Release of lutropin (LH) and follitropin (FSH) in cattle after administration of a new gonadoliberin (GnRH) analogue in comparison with the gonadoliberin decapeptide.

A gonadoliberin (GnRH) analogue nonapeptide (Hoe 766) was administered intramuscularly in concentrations between 2.5 and 50 μg to m?ture cows in order to study the response of lutropin (LH) and follitropin (FSH). Results were compared with those from experiments of the GnRH decapeptide (Hoe 471). Plasma LH and FSH were radio-immunologically determined. Increasing doses of GnRH analogue up to 15–20 μg caused an approximately linear increase in total plasma LH and FSH until the response reached a plateau. With these amounts peak values were about 60 fold higher for LH and 3.5 fold higher for FSH than basal levels about 135 minutes after injection. Higher values lasted for more than 6 h for LH and about 5 h for FSH. The LH response was much greater and more prolonged than for FSH.Doses of the nonapeptide analogue 50 to 70 times lower than the GnRH decapeptide provoked about the same height and duration of LH and FSH response.     

The effects of injecting [D-Ser(But)6] Des Gly-NH2(10) luteinizing hormone releasing hormone ethylamide in early, mid and late seasonal anoestrus on gonadotrophin secretion and luteal function in the ewe

The ability of the luteinizing hormone releasing hormone (LH-RH) analogue [D-Ser(Bu(t))(6)] Des-Gly-NH(2)(10) LH-RH ethylamide to stimulate the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and to induce ovulation and luteal function in seasonally anoestrous ewes was investigated by injecting the analogue at three stages of the anoestrus (day 118, day 182 and day 235 of the year). After injection on day 118, eight of nine ewes ovulated and all of the former secreted progesterone during the subsequent 20 days. After injection on day 182, six of the nine ewes ovulated, of which none showed luteal function. Only two of the nine ewes were not already secreting progesterone on day 235. Both of these responded to the analogue by secreting normal luteal levels of progesterone. The mean LH peak heights in response to injection at the three stages showed no significant differences from one another. The mean FSH peak heightafter injection on day 182 was significantly lower than the mean FSH peak height associated with the other two challenges (P < 0.05). On day 116 of the following year, 20 ewes were treated with the analogue as before. The high progesterone levels confirmed the results of the day 118 challenge in the previous year. However, none of the ewes conceived when inseminated artificially 24 and 36 hours after analogue treatment.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Reaction of o-phthalaldehyde and thiols with primary amines: Fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles

The fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles, formed by the reaction of o-phthalaldehyde (OPTA) and thiols with primary amines, are reported. Variations in thiol and amine substituents and solvent polarity have large effects on the isoindole fluorescence spectra. These parameters, in addition to 3-thiol substitution of the isoindoles, pH, and the use of phosphate vs borate aqueous buffers, were found to have dramatic effects on the corrected relative fluorescence intensity. Low concentrations and nonaqueous solvents apparently stabilized most adducts while aqueous solutions, especially at low pH, caused pseudo-first-order decomposition, probably via hydrolysis to the corresponding 2,3-dihydro-1H-isoindole-1-one. However, 3.3 × 10⁻⁸ solutions of the more intensely fluorescent adducts (total adduct 5 pmol) were readily detected if the fluorescence was determined shortly after adding the isoindole to pH 9.2 borate buffer. The adduct formed using ethanethiol and n-propylamine possessed spectral properties which were the most responsive to changes in solvent polarity and was the most stable under the various conditions employed. Finally, arguments are presented that these isoindoles are the products in several other fluorogenic assays using OPTA.     

