Behavioral fever in anuran amphibian larvae.

Following intraperitoneal injection with killed gram-negative bacteria, $\text{Aeromonas}$$\text{hydrophila}$, tadpoles of $\text{Rana}$$\text{catesbeiana}$ and of $\text{R}$. $\text{pipiens}$ showed significant mean increases in preferred temperature of 2.6°C and 2.7°C, respectively, in an electronic thermoregulatory shuttlebox device. This “behavioral fever” is similar to elevations in preferred temperature previously demonstrated for fishes, reptiles and mammals, although both normal and febrile thermal preferenda vary among vertebrates.     

Kinetics of calcium efflux and exchange from Rana pipiens oocytes immediately following reinitiation of the first meiotic division: Comparison of various meiotic agonists and antagonists

The reinitiation of the meiotic divisions and the release of ⁴⁵Ca from the $\text{Rana}$$\text{pipiens}$ oocyte has been studied as a function of meiotic agonists and antagonists. Each of the meiotic agonists tested (progesterone, insulin, D-600, La³⁺) caused a decreased ⁴⁵Ca uptake and an increased efflux during the first 15 min after exposure. The effects of progesterone, D-600, and La³⁺ are not additive and progesterone will not release additional ⁴⁵Ca in oocytes pretreated with D-600 or La³⁺. Tetracaine inhibits both progesterone-induced release of ⁴⁵Ca and an early step in meiosis (nuclear membrane breakdown). [Tetracaine]_o required for 50% inhibition of nuclear breakdown decreases with decreasing [progesterone]_o suggesting competitive inhibition. The Ca, Mg-ionophore A23187 shows a similar competitive inhibition of progesterone-induced nuclear breakdown and stimulates a rapid release of ⁴⁵Ca within the first 1–3 minutes after exposure to the ionophore. Unlike progesterone, insulin, D-600, or La³⁺, the ionophore A23187 stimulates both uptake and efflux of ⁴⁵Ca by oocytes. These results suggest that both a reduced influx and a selective release of calcium from specific membrane sites is essential for steroid reinitiation of the meiotic divisions in $\text{R.}$$\text{pipiens}$ oocytes.     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Uteroplacental dysfunction and prostaglandin metabolism in zinc deficient pregnant rats

Relationships between perinatal mortality, disrupted uteroplacental function and prostaglandin metabolism have been studied in Zn-deficient rats. Uterine contractility $\text{in vitro}$, placental blood flow $\text{in viro}$, and uterine and placental prostaglandin synthesis from [1?¹⁴C] arachidonic acid $\text{in vitro}$ were investigated at day 22 of pregnancy. High amplitude uterine contractions were almost completely eliminated and utero-placental blood flow was decreased by 85% by Zn deficiency. Synthesis of [1?¹⁴C]-prostaglandin E₂, F_2α and 6-keto-F_1α from [1?¹⁴C] arachidonic acid decreased significantly in uterine tissue but increased in placentae. These possibly inter-related effects may contribute to the high perinatal mortality observed in Zn deficiency.     

Concerning the specificity of heme oxygenase: the enzymatic oxidation of synthetic hemins.

Hemin XIII $\text{4}\text{～}$, hemin III $\text{5}\text{～}$, and iron $\text{1,4-di(β-hydroxyethyl)porphyrin}\text{6}\text{～}$ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX $\text{1}\text{～}$. The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-¹⁴C]$\text{6}\text{～}$ and [α-¹⁴C]$\text{4}\text{～}$ as substrates. The chemical oxidation of $\text{4}\text{～}$, $\text{5}\text{～}$, and $\text{6}\text{～}$ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.     

Temperature dependence of active K+ transport in cation dimorphic sheep erythrocytes

Arrhenius diagrams of K⁺ pump fluxes measured between 15°C and 41°C were discontinuous in high K⁺ but not in low K⁺ sheep red cells. Exposure of low K⁺ cells to anti-L caused a bimodal temperature response of K⁺ pump flux with a transition temperature, $\text{T}\text{c}$, similar to that found in high K⁺ cells but with comparatively higher activation energies above $\text{T}\text{c}$.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

A new category of ovulation inhibitors: linear LH-RH analogues having more than ten residues.

A linear analogue of the luteinizing hormone-releasing hormone, longer than a decapeptide, is described for the first time, which is equivalent in potency to the best known inhibitors of ovulation, and which constitutes an important new lead to the design of inhibitors of even greater potency. At a dosage of 200 μg/rat, the undecapeptide [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH caused 100% inhibition of ovulation. The related analogues, [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH and [(Gly-Pro)¹, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH, were less active, $\text{in}\text{vivo}$. All of these undecapeptides inhibited the action of 0.6 ng/ml of LH-RH by greater than 50% at the very low level of 10 ng/ml.     

Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro

One juvenile hormone was isolated from culture medium containing isolated corpora allata of the grasshopper $\text{Schistocerca vaga}$ (Orthoptera: Acrididae) and was shown by microchemical methods to be methyl (2$\text{E}$, 6$\text{E}$) - (10$\text{R}$) - 10, 11-epoxy-3, 7, 11-trimethyldodeca-2, 6-dienoate. This compound (JH III), which occurs in a sphingid moth $\text{Manduca sexta}$, is the first juvenile hormone identified in an insect order other than the Lepidoptera. Grasshopper organs incorporate both [2?¹⁴C] acetate and [methyl-¹⁴C] methionine into JH III showing $\text{de novo}$ biosynthesis, but no indication of the synthesis of JH I or JH II was seen.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Nitrobenzene reduction and reductive cleavage of azobenzenes in two species of Arachnida

The metabolism of [¹⁴C]-nitrobenzene and [¹⁴C]-4-dimethyl-aminoazobenzene was examined in a tick, $\text{Boophilus microplus}$, and a spider, $\text{Nephila plumipes}$. The reduction of both compounds to aniline is reported in both species. N-Dealkylation of DAB is also recorded.     

Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro

When corpora allata from adult female $\text{Schistocerca}$$\text{gregaria}$ are incubated $\text{in}$$\text{vitro}$ with either ³H-$\text{trans}$, $\text{trans}$ farnesenic acid or ³H-$\text{trans}$, $\text{trans}$, $\text{cis}$ bishomo-farnesenic acid and [$\text{methyl}$-¹⁴C] methionine, they fabricate large quantities of the corresponding double labelled methyl 10, 11-epoxy esters. Radio GLC of these products indicates retention of geometric configuration at the C-2 and C-6 double bonds. Separate analyses of the contents of the glands and medium after incubation show that the epoxy esters are rapidly released from the glands into the medium and that only the glands contain the corresponding unepoxidized esters. We suggest that unepoxidized esters are the intracellular intermediates in the formation of juvenile hormones from the unsaturated acids. Gel filtration shows that the epoxy esters are not released as stable protein complexes but as simple solutes into the medium. Using this method of promoting the synthesis of juvenile-hormone-active compounds, rates of biosynthesis of epoxy esters of up to 33 ng. per pair of glands per hour have been achieved.     

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

¹³C nuclear magnetic resonance (n.m.r.) spectral data for ¹³C reductively methylated N-terminal tryptic glycopeptides and for ¹³C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins A^M and A^N are presented. Their ¹³C chemical shift data are compared with the previously published ¹³C n.m.r. data for ¹³C reductively methylated homozygous glycophorins A^M and A^N in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the ¹³C resonances of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glyco-octapeptide A^N and of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of glyco-octapepti A^M indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of ¹³C reductively methylated glycophorin A^M is lost when the glyco-octapeptide A^M is produced. The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glycooctapeptide A^N titrated with a $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of 7.7 (Hill coefficient $\text{～ 1}$). The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for ¹³C-labelled glycooctapeptide A^M molecule, the pH dependence of the chemical shift of the ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.     

Metabolic sequestration of putrescine in Neurospora crassa

The metabolic fate of putrescine labelled $\text{in}\text{vivo}$ was investigated after administration of a trace (10^?7 M) of L-[¹⁴C]ornithine to exponentially growing mycelia of $\text{Neurospora}\text{crassa}$, followed by a large chase (2 mM) of L-[¹²C]ornithine. The specific radioactivities of putrescine and spermidine were determined during the chase period by reaction with [³H]dansyl chloride of known specific radioactivity and isolation of the dansyl-derivatives by thin-layer chromatography. Radioactivity remained in the putrescine pool for over 2 h during the chase period. This suggests that putrescine is largely sequestered (80% or more) $\text{in}\text{vivo}$. The metabolic sequestration of polyamines may be a significant factor in the regulation of polyamine synthesis.     

Purification and nucleoside composition of a Q* - containing aspartic acid tRNA from Drosophila.

One form of aspartic acid tRNA from $\text{Drosophila}$,$\text{melanogaster}$ (tRNA_2δ^Asp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of $\text{Drosophila}$, it therefore appears that tRNA^Asp contains the more highly modified nucleoside, Q¹ (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNA_2δ^Asp to NaIO₄ treatment. Utilizing Con A-Sepharose chromatography, tRNA_2δ^Asp from $\text{Drosophila}$ was purified and its nucleoside composition determined by chemical tritium labelling. In addition to the major nucleosides, this tRNA contains rT, hU, m⁵C, ψ, and Q¹, but no other modified nucleosides. Its nucleoside composition is very similar to yeast tRNA^Asp.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

Structural studies of the M blood group determinant

Structural studies of homozygous glycophorin A^M were undertaken by monitoring the ¹³C methyl resonances of ¹³C reductively methylated glycophorin A^M (contains five $\text{N}{}^{\mathrm{?}}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ Lys residues, and the N-terminal $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C]}\text{dimethyl}$ Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin A^M and deglycosylated glycophorin A^M indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated A^M.     

Inhibition by zinc of the binding of C1 to antigen-antibody complexes

Zinc sulphate in the range of 10^?4 to 2×10^?5 M prevents the binding of $\text{C}\text{1}$ to antigen antibody complexes, and the initation of the cascade of events in the classical complement pathway leading to cell lysis. Other heavy metals, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, or Mn⁺⁺ were without effect in this concentration range. Zinc was ineffective when added after $\text{C}\text{1}$ was bound and failed to displace $\text{C}\text{1}$ which was already bound to antigen antibody complexes. The ability of zinc to regulate the binding of the zymogen or activated form of $\text{C}\text{1}$ to antigen-antibody complexes represents a new method of controlling the initiation of the classical complement pathway.