Demonstration of cytoplasmic receptors for the molting hormones in crayfish

Summary From in vitro experiments using different binding assays it is in crayfish demonstrated that the cytosol of target tissues is able to bind both ecdysone and ecdysterone. The ability to bind ecdysteroids is destroyed by heating and by treatment with -chymotrypsin and N-ethyl-maleinimide (NEM) (Figs. 4, 5). In target tissues there is a strong positive correlation between protein content and binding (Fig. 6). The association of the hormone-protein-complex is rapid, taking only a few min even at 5° C (Fig. 3). The binding of the two hormones to the cytosol is both specific and saturable. The association constants for the cytoplasmic receptors from hypodermis, hindgut and midgut gland are in the range of 3–6×10⁷ M^–1 for ecdysone and 5–7×10⁸ M^–1 for ecdysterone (Fig. 8). The data suggest the existence of cytoplasmic ecdysteroid receptors.     

High-pressure, anion-exchange chromatography of proteoglycans

Although high-performance liquid chromatography has been used extensively to characterize the glycosaminoglycan chains of proteoglycans, very few researchers have reported the use of this technology for the separation of intact proteoglycan species. The high molarity denaturing buffers required for proteoglycan disaggregation and separation are often not compatible with the low back-pressure limitations imposed by many of the HPLC systems designed for the separation of biological macromolecules. In this study, heparan sulfate and dermatan sulfate proteoglycans, obtained by the metabolic labeling of cultured corneal endothelial cells, were rapidly and completely separated in less than an hour in a high-pressure liquid chromatography system. The separation, which used a Dionex BioLC system equipped with a Pharmacia Superloop and a ProPac PA1 column, also effected a greater than 10-fold concentration of the proteoglycans during the separation procedure. All buffers were 8 M in urea, and the back-pressures generated during the separation were well below the limit of the system. The pooled fractions from the ion-exchange column were subsequently analyzed for glycosaminoglycan composition and molecular size. The system was able to resolve dermatan sulfate-substituted species from heparan sulfate-substituted species in a single chromatographic step. The proteoglycan nature of the recovered products was established by Sepharose CL-4B chromatography and gel electrophoresis.     

High-pressure liquid chromatography of plasma free acid porphyrins

The separation and quantitation of plasma free acid porphyrins by high-pressure liquid chromatography and fluorescence is described. Porphyrins were extracted from plasma in a simple manner with a recovery >90%. They were separated by high-pressure liquid chromatography on a silica gel (10 μm) column, using a gradient of acetone:dilute acetic acid. Resolution of seven free acid porphyrin standards including coproporphyrins I and III, but not uroporphyrins I and III, was achieved in 12 min at picomolar concentrations. Plasma of patients with erythropoietic protoporphyria displayed protoporphyrin. Uroporphyrin was the only porphyrin found in plasma of eight patients with porphyria cutanea tarda. Normal plasma contained small amounts of uroporphyrin and/or traces of protoporphyrin.     

High-pressure liquid chromatography of alpha-keto acid 2,4-dinitrophenylhydrazones

Various α-keto acids were separated as their 2,4-dinitrophenylhydrazine derivatives by ion-pair, reverse-phase, high-pressure liquid chromatography. Excellent baseline resolution was obtained for a seven-component homologous series of α-keto acid dinitrophenylhydrazones at increasing carbon-chain length. Branched-chain keto acids were also separated. Resolution of syn and anti isomers of the α-keto acid derivatives was possible. Pyruvate from biological material was located and identity confirmed by an enzymic peak shift technique. Monitoring at 366 nm permits low-level (nanogram) amounts of keto acids to be detected. Ion pair versus ion exchange is discussed with regard to the mechanism of chromatographic separation.     

High-pressure liquid chromatography of cobalamins and cobalamin analogs

High-pressure liquid chromatography has been used to separate, identify, and quantitate 37 different cyanocobalamin analogs, including the most commonly occurring analogs that result from bacterial synthesis. This technique has also been used to simultaneously separate, identify, and quantitate five naturally occurring cobalamins that differ in their upper axial ligands: methylcobalamin, adenosylcobalamin, hydroxocobalamin, cyanocobalamin, and sulfitocobalamin. This method permits rapid quantitative detection and identification of cobalamins and naturally occurring and synthetic cobalamin analogs in complex mixtures.     

Past and present studies with ponasterones, the first insect molting hormones from plants.