Ovarian hormones and the duration of sexual receptivity in the female golden hamster

The induction of sexual receptivity and its maintenance after copulation in ovariectomized female golden hamsters (Mesocricetus auratus) was found to be a function of the levels of ovarian hormones administered. Various combinations of estradiol benzoate (between 0.6 and 666 μg) and progesterone (between 0.05 and 5.0 mg) were administered in two experiments. Although some animals responded at 0.6 μg, higher levels of estradiol benzoate (1–6 μg or more) were more effective in inducing levels of lordosis equivalent to those seen in intact females in natural estrus. After mating, a depression in lordosis was observed in both ovariectomized and intact females. However, in ovariectomized females (excluding animals that did not respond initially) the duration of postcopulatory receptivity was a function of the level of progesterone administered. High levels of progesterone tended to prolong slightly the duration of postcopulatory receptivity.     

Lipid-protein interactions of erythrocyte membranes: comparison of normal O, Rh (D) positive with the rare O, Rh null

Phospholipase A₂ modification of lipid-protein interactions of normal O,Rh(D) positive erythrocyte membranes increased the fluorescence intensity of the membrane bound probe, 1-anilinonaphthalene-8-sulfonate (ANS) and increased the N-1-[¹⁴C]-ethyl maleimide ([¹⁴C]-NEM) labeling of sulfhydryl groups in two proteins of molecular weight >200,000. In marked contrast, phospholipase A₂ modification of the rare phenotype O,Rh_null membranes resulted in no significant increase in ANS fluorescence or labeling of sulfhydryl groups by [¹⁴C] NEM. Since the O,Rh_null erythrocytes demonstrated an increased osmotic fragility and decreased survival time, the fluorescence and sulfhydryl labeling data support the conclusion that hydrophobic bonding between β-fatty acid side chains and non-polar regions of asymmetric proteins is necessary for maintaining the native structure of the O,Rh(D) positive membrane. Comparative studies with phospholipase C or D implied that ionic bonding played a similar though less important structural role in both membranes.     

Analysis of double-helix motions with spin-labeled probes: binding geometry and the limit of torsional elasticity

The e.p.r.⁵ spectra of a family of spin-labeled probes non-covalently bound to DNA have been measured as functions of helix orientation, packing density and temperature. The spectra are interpreted in terms of the geometrical relations between the helix axis and the orbital containing the unpaired electron and in terms of the motions of the helix. Torsional and flexural motions can be distinguished.Spectra from well-ordered helices have been obtained using fully hydrated DNA fibers that are in thermodynamic equilibrium with unbound probe in dilute salt solution. The binding equilibria are similar to the equilibria in dilute DNA solution. The spatial relations between the spin label and the helix, inferred from the spectra, correspond closely to the structure expected on the basis of intercalation perpendicular to the helix axis and a sterically hindered amide bond between the spin label and the intercalating moiety of the probe. Viscometric measurements with one probe also indicate intercalation.Linear e.p.r. spectra of solutions, randomly condensed DNA, and fibers show substantial torsional motion but no detectable flexure on the linear e.p.r. time scale (> 300 ns). The correlation time of a propidium-based probe is much longer than that of aminoacridine intercalators. The probes with short correlation times are considered to be too weakly coupled to the adjacent base-pairs to be reliable indicators of DNA dynamics. For the propidium probe the correlation time, 30 nanoseconds, and its temperature dependence are compared with the properties expected according to four models: tight rotational coupling along the entire length of the helix; swivels at fixed intervals; a two-state exchange; and elastic rotational coupling between adjacent nucleotide pairs. In terms of the fourth model, the results suggest that each nucleotide pair undergoes random oscillation with an r.m.s. amplitude of not more than 4 ° to 5 ° at room temperature. That value agrees with estimates made in other ways.     

