradients migrates as 26S on formamide-polyacrylamide gels. The relative amount of this RNA increases dramatically at the time of terminal differentiation.