The search for antitumor compounds from Southeast Asian plants led to ponasterones, the first phytoecdysteroids, just a year after structure determination of ecdysone and 20-hydroxyecdysone. An independent study of Chinese herb constituents by Takemoto et al. at Tohoku University led to the simultaneous and totally independent discovery of phytoecdysteroids. These findings greatly facilitated research in insect and crustacean physiology. The original structural studies on various phytoecdysteroids have led to interdisciplinary bioorganic studies in the area related to ecdysone receptor, ecdysone biosynthetic precursor (or its storage form), crustacean molt inhibitory factors, and so on.     

Role of gonadal and adrenal steroids and thyroid hormones in the regulation of molting in domestic goose

Plasma levels of testosterone (T), 17-β-estradiol (E2), progesterone (P4), dehydroepiandrosterone (DHEA), corticosterone (B), thyroxine (T4) and triiodothyronine (T3) were monitored during postnuptial and the prenuptial molt in domestic goose (Anser anser domesticus) in both sexes. 1. At the beginning of postnuptial molt (when the old, worn dawny-, and cover feathers' loss starts) in ganders, the levels of T, E2, P4 decrease while DHEA and B significantly increase. The elevated levels of T4 and low T3 concentrations characteristic of the last phase of the reproduction, remain unchanged. In layers, similar changes were observed, however, B decreases. 2. In the early phase of outgrowth of wing and cover feathers, plasma levels of T, E2 and P4 are low. Elevated B, DHEA and T4 concentrations decrease in ganders, while in layers DHEA increases and B and T4 levels remain unchanged. T3 increases in both sexes. 3. The subsequent intensive outgrowth period of wing- and cover feathers both in ganders and in layers is characterized by very low levels of T, E2, DHEA and T4, but P4 increased, and T3 concentration remain high. 4. At the end of postnuptial molt - when the outgrowth of dawny, cover-, and wing feathers stops - very low T, E2, P4, DHEA and T4 levels and and high T3 plasma levels were found in both sexes. Fast increase of plasma B was detected in ganders, while in geese, B concentration remain high. 5. During prenuptial molting (outgrowth of contour and tail feathers) low E2, P4 and T4, increasing T and DHEA, but very high T3 and B plasma concentration were measured in ganders. In layers, very low T, E2, P4, DHEA and T4 levels, and very high B and T3 levels were found. 6. At the beginning of the fall-winter sexual repose (postmolting stage) T, E2, P4, DHEA and T4 levels increase, T3 and B declines in both sexes. 7. In the subsequent phase of fall-winter period (preparatory stage) there is a further increase in T, P4 and T4, a fast increase of B and a decrease of E2, DHEA and T3 in ganders. In layers, T, P4 and DHEA decrease, B increases and the T4 and T3 do not change. 8. At the beginning of reproduction high T level, unchanged DHEA, slightly declined P4, and decreased E2, T4, T3 and a strong decline of B concentrations occur in ganders. In layers, T is further increased, E2 and P4 shows high levels, and, at the same time DHEA and T3 remain unchanged, while B and T4 decrease.     

High-pressure liquid chromatography for isolation of clastogenic agents from urine

Small columns of XAD-2 resin have been widely used to extract and concentrate mutagenic materials from urine. Using analytical HPLC and assays for clastogenicity with Chinese hamster ovary cells, we found that small columns of XAD-2 resin (1.5 ml bed volume) retain only a small percentage of organic material and undetectable amounts of genotoxic activity in urine samples. Increasing the size of the XAD resin bed resulted in better recoveries, but much organic material was still lost by overloading of the column. In contrast, when urine was acidified and chromatographed by preparative reversed-phase HPLC using large-bed-volume (500 ml) commercial columns, retention of hydrophobic organic material from urine was excellent. Subsequent stepwise elution of the column with increasing concentrations of acetone produce 3 fractions of organic material of increasing hydrophobicity. When urine from smokers was analysed, all 3 fractions contained material which was clastogenic to Chinese hamster ovary cells. The procedures developed are suggested as a new general purpose approach to the isolation of genotoxic materials from urine.     

High-pressure liquid chromatography of neurotoxic phenylphosphonothioate esters and related compounds

It has been suggested that perfluorooctanoic acid occurs in human plasma; however, no method of analysis for this compound in biological samples has been published to date. A method is presented for the analysis of perfluorooctanoic acid in plasma, urine, and liver tissue based on conversion of the acid to its methyl ester followed by separation and quantitation by gas chromatography.     

The molting hormones from the embryonated egg of the tobacco hornworm, Manduca sexta (L.)

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.     

