LARGE-SCALE ISOLATION AND FRACTIONATION OF ORGANS OF DROSOPHILA MELANOGASTER LARVAE

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.     

FRACTIONATION OF SPINACH CHLOROPLASTS BY FLOW SEDIMENTATION-ELECTROPHORESIS

A separation of spinach chloroplasts in vitro into fractions according to size (volume) and activity (light-dependent shrinkage and NADP reduction) has been achieved by stable-flow free boundary sedimentation-electrophoresis. The salient features of this chloroplast study are: (a) separation is achieved within 30 min; (b) only small density gradients are required, thus minimizing osmotic effects; (c) the fractions are collected continuously, with size fractionation being evidenced; and (d) particles are separated into fractions of higher and lower activities as compared with the control population.     

Phosphorylation of proteins of ribosomes and nucleolar preribosomal particles from Novikoff hepatmoa ascites cells

After labeling for two hours in vivo with ³²P-labeled orthophosphate, proteins from cytoplasmic ribosomes and nucleolar preribosomal particles of Novikoff hepatoma ascites cells were analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Five proteins (B2, B3, B6, B32 and B35P) were phosphorylated in the ribosomes. Approximately 19 proteins were phosphorylated in the nucleolar preribosomal particles; although four of these were ribosomal proteins, they were different from the proteins labeled in the ribosomes. The 15 additional phosphorylated nucleolar preribosomal particle proteins were non-ribosomal. These results suggest that phosphorylation of proteins of the nucleolar preribosomal particles is independent of phosphorylation of the cytoplasmic ribosomal proteins and may be a part of the maturation process of preribosomal particles.     

Geranylated carbazole alkaloids with potential neuroprotective activities from the stems and leaves of Clausena lansium

Clausena lansium (Lour.) Skeels is an evergreen small tree or shrub with great economic value, which belongs to the genus Clausena of the Rutaceae family. C. lansium is indigenous to Southern China, while currently widely cultivated in subtropical and tropical regions not only for the nutritional value and pharmacological uses of its fruits but also as a medicinal and ornamental plant. In this study, a systematic phytochemical study on the stems and leaves of C. lansium caused the separation and identification of two new geranylated carbazole alkaloids, clauselansiumines A (1) and B (2), as well as 10 known geranylated carbazole alkaloids (3–12). The chemical structures of these isolated geranylated carbazole alkaloids (1–12) were unambiguously determined based on comprehensive spectral data analyses. All these isolated geranylated carbazole alkaloids were tested for their neuroprotective effects against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells in vitro. Compounds 1–12 displayed remarkable neuroprotective effects holding the EC₅₀ values ranging from 0.48 ± 0.04 to 12.36 ± 0.16 μM. These research results disclosed that the separation and purification of these geranylated carbazole alkaloids possessing remarkable neuroprotective effects separated from C. lansium could be extremely important to the discovery of new agents for the treatment and prevention for Parkinson's disease.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Rapid filter method for the microfluorometric analysis of DNA.

A rapid filter method for the microfluorometric analysis of DNA is reported in this communication. Cells collected on cellulose filters are subject to an assay sequence developed from a fluorometric method initially described by J. M. Kissane and E. Robins ((1958) J. Biol. Chem.233, 184–188) for DNA quantitation. The assay is one of high specificity requiring no separation of DNA from other major cellular components. Samples containing as little as 0.2–0.3 μg of DNA have been analyzed by this filter method with a coefficient of variation for replicate standard samples of 3.3%. The DNA content of a number of cell types having different physiological characteristics has been determined by the use of this filter technique. The data obtained for Chlorella, Chlamydomonas, Ochromonas, and Stronglyocentrotus eggs were well within the values reported for these cells by other investigators using classical macromethods. We have taken advantage of the high specificity, sensitivity, and accuracy of this filter assay to determine DNA content during the synchronous growth cycle of the wall-less alga Olisthodiscus luteus using a single small volume culture as a sample source.     

Chemical constituents from the stems of Alyxia schlechteri

The extraction of the stems of Alyxia schlechteri and then using chromatographic separation and recrystallization afforded 2 new compounds, a benzyl coumarin derivative named alyterinin (1) and a germacrane sesquiterpene named alyterinone (2). In addition, 20 known compounds were discovered from the stems of this plant. Mosher's method was used to identify the absolute configuration of 2. Ficusequilignan A (12) showed strong antifungal activity against Pythium insidiosum. Structural determination of all compounds was accomplished by 1D and 2D NMR, IR and MS spectrometry.     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