Comparison of the acid denaturation of several hemoglobins which differ in amino acid sequence

There are pronounced differences in kinetic and thermodynamic stability between human and horse hemoglobins. Since the amino acid sequences of the α, β dimers of horse and human hemoglobins differ in 61 locations, it is difficult to account for them in terms of specific direct or indirect effects of the sequence differences. Rhesus hemoglobin differs from human in only 12 locations and its stability resembles that of human more closely than does horse, although pronounced differences remain. The stabilities of rhesus ferrihemoglobin and deoxyhemoglobin (Hb⁺ and Hb °) are intermediate between those of the corresponding high-spin forms of horse and human hemoglobin; but there are only small or negligible differences between the low-spin forms (carbonylhemoglobin and oxyhemoglobin) of the two species. The equilibrium isotherm between native and acid unfolded forms of rhesus Hb⁺ resembles that of horse more than that of human, but it is slightly more stable and slightly less cooperative. The effects of octanol on the rates of unfolding of rhesus ferrihemoglobin are only slightly smaller than with human. There is no effect of octanol on the unfolding rate of any of the CO hemoglobins. Unlike the equilibria of horse and human, octanol is also without effect on the unfolding equilibrium of rhesus ferrihemoglobin, and thus qualifies as a true catalyst of the initial stage of the acid unfolding reaction of the monkey ferriprotein. Differences in stability are tentatively attributed to a limited number of the 12 differences between the two proteins.     

On the chemical structure of the apical droplet from eggs of Culex pipiens

The chemical nature of the apical droplet from eggs of Culex pipiens was investigated by chromatographic techniques. Results indicated that the hydrolysate of the apical drop contains C-12, C-14, C-16, and C-18 straightchain aliphatic fatty acids. A C-12β-hydroxy fatty acid was also found, but the largest component of the fatty acid mixture of the apical drop was shown to be a C-14β-OH fatty acid. Two other fractions appear to be unsaturated fatty acids, probably C-12 and C-14. Quantitative estimation of the percentage of each fatty acid in the mixture showed that about 85 per cent of the fatty acid content of the apical drop consisted of hydroxy fatty acids. By thin-layer chromatography, the largest component coincided with β-OH myristic acid.Glycerol was confirmed to be present in the hydrolysate. Feeding studies with radioactive ³²PO₄^?3 and ³⁵SO₄^?2 showed no significant incorporation of phosphorus, but a sulphur-containing anionic compound could be detected in the apical drop. Infrared analysis showed the presence of an ester group, double bond, primary and secondary alcohol groups, suggesting the presence of hydroxy-, unsaturated-, saturated straight-chain fatty acids, as well as mono-and diglycerides. The structural evidence explains in part the surfactant properties of the apical drop.     

Avoidance learning at two levels of shock in rats receiving MSH1,2

Rats receiving MSH or diluent were trained in a two-way shuttle box to avoid shock presented at two levels of intensity. At the low level MSH improved acquisition; at the high level rats given MSH tended to perform more poorly, but were not statistically different from controls. No differences were found in measures of activity or excitability, but higher defecation rates, which may indicate more emotionality, were found in groups receiving high levels of current and in groups receiving MSH. The study suggests that MSH may facilitate learning at low, but not at high, levels of shock.     

An analysis of sandbathing and grooming in the kangaroo rat (Dipodomys merriami)

The sandbathing and grooming behaviour of ten kangaroo rats (Dipodomys merriami) were recorded on sand and woodchip substrates after periods of 0,1,5 and 10 days without sand. Sandbathing is restricted to the sandy substrate. Grooming occurs on both, but with a higher frequency on sand. Increases in both grooming and sandbathing occur with increasing sand deprivation, but the temporal patterning does not change. D. merriami tends to alternate sandbathing components in contrast to other Dipodomys species. Lipid on the pelage increases noticeably with sand deprivation and decreases during a sandbathing bout; sand appears to be removed from the pelage by shaking and grooming. These findings suggest a three-process system for care of the body surface.     

Solid-phase tuftsin (Thr-Lys-Pro-Arg) synthesis: deprotection and resin cleavage with trifluoromethane sulfonic acid.

A method is described for the synthesis of the naturally occurring tetrapeptide tuftsin (Thr-Lys-Pro-Arg). This stimulates phagocytosis of granulocytes and macrophages. Trifluoromethanesulfonic acid is used to cleave the tetrapeptide from its supporting resin in solid-phase synthesis. This reagent also causes deprotection of several protecting groups in bifunctional residues. The most significant is the complete removal of the tosyl group from N^G-tosyl-arginine-resin ester.     