The apparent requirement of two hormones, alpha- and beta-ecdysone, for molting induction in insects

Puff formations at loci I-18-C and IV-2-B of the salivary gland chromosomes are early indications of a beginning molting process in Chironomus tentans larvae. The effectiveness of the two ecdysone analogs, α- and β-ecdysone, in inducing these puffs was compared. Incubation of salivary glands in vitro with β-ecdysone causes only puff IV-2-B to appear; incubation with α-ecdysone stimulates initially puffing at only I-18-C. After an injection of α-ecdysone, puffing at I-18-C begins within less than 15 min, whereas puffing at IV-2-B is delayed for more than 30 min. Following an injection of β-ecdysone, puffing at IV-2-B begins within less than 15 min, whereas puffing at I-18-C is delayed. Injected ³H-α-ecdysone is converted to β-ecdysone and a polar compound. Injected ³H-β-ecdysone is converted to a compound less polar than α-ecdysone and a polar metabolite which stimulates puffing at I-18-C, like α-ecdysone. It is suggested that the two ecdysones have different targets in the cell, that they can be rapidly converted to compounds with the activity of the other analog, and that the induction of a complete molt requires the action of both hormones.     

High-pressure liquid chromatography quantitation of cytochrome c using 393 nm detection

The release of cytochrome c from the mitochondrial intermembrane space can induce apoptotic cell death. Previous methods to detect cytochrome c release from mitochondria have relied upon immunoblotting, a procedure that can be limited by nonlinearity of signal, epitope masking, and impracticality for large numbers of samples. In order to circumvent these limitations, we have developed a reverse-phase high-pressure liquid chromatography method for cytochrome c detection and quantitation by taking advantage of a novel acid-induced absorbance maximum at 393 nm for cytochrome c in buffer containing 0.1% trifluoroacetic acid. Using a C4 reverse-phase analytical column, this assay had a quantitation limit of 10 ng (0.8 pmol) of cytochrome c. We demonstrated the detection and quantitation of cytochrome c from isolated mitochondria. This method of cytochrome c analysis may be useful for the study of agents that cause mitochondrial dysfunction and apoptotic cell death.     

Purification of growth hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) on a column of Radial-Pak C₁₈ cartridge was utilized for the purification of a variety of growth hormone (GH) proteins from mammalian, avian, amphibian and fish pituitary glands. Recovery of GH from pituitary glands of up to 0.43% of total protein was obtained with a high degree of homogeneity as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The HPLC-purified GHs show reactions of identity or near identity by immuno-diffusion studies on agar gel. This method offers a convenient and rapid purification of vertebrate GH on an analytical or preparative scale.     

High-pressure liquid chromatography of polycyclic aromatic hydrocarbons and some of their derivatives.

The separation of polycyclic aromatic hydrocarbons and their derivatives by means of high-pressure liquid chromatography on Permaphase ODS is described. The method consists of the (isocratic) elution of compounds from the column with a methanol-water mixture of constant composition and is particularly suited to the identification of metabolic products of polycyclic hydrocarbons.     

Analysis of pancreatic peptide hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) is examined as a method for separating pancreatic peptides. The method was based on gradient elution with acetonitrile in an acid phosphate buffer (pH 3.10). Apart from human and porcine insulin all the other peptide standards tested (thyrotropin-releasing factor, vaso-active intestinal polypeptide, human C-peptide, porcine C-peptide, somatostatin, porcine glucagon, porcine proinsulin and porcine pancreatic polypeptide) could be separated simultaneously in 40 minutes with a binary gradient composed of five linear segments and increasing from 0 to 60% acetonitrile. Human and porcine insulin could be almost completely resolved by a minimal reduction in the steepness of the acetonitrile gradient. Repeated injections of human C-peptide and porcine insulin resulted in a coefficient of variation of less than 1.5% in the retention times. The use of 125I-labelled peptides gave recoveries exceeding 90%. HPLC of acid ethanol extracts of autopsy pancreases from three infants showed that the immunoreactivity of the peptides measured remained unaffected by the chromatography. Both immunoreactive C-peptide and immunoreactive insulin (IRI) were recovered in two peaks, the second common peak representing proinsulin and amounting to 6.5 to 8.4% of total IRI. Immunoreactive glucagon was eluted in a single peak. Chromatography of plasma extracts from two infants of diabetic mothers demonstrated that proinsulin accounted for 59-63% of total IRI, while insulin was separated into two peaks corresponding to the standards of human insulin and porcine insulin. These results indicate that reversed -phase HPLC is a method with a good reproducibility and a high recovery applicable to the rapid and effective separation of pancreatic peptides from biological extracts.