UPDATE ON THE CLINICOPATHOLOGY OF PITUITARY ADENOMAS

Objective: Pituitary adenomas are the third most common central nervous system tumors and arise from the anterior pituitary within the pituitary fossa.Methods: Literature review and discussion.Results: The signs and symptoms of patients with pituitary adenomas vary from ‘mass effects’ caused by a large adenoma to features secondary to excess pituitary hormones produced by the functioning pituitary adenoma. Detailed histopathologic assessment, based on novel classifications and the latest World Health Organization guidelines, helps to categorize pituitary adenomas into different subtypes and identify features that, in some cases, help to predict their behavior. Most of the pituitary tumors occur sporadically without known genetic predisposition, but in a significant minority of cases, somatic mutations can be identified in the GNAS and USP8 genes. A small proportion of the cases have germline genetic defects or embryonic mutations leading to mosaicism. Genes with germ-line mutations predisposing to pituitary adenomas include AIP, GPR101, MEN1, CDKN1B, PRKAR1A, PRKAR2A, DICER1, NF1, and SDHx, whereas more recently, CABLES1 has also been implicated.Conclusion: Understanding the pathogenesis of pituitary adenomas will allow clinicians to correlate the pathologic and genetic features with clinical data, helping decisions on the best management of these tumors.Abbreviations: ACTH = adrenocorticotropic hormone; AIP = aryl hydrocarbon receptor-interacting protein; αSU = alpha-subunit; EGFR = epithelial growth factor receptor; ER = estrogen receptor; FSH = follicle-stimulating hormone; GH = growth hormone; GHRH = growth hormone–releasing hormone; IGF-1 = insulin-like growth factor 1; LH = luteinizing hormone; MEN1 = multiple endocrine neoplasia 1; MRI = magnetic resonance imaging; NFPA = nonfunctioning pituitary adenoma; PRL = prolactin; TSH = thyroid-stimulating hormone; USP8 = ubiquitin-specific peptidase 8; WHO = World Health Organization     

Lactones 39. Chemical and microbial synthesis of lactones from (−)-α- and (+)-β-thujone

The effective method of isolation, separation and purification of (?)-α- and (+)-β-thujone (1a and 1b) from Thuja occidentalis was elaborated. Chemical (m-CPBA) and microbial Baeyer–Villiger oxidation of (?)-α- and (+)-β-thujone was carried out. Four new bicyclic δ-lactones (2a, 2b, 3a and 3b) with condensed cyclopropane ring were obtained.     

Regulation of purine metabolism adenylosuccinate synthetase from novikoff ascites tumor cells

Adenylosuccinate synthetase has been partially purified from Novikoff ascites tumor cells. The properties of the protein are quite different from the enzyme from rat liver in that the K_m for aspartate is higher and the K_I for the feedback inhibitor AMP is also higher. The antibiotic hadacidin has a preferential inhibitory effect on the tumor enzyme. These results suggest that the Novikoff ascites tumor enzyme is less sensitive to normal feedback controls but may be more sensitive to specific antitumor drugs.     

Bioactive butylphthalide derivatives from Ligusticum chuanxiong

Seven new butylphthalide derivatives, ligusticumolide A-G (1–7), together with two known butylphthalide derivatives (8–9) were isolated from an ethanol extract of Ligusticum chuanxiong Hort. The structures of these derivatives were elucidated from analysis of 1D/2D NMR, UV, IR and HRESIMS data. The absolute configurations of these derivatives were determined by electronic circular dichroism (ECD) calculations and Mosher′s method. Ligusticumolide A (1) and ligusticumolide B (2) are enantiomers that were obtained by chiral separation. Ligusticumolide C (3) and ligusticumolide D (4) are diastereomers. All of the compounds were evaluated for their hepatoprotective activity against N-acetyl-p-aminophenol-induced HepG2 cell injury. Compounds 4, 5, and 7–9 showed more significant hepatoprotective activity than that of the positive control drug (bicyclol) at a concentration of 10 μM (p < 0.01).     

Evidence against protein-induced `internal pressure' in biological membranes. Partition of 8-anilinonaphthalene-1-sulphonate into Triton X-100 micelles and submitochondrial particles

It has recently been proposed that although small amphiphilic molecules partition into phospholipid vesicles this partition is reduced by a factor of 10³–10⁴-fold by the presence of proteins in biological membranes [Conrad & Singer (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5202–5206 and (1981) Biochemistry 20, 808–818]. However, the affinity with which 8-anilinonaphthalene-1-sulphonate partitions into, or binds to, Triton X-100 micelles and submitochondrial particles is very similar and therefore does not support this proposal.     

