Actin Degradation in the Metamorphosing Bullfrog Tadpole Tail

Degradation of tail muscle proteins was investigated during metamorphosis of Rana catesbeiana , tadpole. Regressing tail muscle contained actomyosin which was comparable to that of non-regressing tail muscle in its physico-chemical character, althouth the actomyosin content of the former tissue decreased as compared to the latter. However, when muscle proteins were extracted in the SDS-containing medium (TSM) and analyzed by SDS-polyacrylamide gel electrophoresis, we found that the protein band corresponding to actin disappeared completely during the late climax stage of metamorphosis. Detailed studies on this phenomenon showed that the apparent absence of actin on SDS-polyacrylamide gel electrophoresis was dependent upon the metamorphic stages of the tadpoles investigated. When TSM extract from the premetamorphic tadpole tail muscles which contained actin was incubated with the same extract from tadpoles of the climax stage, actin derived from premetamorphic tadpole disappeared on gel electrophoresis, indicating that tail muscle tissues of the climax stages contain the actin-degrading enzyme. Characterization of the enzyme was performed with a crude extract using actin prepared from rabbit thigh muscle as a substrate. Actin degrading activity showed incubation time- and temperature-dependency and the activity decreased gradually when the extract was preheated at increasing temperatures with the complete inactivation at 100°C. The major degradation products of actin hydrolysis by the enzyme had a Mr=28,000 and 14,000 which indicated the enzyme splits actin at a specific point. The activity had an optimum pH of 7.5 and was inhibited by leupeptin and iodoacetate and required the presence of a thiol reagent.     

Isolation and Characterization of Mesenchymal Cells from the Tail of Bullfrog Tadpoles*

Mesenchymal cells were separated with the aid of EDTA, dispase, collagenase and hyaluronidase from tail fins of Rana catesbeiana tadpoles and were cultured to examine their morphology and response to thyroid hormone, which controls metamorphosis. The mensenchymal tissue consisted of two main types of cells, macrophage-like cells (M cells) and fibroblastic cells (F cells), distinguishable by their morphological and biochemical characteristics. M cells were preferentially located near the boundary between the mesenchyme and the epidermis. They were more adhesive to the plastic culture dishes than F cells. The M cells developed a bizarre rod-like branching process during one week of culture, which appeared to be identical to the process formed by mammalian macrophages undergoing clasmatosis. M cells cultured for more than one week tended to fuse together forming multinuclear giant cells. F cells were found uniformly in the tissue and were active in collagen biosynthesis. Thyroid hormone at a physiological concentration greatly reduced the life span of F cells, but did not reduce that of M cells.     

The Presence of Intramitochondrial Yolk-crystals in Oocytes of Hypophysectomized Bullfrog Tadpoles*

Ovaries of hypophysectomized Rana catesbeiana tadpoles. weighing I to 14 g, were prepared for electron microscopic study. The oocytes are at the growth phase, ranging from 50 to 190 μm in diameter. The observation on these oocytes has revealed the presence of intramitochondrial yolk-crystals but not cytoplasmic yolk platelets. The crystalline structure, situated within the intracristal space, consists of a hexagonal array of dense particles about 50 Å in diameter and 72 Å in periodicity. Our data agree with those reported in oocytes of intact ranid species. According to literatures, crystals of intramitochondrial yolk and of cytoplasmic yolk platelets show similar ultrasturctures. The precursor of cytoplasmic yolk platelets in adult Xenopus oocytes is known to be synthesized in the estrogen-stimulated liver and incorporated via circulation into oocytes by gonadotropin-dependent micropinocytosis. The present finding suggests that the intramitochondrial yolk could be formed within oocytes, independently of the pituitary control.     