The in vivo effect of benzamide and phenobarbital on liver enzymes: poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.     

Male sexual behavior in deer mice (Peromyscus maniculatus) following castration and hormone replacement

Sexually experienced male deer mice (Peromyscus maniculatus bairdi) were castrated and tested for male sexual behavior. In the weeks following castration male sexual behavior decreased. Ejaculation disappeared first, followed by intromission and, finally, mounting. Castrated males failing to copulate were assigned to one of four treatment groups: 200 μg testosterone propionate (TP); 200 μg dihydrotestosterone propionate (DHTP); 2 μg estradiol benzoate (EB); or sesame oil (OIL). TP and DHTP were equally effective in restoring the complete male sexual behavior pattern. In contrast, EB was effective in stimulating mounting and minimally effective in stimulating intromissions (vaginal penetration), but did not stimulate ejaculatory responses. These data indicate that in deer mice testosterone may mediate male sexual behavior through reduction to dihydrotestosterone rather than through aromatization to estradiol.     

Computer simulation of cellular movements: cell-sorting, cellular migration through a mass of cells and contact inhibition

The motility rules for cellular movement proposed earlier by Goel &; Rogers for engulfment of two or more intact embryonic tissues have been used to simulate on a computer the phenomena of cell-sorting, migration of individual cells through a mass of cells and contact inhibition of overlapping. These simulations in the most part are found to be consistent with the observations with real cells.     

Changes in enzyme pattern of ehrlich ascites tumor cells following serial cultivation in media with increased (hypertonic) NaCl content

Adaptation of Ehrlich ascites tumor cells to serial cultivation in media with progressively elevated (hypertonic) NaCl content (“high NaCl”-tolerant cells) has resulted in progressive increases of the cellular activities of NAD-dependent glycerol-3-phosohate dehydrogenase (EC 1.1.1.8), NAD-dependent malate dehydrogenase (EC 1.1.1.37), glutamate—oxalacetate transaminase (EC 2.6.1.1.), NAD(P)-dependent glutamate dehydrogenase (EC 1.4.1.3), NADP-dependent malate dehydrogenase (EC 1.1.1.40, “malic enzyme”) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42). The activities of glutamate—pyruvate transaminase (EC 2.6.1.2.) and of glycolytic enzymes as phosphofructokinase (EC 2.7.1.11), glyceradehydephosphate dehydrogenase (EC 1.2.1.12) and lactate dehydrogenase (EC 1.1.1.27) were only slightly and not in progressive manner (in response to the progressive increase of the environmental NaCl concentration) affected. These changes are discussed with respect to a metabolic pattern of these “high NaCl”-tolerant cells which is compatible with increased energy requirements, especially for active cation transport. It is suggested that these increased cellular enzyme activitees reflect an increased transfer of reducing equivalents across mitochondrial membranes (via the “glycerophosphate cycle and the malate—aspartate shuttle”) and possibly a stimulated lipid metabolism. These alterations in the level of enzyme activities must be regarded as an adaptive cellular response to the “high NaCl” enviromment, since readaptation to growth in regular isotonic media resulted in a reversion to the enzyme pattern characteristic of the parent cells.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Reactivity of amino acids in the azo coupling reaction: I. Dependence of their reactivity on pH

Azo coupling reactions of N-α-acetylhistidine, N-α-acetyltyrosine, and N-α-acetyllysine with p-methylbenzenediazonium ion were investigated as model reactions to obtain information on the relative reactivity of the histidine, tyrosine, and lysine moieties of protein, separated from structural effects. The azo coupling yields of the amino acids increased as the pH of the reaction medium was increased, indicating that the ractive species are the imidazole anion of histidine, the phenolate anion of tyrosine, and the neutral ε-amino group of lysine. It was calculated, based on percentage yields of the azo products, that the imidazole anion is more reactive than the phenolate anion and the ε-amino group, respectively.