Chemical constituents from the seeds of Cassia obtusefolia and their in vitro α-glucosidase inhibitory and antioxidant activities

Basing on chromatographic separation techniques, fifteen aglycones (1–15), including two new anthraquinone aglycones (1, 2) and thirteen known compounds (3–15), were isolated from the small polar fraction of Cassia obtusefolia (petroleum ether extract). Structural elucidations were performed by 1D/2D NMR spectroscopy and mass spectrometry. The in vitro antioxidant and α-glucosidase inhibitory activities of these fifteen compounds were determined. Except compounds 12 (IC₅₀ 3.03 ± 0.31 μg/mL, stronger than ascorbic acid, which IC₅₀ was 6.48 ± 2.30 μg/mL) and 13 (IC₅₀ 78.40 ± 2.39 μg/mL), the free radical scavenging capacities of other compounds were weak. Compounds 4, 5, 6 and 13 exhibited inhibitory activities on α-glucosidase with IC₅₀ values of 50.60 ± 1.10, 22.57 ± 0.07, 60.09 ± 1.40, and 80.01 ± 2.66 μg/mL separately, however, all the α-glucosidase inhibitory activities were weaker than positive control (acarbose).     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Enantiomeric chromones from Harrisonia perforata

Six new chromones (1–3 and 7–9), along with 10 known ones, were isolated from the ethanol extract of Harrisonia perforata. The structures of the new compounds were elucidated by extensive spectroscopic and single crystal X-ray diffraction analyses. Compounds 1–3 and 8–9 are all racemates, and the corresponding enantiomers [(+)-1/2/3/8/9 and (−)-1/2/3/8/9] were obtained by chiral HPLC separation, respectively. The absolute configuration of (+)-1 was deduced by Mosher's method.     

Design, Syntheses, and Characterization of a Sterically Encumbered Dioxo Molybdenum (VI) Core

Dioxo-Mo^VI complexes of general formula Tp^∗MoO₂(p-SC₆H₄Dn) (6a-6c) (where Tp^∗ = hydrotris(3,5-dimethyl-pyrazol-1-yl)borate and Dn = dendritic unit) have been synthesized and characterized by spectroscopy and mass spectrometry. ¹H NMR spectra of the metal complexes indicate that the C_s local symmetry about the metal core does not change by the incorporation of dendritic functionality at the thiophenolato ring. Electrochemical data show a ∼20 mV change in the redox potential in the complexes with dendritic ligands suggesting a very small perturbation in the redox orbital, which is also supported by small changes in the electronic spectra. The peak-to peak separation (ΔE_p) increases from 125 mV in 6(a) to 240 mV in 6(c), suggesting sluggish electron transfer in molecules with larger dendritic ligands.     

Cytoplasmic Ribonucleoprotein Components of the Novikoff Hepatoma

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA, very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an s20, _w of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 2:1. ATP and pyrophosphate were equally effective in causing dissociation. ADP was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mµ in diameter. The 46 and 28 S subunits also appeared spheroidal.     

Characterization of the peptidases of Calliphora: Many features allow the utilization of small peptides as an amino acid reservoir

Electrophoretic separation of whole flies and of haemolymph indicates the presence of four peptidases, named dipeptidase A, B and C (Dip A, B and C) and leucine amino peptidase (LAP) after enzymes of similar substrate specificities and electrophoretic mobilities found in Drosophila (Laurie-Ahlberg, Biochem. Genet.20, 407–424, 1982; Walker et al., Insect Biochem.10, 535–541, 1980). Prominent in both tissues and haemolymph, dipeptidase A and B together hydrolyse a variety of dipeptides in vitro and probably most of the fly's small peptide component in vivo. Though Dip A and Dip B hydrolyse many of the same substrates, their activities differ in at least several respects. Dip A's K_ms are higher than Dip B's K_ms and hence in vivo the two enzymes together are likely to provide peptide hydrolysis through a wide range of substrate concentration. Dip A's unique hydrolyses are of peptides with biosynthesized amino acids in the N-terminal position and Dip B's unique hydrolyses are of peptides with essential amino acids in the N-terminal position. Dip B, but not Dip A, is inhibited by free amino acid. It is inhibited non-competitively and most strongly by essential amino acids. In cell-free haemolymph Dip B's activity is more stable than Dip A's. The accumulation and maintenance of small peptides in times of dietary sufficiency and the utilization of the small peptides as a source of amino acid in times of dietary scarcity (Collett, Insect Biochem.6, 179–185, 1976a; J. Insect Physiol.22, 1433–1440, 1976b) may be attributed to these features.     

Antineoplastic agents 582. Part 1: Isolation and structure of a cyclobutane-type sesquiterpene cancer cell growth inhibitor from Coprinus cinereus (Coprinaceae)

Bioassay-guided (murine P388 lymphocytic leukemia and human cancer cell lines) separation of an ethyl acetate extract prepared from the inky cap fungus Coprinus cinereus led to the isolation of three new sesquiterpenes, 7,7a-diepicoprinastatin 1 (1), 14-hydroxy-5-desoxy-2S,3S,9R-illudosin (2), and 4,5-dehydro-5-deoxyarmillol (3), together with the known armillol (4). The structure and relative configuration of 1 was determined by single-crystal X-ray diffraction experiments. The structures of compounds 2, 3, and 4 were each deduced by a combination of HRMS and 1D and 2D NMR techniques. Cyclobutane 2 led to modest inhibition of the murine P388 leukemia cell line.