Aplysianin-A,an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare,Aplysia kurodai

Summary Aplysianin-A, an antibacterial and antineoplastic factor in the albumen gland of the sea hareAplysia kurodai, was isolated. It had a molecular weight of approximately 320 kD and consisted of subunits with a molecular weight of 85 kD. It contained 9.8% neutral sugar. Aplysianin A showed 50% inhibition ofBacillus subtilis growth at a concentration of 4 g protein/ml and 50% lysis of murine MM46 tumor cells at 14 ng protein/ml. A partial identity of antigenic specificity of the purified specimen with an antineoplastic factor fromAplysia eggs was observed in immunodiffusion tests.Acknowledgment. We are indebted to the staff of Fisheries Research Laboratory, Faculty of Agriculture, University of Tokyo, Maisaka, for the collection of sea hares.     

Isolation and characterization of a new hemagglutinin from the red alga Gracilaria bursa-pastoris

A new agglutinin has been isolated from the red alga Gracilaria bursa-pastoris by affinity chromatography on a yeast mannan-Cellulofine column. This agglutinin was isolated as a monomeric glycoprotein with a relatively low molecular weight. It had an isoelectric point of 4.7 and contained large amounts of Gly, Asx and Glx. It agglutinated trypsin-treated rabbit erythrocytes at the low concentration of 30 ng/ml. The activity was inhibited only by glycoproteins bearing N-glycans. This agglutinin also showed mitogenic activity for mouse splenic lymphocytes.     

Isolation and characterization of a new hemagglutinin from the red algaGracilaria bursa-pastoris

Summary A new agglutinin has been isolated from the red algaGracilaria bursa-pastoris by affinity chromatography on a yeast mannan-Cellulofine column. This agglutinin was isolated as a monomeric glycoprotein with a relatively low molecular weight. It had an isoelectric point of 4.7 and contained large amounts of Gly, Asx and Glx. It agglutinated trypsin-treated rabbit erythrocytes at the low concentration of 30 ng/ml. The activity was inhibited only by glycoproteins bearing N-glycans. This agglutinin also showed mitogenic activity for mouse splenic lymphocytes.     

Physicochemical properties of an extracellular polysaccharide isolated from culture media of Bacillus amyloliquefaciens]

A new extracellular polysaccharide has been isolated by chromatography on anion exchanger of a fraction obtained from highly viscous culture media of Bacillus amyloliquefaciens. This polysaccharide is characterised by high molecular weight (1,000,000 dalton) and intrinsic viscosity (323 ml/g). It contains 24% neutral sugar (galactose and mannose 5:1), 35% glucuronic acid and 51.5% N-acetylhexosamines (N-actylglucosamine, N-acetylgalactosamine and N-acetylbacillosamine 6:9:1).     

Endogenous pyrogen formation by human blood monocytes stimulated by polyriboinosinic acid:Polyribocytidylic acid

The pyrogenic response to supernatants from human blood monocytes stimulated with polyriboinosinic acid:polyribocytidylic acid (poly I:C) was characteristic of a response to endogenous pyrogen in that it was brief and monophasic, and was destroyed by heating the supernatants at 70°C for 30 min. Pyrogen production was unimpaired when the incubations were carried out in the presence of cycloheximide (50 g/ml; an inhibitor of protein synthesis) or indomethacin (50 g/ml; an inhibitor of prostaglandin synthesis). Also, neither interferon, interleukins, tumor necrosis factor nor prostaglandin E₂ were detectable in the supernatants from the poly I:C-stimulated human monocytes.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells

Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

A factor potentiating serotonin in the induction of germinal vesicle breakdown in surf clam oocytes

Summary Simultaneous addition of an aliquot of body fluid obtained from the surf clam,Spisula solidissina, enhanced oocyte germinal vesicle breakdown induced with serotonin but not with KCl. When the body fluid and serotonin were added sequentially to the oocytes, potentiation did not occur. Body fluids of both males and females were effective at a 200-fold dilution. The factor is stable when treated with heat, acid, base, trypsin and pronase. It is hydrophobic and not dialyzable through tubing with a molecular weight cutoff of 1000 daltons. The factor is probably not a protein.17 November 1986Acknowledgments. This work was supported by the U.S.-Japan Cooperative Science Program, NSF INT-8211350, and Rockefeller Foundation Grant GAPS 8506. The authors thank Drs H. Shirai, T. Kishimoto, K. Sano, E. Sato and H. Ueno for valuable discussion.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages of Plasmodium falciparum and some tumor cell lines