Immunological Characterization of Nitrate Reductase in Different Tissues of Spinach Seedlings

In spinach seedlings and roots, NADH-nitrate reductase (NR)activity (per g fresh weight) decreased as the seedlings aged.Experiments using double immunodiffusion analysis and immunotitrationshowed no differences in the immunological properties of NRfrom spinach seedlings at various stages of aging. Comparisonof spinach leaf to the spinach root enzyme using the Ouchterlonydouble diffusion technique revealed a high degree of similaritybetween them. (Received November 6, 1985; Accepted March 4, 1986)     

Biochemical Characterization of Myelin Isolated from the Central Nervous System of Xenopus Tadpoles

Abstract: Myelin isolated from the central nervous system of Xenopus tadpoles was characterized biochemically and compared with Xenopus frog and mammalian myelins. Xenopus tadpole myelin contains the characteristic protein and lipid components of mammalian myelin, although quantitative differences exist. The biochemical composition of Xenopus tadpole myelin suggests that it is an immature form of XePnopus frog myelin. Basic protein and proteolipid protein are prominent components of Xenopus myelin, but isolated tadpole myelin contains a greater proportion of higher molecular weight proteins than Xenopus frog or mature mammalian myelin. The basic protein has a higher apparent molecular weight than mammalian myelin basic protein. The levels of 2',3'-cyclic nucleotide 3'-phosphodiesterase are significantly higher in whole tadpole brain homogenate and purified myelin than in similar mammalian preparations. Tadpole myelin lipids contain a higher proportion of phospholipids and less galactolipid than mammalian myelin. Tadpole myelin galactolipids include a high (16%) percentage of monogalactosyl diglyceride, a component found in only trace quantities (0.9%) in bovine myelin.     

Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

Background

Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.

Principal Finding

In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10⁶ the tissue culture''s infectious dose (TCID₅₀) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10⁻⁵ was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.

Conclusion

These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.     

Selenium:Mercury Molar Ratios in Bullfrog and Leopard Frog Tadpoles from the Northeastern United States

Vertebrates experience adverse effects from methylmercury, largely obtained through their food. Selenium has the potential to reduce the toxic effects of methylmercury (and vice versa). In this paper, we examine the selenium:mercury molar ratios in tadpoles (Lithobates sphenocephalus, Lithobates catesbeianus (formerly Rana), and a newly documented leopard frog species currently referred to as R. sp. nov.) and fully formed leopard frog metamorphs. There were no significant differences in metal levels between the two leopard frog species, and data were therefore combined. Selenium:mercury molar ratios varied from 19 to 38 for bullfrog tadpoles, from 16 to 330 for leopard frog tadpoles, and from 7 to 17 for leopard frog metamorphs. Leopard frog tadpoles with less than 45 days exposure to field conditions had significantly higher molar ratios than other tadpoles and leopard frog metamorphs. There were significant locational differences for the molar ratios of bullfrogs, and leopard frog tadpoles with more than 45 days of field exposure. At the sites where we were able to sample both leopard frog tadpoles and leopard frog metamorphs, there were significant differences between the two distinct life stages. Most of the variation in the ratio was accounted for by selenium levels, field sites, and exposure period.     

Biochemical and Histochemical Studies on Catecholamines in Bullfrog Spinal Ganglia

Abstract: Bullfrog spinal ganglia were subjected to two types of examination to determine whether catecholamines were present. A biochemical microassay developed by Kissinger and co-workers and a histofluorescence technique for cellular demonstration of biogenic monoamines developed earlier by Falck and co-workers were used. Bullfrog spinal ganglia and dorsal roots were found to contain catecholamines, primarily adrenaline.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1.26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively.These results provide a substantial basis for the further studies of the acid invertases in apple fruit.     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.     

Use of Bullfrog Tadpoles (Rana catesbeiana) to Examine the Mechanisms of Lead Neurotoxicity

Sublethal exposure to lead elicits changes in behavior, particularlylearning. Previously, we had shown that bullfrog tadpoles exposedto 1 mg Pb/liter for 7 days exhibit learning deficits in a discriminateavoidance learning assay. The precise mechanisms involved inthese lead-induced learning deficits are not understood, butCNS monoamine neurotransmitter systems have been implicatedin the learning process. In the present study, we exposed bullfrogtadpoles to 1.7± 0.2 mg Pb/liter for 7 days and thencompared concentrations of neurotransmitters from whole brainsamples with those of controls. Serotonin or 5-hydroxytryptamine(5-HT) was significantly decreased in the lead exposed groupwhile the serotonin metabolite, 5-hydroxyindoleacetic acid,(5-HIAA) was similar to controls. No changes were observed inthe catecholamines epinephrine and norepinephijine followinglead exposure. The ratio of 5-HIAA/5-HT was significantly increasedin lead exposed animals as a result of the decrease in 5-HT,suggesting a decrease in 5-HT biosynthesis rather than an increasein 5-HT metabolism. These findings are the first to suggestthat lead exposure in bullfrog tadpoles affects the monoamineneurotransmitters that are implicated in the learning process.The results of the present study, in conjunction with previousevidence of learning deficits following lead exposure, offerthe possibility of correlating lead exposure with learning deficitsand alterations in CNS neurotransmitters in bullfrog tadpoles.The use of this tadpole model shows promise as a means to examineand understand the mechanisms involved in lead neurotoxicity.     

