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.     

The ventromedial nucleus of the hypothalamus and the hormonal arousal of sexual behaviors in the female rat

Bilateral lesions of the ventromedial nucleus of the hypothalamus interfered with the estrogenic induction of sexual receptivity in the female rat, but seemingly did not affect the ability of female rats to show lordosis following combined stimulation with estrogen and progesterone. In addition, ventromedial hypothalamic lesions did not affect the ability of females to show male-like sexual activity in response to exogenous androgenic stimulation.     

Role of hypothalamic serotonergic receptors in the control of lordosis behavior in the female rat

The role of serotonin in mediating hypothalamic control of sexual behavior in estrone-primed ovariectomized (OVX) rats was studied by comparing the lordotic patterns following medial preoptic (MPOA) and arcuate-ventromedial (ARC-VM) infusions of serotonin (5-HT), methysergide (MS), and vehicle. In the initial experiments, low receptivity (preinfusion receptivity: mean lordosis/mount ratio = 0.164) was maintained by priming each animal with a low dose of estrone 48 hr prior to mating. The infusion of MS in either the MPOA or ARC-VM area resulted in a significant enhancement of lordotic behavior from initial low receptivity, 5-HT infusions were found to have no statistically significant effect upon lordotic behavior. In order to corroborate the findings observed in the low preinfusion receptivity protocol, OVX rats were primed with higher doses of estrone to maintain a high level of receptivity (preinfusion receptivity: mean lordosis/mount ratio = 0.787). Using this protocol, significant depressions in lordotic behavior were observed following MPOA or ARC-VM infusions of 5-HT, It was thus proposed that serotonergic receptors within the MPOA or ARC-VM areas have inhibitory effects upon lordotic behavior. In addition to the effects of 5-HT upon estrogen-induced sexual receptivity, serotonergic influences upon luteinizing hormone-releasing hormone (LRH)-facilitated mating behavior were also evaluated. Comparisons were made between the lordotic responses following MPOA or ARC-VM infusions of vehicle, LRH, or LRH with 5-HT in OVX rats primed with low doses of estrone. The infusion of LRH into the MPOA or ARC-VM significantly enhanced lordotic behavior above vehicle levels. However, the addition of 5-HT to the LRH infusate abolished this behavioral enhancement. These findings indicated that LRH and 5-HT have opposing effects within forebrain areas known to be important for the control of lordotic behavior.     

Assay of mono ADP-ribosyltransferase activity by using guanylhydrazones

Guanylhydrazones of p-nitrobenzaldehyde and methylglyoxal serve as acceptors of ADP-ribosyl groups for the reactions catalyzed by cholera toxin. The absorption spectrum of the ADP-ribosylated p-nitrobenzylidine aminoguanidine is similar to that of a 1:1 mixture of ADP-ribose and p-nitrobenzylidine aminoguanidine. Results from fast atom bombardment mass spectrometry prove that the product is mono-ADP-ribosylated. ADP-ribosylation lowers the pKa of the p-nitrobenzylidine aminoguanidine by 0.7-0.8 pH unit. Assay methods are developed for measuring the ADP-ribosyltransferase reaction by following the rate of disappearance of p-nitrobenzylidine aminoguanidine by high-performance liquid chromatography or spectrophotometrically by monitoring the absorbance increase at 370 nm accompanying ADP-ribosylation of p-nitrobenzylidine aminoguanidine. The high-performance liquid chromatographic system can be utilized to measure ADP-ribosyltransferase activity in animal tissues. By using this procedure, the presence and quantitation of an ADP-ribosyltransferase in a homogenate of rabbit skeletal muscle is reported.     

Demasculinization of male Japanese quail by prenatal estrogen treatment

Genetic male Japanese quail were administered sex hormones or the oil vehicle on Day 10 of incubation and were caponized 3 weeks after hatching. As adults, the capons were injected with testosterone propionate daily for 2 weeks and then were tested for masculine sexual behavior in response to sexually receptive females. Males that had received as little as 2 μg of estradiol-17β in ovo failed to exhibit the head grabbing and mounting typical of the normal masculine sexual response to females. In a second experiment, this demasculinization was produced by prenatal treatment with 2 μg of estradiol-17α, estrone, estriol, or diethylstilbestrol, but not by this quantity of testosterone. These data suggest that an estrogen is the agent of behavioral demasculinization in the normal female, and that endogenous testosterone poses no difficulty for proper sexual development in the normal male.     