In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages of P. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 micrograms/ml respectively which have no effect on the growth of P. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain of Candida remained below 2 micrograms/ml. These experiments suggest that 5-FC (at 50 micrograms/ml) alone or in combination with KTZ (at 1 microgram/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

Pinitol, a larval growth inhibitor for Heliothis zea in soybeans

A search for insect growth inhibitors in methanol extracts of soybean leaves resulted in isolation of pinitol. Pinitol caused a 50% reduction in weight gain (ED50) of Heliothis zea larvae at about 0.7% when added to a synthetic diet. Although myo-inositol is a normal component of the insect diet, it also caused growth inhibition at higher concentrations; ED50 4%.     

Specific phosphorylation of a calf serum protein by a kinase from the cell surface]

A protein of molecular weight approximately 200,000, which exists in trace amounts in calf serum, is specifically phosphorylated by an enzyme located at the surface of cultured cells. The emzymatic reaction utilizes ATP, is enhanced by Mg++ and Zn++ ions, and is not dependent on cyclic AMP. This kinase activity is associated with normal growing fibroblasts but disappears when they are contact-inhibited. It remains high, however, in transformed and malignant cells, whatever their growth state.     

Pinitol,a larval growth inhibitor forHeliothis zea in soybeans

Summary A search for insect growth inhibitors in methanol extracts of soybean leaves resulted in isolation of pinitol. Pinitol caused a 50% reduction in weight gain (ED₅₀) ofHeliothis zea larvae at about 0.7% when added to a synthetic diet. Although myo-inositol is a normal component of the insect diet, it also caused growth inhibition at higher concentrations; ED₅₀4%.The authors are indebted to R.J. Molyneux for a reference sample of pinitol and to J. Baker for aid with the bioassay. Reference to a company and/or product named by the Department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.     

Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg.

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Isolation of the major toxic protein from the skin venom of the crested newt, Triturus cristatus

A new protein, of molecular weight 160,000, was isolated from the skin venom of the crested newt and partially characterized by bioassays and biochemical methods. This protein shows the same functional properties as the crude venom, which produces convulsions in mice and is cytotoxic.     

Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Metallothionein induced in the frogXenopus laevis

Summary A low molecular weight cadmium- and copper-binding protein, induced in the liver of the frog,Xenopus laevis, by the administration of cadmium, was shown to consist of a single isoprotein and was characterized as an amphibian metallothionein based on its high cysteine and metal content, its low molecular weight, and the lack of aromatic amino acids.The authors thank Dr K. Kubota for his encouragement.     

Aggrecan, aging and assembly in articular cartilage

The primary function of articular cartilage to act as a self-renewing, low frictional material that can distribute load efficiently at joints is critically dependent upon the composition and organisation of the extracellular matrix. Aggrecan is a major component of the extracellular matrix, forming high molecular weight aggregates necessary for the hydration of cartilage and to meet its weight-bearing mechanical demands. Aggregate assembly is a highly ordered process requiring the formation of a ternary complex between aggrecan, link protein and hyaluronan. There is extensive age-associated heterogeneity in the structure and molecular stoichiometry of these components in adult human articular cartilage, resulting in diverse populations of complexes with a range of stabilities that have implications for cartilage mechanobiology and integrity. Recent findings have demonstrated that aggrecan can form ligands with other matrix proteins. These findings provide new insights into mechanisms for aggregate assembly and functional protein networks in different cartilage compartments with maturation and aging.     

Nucleolar localization and circadian regulation of Per2S,a novel splicing variant of the Period 2 gene

In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.     

A study of macromolecular diffusion through native porcine mucus.

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueous and mucus layers. Over the molecular weight range of solutes investigated (126-186,000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (greater than 30,000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.     

The effect of actinomycin D on the production of viruses by the protoplasts of Chinese cabbage infected in vitro by the yellow mosaic virus of turnips]

Chinese Cabbage (Brassica sinensis L. var. Cantonner) protoplasts were infected by Turnip yellow mosaic virus (TYMV) and inoculated in the presence or absence of actinomycin D. Virus production was determined 40 hrs. after inoculation, the time required for the virus replication cycle to be terminated. While actinomycin D had no effect on TYMV production when present at a concentration of 1 microng/ml, a 50 to 80% inhibition of virus production was noticed at concentrations of the order of 5 to 10 microng/ml, and the inhibition reached 90% with 25 microng/ml.