Nitrate Reductase from the Marine Diatom Skeletonema costatum (Biochemical and Immunological Characterization)

Assimilatory nitrate reductase (NR) was purified from the marine diatom Skeletonema costatum (clone Skel) using Cibacron blue-Sepharose affinity chromatography. The single-step purification scheme yielded a 103-fold purification of specific activity with an overall recovery of 40.8%. Only NADH-dependent NR activity (form EC 1.6.6.1) was observed in this species. Kinetic analysis revealed that this form had apparent Michaelis constants of 3.6 [mu]M for NADH and 295 [mu]M for NO3- when purified from cells grown in NO3--enriched seawater. The S. costatum NR exhibits a pH optimum of 7.4, a temperature optimum of 14[deg]C, and enzyme activity not sensitive to Mg2+ inhibition. The strong temperature dependence of NR activity in S. costatum may contribute to the seasonal and latitudinal distributions and abundances of this bloom-forming species. Chromatographically isolated NR was further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single polypeptide with an apparent molecular mass of 110 kD. The 110-kD polypeptide was used to generate polyclonal antibodies. The antiserum recognized a single 110-kD polypeptide in western blots of total proteins from S. costatum, as well as the native enzyme. Western blot analysis also revealed an antigenic similarity of NR from two additional diatom species, whereas no cross-reactivity was observed with NR from other phytoplankton taxa, including prymnesiophytes, dinoflagellate, cyanobacterium, and green alga. This result suggests a structural diversity of NR in phytoplankton and identifies the potential for development of taxon-specific NR antisera for ecological studies.     

Biochemical and Immunological Studies on Aspergillus

Two kinds of polysaccharides, glucan and glycopeptide (galactomannan-peptide), were obtained from Aspergillus fumigatus (IFO 5840) by extraction with 50% pyridine and were purified by fractional precipitation with acetone, by a column chromatography on Dowex-50 and DEAE-cellulose and by the gel filtration on Sephadex G-50 and G-200. The glycopeptide, designated APSK-66 fraction, showed both an Arthus and delayed type (tuberculin type) skin reactions in sensitized rabbits and guinea pigs. By treatment with proteolytic enzymes, the delayed type skin reactivity of APSK-66 fraction was reduced but the Arthus type skin reactivity was not affected. However, the Arthus type skin reactivity of APSK-66 fraction was completely lost by periodate oxidation, and the delayed type skin reactivity of APSK-66 fraction was retained. The APSK-66 fraction showed precipitation, complement fixation and passive hemagglutination reactions with rabbit antisera against A. fumigatus. Glucan, designated as APSK-33 fraction, did not show any immunological activity when tested in the present experiment. The chemical structures of the glucan and galactomannan were discussed.     

Biochemical and Immunological Characterization of Truncated Fragments of the Receptor-Binding Domains of C. difficile Toxin A

Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases.     

Ultrastructural and Biochemical Changes in Cotyledon Reserve Tissues3

Changes in major protein, lipid and carbohydrate reserves duringthe germination of Citrus limon (L.) Burm. f. seeds have beenstudied. The rate of release of amino acids and soluble sugarshas been evaluated. Mobilization of protein reserves began 4 d after the onset ofimbibition. The main period of hydrolysis occurred between 8d and 24 d after the start of germination. Ultrastructural studies showed the presence of protein bodiesin quiescent cotyledon cells. These bodies virtually disappeared14 d after the start of germination. The nitrogenous compoundsthat were liberated and subsequently translocated were predominantlyin the form of asparagine, arginine, and proline. The cotyledonshad a lipid content representing 51.7% of their dry weight.Lipid reserves in quiescent cotyledons were laid down in theform of oil-bodies. These organelles rapidly disappeared asgermination progressed, and were replaced by vacuoles. The starch content of quiescent cotyledons is very low, butit increased considerably up to 20 d after germination started. Key words: Proteins, lipids, carbohydrates