Inhibition of mitogenesis in bovine lymphocytes by juvenile hormones

Phytohemagglutinin and the co-carcinogenic phorbol ester, 12-0- tetradecanoyl-phorbol-13-acetate, act synergistically in cultures of bovine lymphocytes to induce a mitogenic response. Insect juvinile hormones appear to act selectively to block or counteract an early hormones appear to act selectively to block or counteract an early event in the induction of DNA and nuclear replication while allowing other blastogenic events such as increases in RNA and protein synthesis and cell size to occur.     

Activation of helper T cells by immune complexes.

Spleen cells from mice primed with dinitrophenylated human γ-globulin (DNP-HGG) did not mount a secondary anti-DNP response in diffusion chamber cultures upon stimulation with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH). The same cells, however, responded to stimulation with DNP-KLH complexed with anti-KLH antibody of rabbit or mouse origin. There is an optimal antigen:antibody ratio at which the immune complexes (IC) must be formed for maximal activity. T cells are required for the immunogenic activity of IC, since T-cell-depleted cultures did not respond. It was found that IC made with carrier and anticarrier antibody stimulated the development of carrier-specific helper T cells in cultures of spleen cells, thymocytes, and nylon wool nonadherent spleen cells from nonimmune mice. In contrast, free carrier did not elicit helper T cells. IC made with carrier and the F(ab′)₂ fragment of anticarrier antibody were immunogenic, but those made with carrier and the Fab′ fragment of anticarrier antibody were not, suggesting that helper T-cell activation is triggered by crosslinking of antigen-specific surface receptors.     

Conformational change of pepsin induced by its substrate N-acetyl-L-phenylalanyl-L-3,5-diiodotyrosine

Specific contributions of tyrosyl and of tryptophanyl residues can be distinguished in the near-ultraviolet circular dichroism spectrum of porcine pepsin. Upon addition of the dipeptide substrate, N-acetylphenylalanyl-l-3,5-diiodotyrosine, at pH values below 4.0, a change in the circular dichroic spectrum results, suggesting that in the presence of substrate the asymmetric environment of certain aromatic amino acid residues of the enzyme is altered. The changes observed are discussed in relation to the enzymatic function of pepsin.     

Statistical evaluation of ways to analyze nonideal systems by sedimentation equilibrim

Three equations describing sedimentation equilibrium are examined and tested for their ability to analyze data. The testing procedure using simulated data is similar to that described previously (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) and used with another equation. The equations examined here are found to be of much less statistical reliability and of a more restricted range of application than the previously examined equation. The equation described previously, (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) is also used here to examine the conditions necessary to detect isodesmic systems of more than four components. The self-association of lysozyme reported previously (Sophianopoulos, A. J., and Van Holde, K. E. (1964) J. Biol. Chem.239, 2516–2524) is reexamined at pH 8.2, 0.15 ionic strength, and 13°C. The tentative conclusion is that the system is mainly a monomer-dimer, with a small, uncertain amount of tetramer possibly present. Under the above conditions the second virial coefficient, B, is estimated to lie in the range 0–4.4 × 10^?6 mole·dl·g^?2, the dimerization constant. K₂₁, lies in the range 2.3–2.7 × 10^?3m, and the tetramerdimer constant, K₄₂, is in the range 1.5–15 × 10^?3m.     

Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes.

Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection of the slice diffusates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem.75, 314–324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from a electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%, with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.     

The biosynthesis of progesterone by cultured mouse midgestation trophoblast cells.

The ability of cultured midgestation mouse trophoblast cells to synthesize progesterone from pregnenolone has been monitored by radioimmunoassay or chromatography and crystallization. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of Δ⁵,3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Since there is little or no further metabolism of the progesterone formed, the ability of trophoblast cells to convert pregnenolone to progesterone in vitro is an accurate reflection of the activity of 3β-HSD in these cells.Midgestation cultures of giant trophoblast cells have a substantially higher 3β-HSD specific activity than the smaller ectoplacental cone cells. Neither giant trophoblast nor ectoplacental cone cell cultures show an increased 3β-HSD specific activity in response to a variety of hormones, including gonadotrophins. In fact, regardless of the gestation age at which the trophoblast cultures are initiated, 3β-HSD activity inevitably follows the same temporal pattern observed in vivo. Taken together, these facts suggest that the levels of 3β-HSD in trophoblast cells are intrinsically controlled and that, unlike the ovary, progesterone production by trophoblast cells in vivo is not influenced by gonadotrophic hormone levels.     

Sequence organization in Xenopus DNA studied by the electron microscope.

Xenopus laevis DNA was extracted from red blood cells and sheared to a mean length of 2780 nucleotides. The DNA was stripped of foldback-containing fragments and incubated to C₀t 10 (mol · s · l⁻¹), allowing most repetitive sequences to form duplex structures. Duplex-containing fragments were eluted from an hydroxylapatite column and visualized for electron microscopy by spreading from 57% formamide according to the modified Kleinschmidt technique of Davis et al. (1971). The mean length of the fragments observed was 2445 nucleotides. A total of 1700 DNA strands were photographed and studied. Less than 5% of the total strand length was in uninterpretable structures. Every molecule falling within the confines of the plates was included in the sample. Over 50% of the total strand length in the sample was found in structures bearing at least one interspersed repetitive sequence duplex terminated by four single-strand regions. The fraction of DNA present in duplex regions was almost exactly that predicted if the duplex regions represent all the interspersed middle repetitive sequence in the Xenopus genome. Direct measurement of visualized duplexes shows that the mean length of interspersed repetitive sequence elements in this genome is 345 nucleotides. Duplex length was shown to be independent of the length of the strands bearing the duplexes. These observations provide direct confirmation of the length of approximately 300 nucleotides indicated for interspersed repetitive sequences by earlier physical-chemical studies 011 Xenopus DNA. In strands carrying two duplexes terminated by single-strand regions the interduplex, or single-copy sequence element length could be measured. Sequence interspersion curves generated from these data are roughly consistent with those derived earlier from measurements of hydroxylapatite binding as a function of fragment length.     

Unsaturated fatty acids as second messengers of superoxide generation by macrophages

Chemically elicited guinea pig peritoneal exudate macrophages respond by superoxide (O2-) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O2- production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca2+. O2- production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O2- production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O2- production by stimulants or fatty acids. On the contrary, O2- formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O2- generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O2- production; native and lipoxygenase-treated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O2- production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O2- generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

Synthesis of 2,3,4-trihydroxy-L-phenylalanine from s-methyl-L-cysteine and pyrogallol by L-tyrosine phenol-lyase.

A trihydroxy derivative of phenylalanine was synthesized from S-methyl-L-cysteine and pyrogallol by the crystalline tyrosine phenollyase (L-tyrosine phenol-lyase (deaminating) EC 4. I. 99.2 formerly known as β-tyrosinase) from Escherichia intermedia. The product was isolated as its N-acetyl-triacetoxymethylester and identified as 2,3,4-trihydroxy-L-phenylalanine by the analyses of NMR, MS spectra and optical rotation.     

Reversal of immunological tolerance by aclacinomycin through inhibition of suppressor cell activity

The elimination of suppressor cells by aclacinomycin, which could be the mechanism by which immune responses are enhanced after its administration, was studied in mice in which tolerance had been induced by the injection of high doses of sheep red blood cells (SRBC). We observed that tolerance could not be induced in aclacinomycin-treated mice, and that aclacinomycin inhibited the expression of tolerance to SRBC. This drug also diminished the capacity of spleen cells from SRBC-tolerant mice to inhibit the response of normal animals upon adoptive transfer, indicating that suppressor cells had been eliminated from the tolerant spleen cell population. The efficiency of the elimination of suppressor cells for DTH reactions appears greater than that of suppressor cells for plaque-forming cell responses.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.