Purification and characterization of calcium-binding conchiolin shell peptides from the mollusc,Haliotis rufescens,as a function of development

Summary Conchiolin peptides of the molluscan shell are believed to determine structural organization and facilitate calcification during shell formation. Changes in patterns of conchiolin synthesis during development, and the possible contribution of these peptides to shell formation, have been investigated by purification and characterization of the soluble peptides extracted from the shell of the gastropod mollusc,Haliotis rufescens (red abalone), at various stages of development. Shell peptides were purified from young post-larvae, juveniles and adults by gel-filtration column chromatography in aggregation-reducing bicarbonate buffers. Calcium-binding domains were detected spectrophotometrically after reaction with a cationic carbocyanine dye. Juvenile and adult shell peptides were found to be heterogeneous, and rich in aspartic acid and glycine residues; in contrast, post-larval shells were found to contain one major glycine-rich component. The juvenile shell peptide population shares components from each of the other two populations, suggesting that the synthesis of the different shell peptides results from the differential expression of a multi-gene family, in a developmentally controlled progression. Enzymatic analyses suggest that calcium binds to the aspartic acid residues of the peptide core, rather than to satellite groups such as phosphate, sulfate or carbohydrate. The possibility is discussed that the aspartic acid residues found in shell peptides may play an important role in the calcification of the abalone shell matrix. The methods demonstrated here also should prove useful for the purification, characterization, and comparative analysis of calcium-binding proteins of connective tissues, extracellular matrices and support structures in many other systems.Abbreviations Asp aspartic acid - BSA bovine serum albumin - Da daltons - EDTA ethylenediaminetetraacetic acid - GABA -aminobutyric acid - HPLC high-pressure liquid chromatography - ODS octadecylsilane - OPA o-phthaldialdehyde - SDS sodium dodecyl sulfate     

Expression profile of rrbp1 genes during embryonic development and in adult tissues of Xenopus laevis

Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19–39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.     

Expression analysis of nel during zebrafish embryonic development

Nel is a multimeric extracellular glycoprotein which predominantly expressed in the nervous system and play an important role in neural development and functions. There are three nel paralogues included nell2a, nell2b, and nell3 in zebrafish, while systematic expression analysis of the nel family is still lacking. In this study, we performed a phylogenetic analysis on 7 species, in different species the nell2a are highly conserved, as is nell2b. Then, the expression profiles of nell2a, nell2b and nell3 were detected by in situ hybridization in zebrafish embryo, and the result showed that nel genes highly enriched in the central nervous system, but distributed in different regions of the brain. In addition, nell2a is also expressed in the olfactory pit, spinal cord, otic vesicle and retina (ganglion cell layer), nell2b was detected to express in gill arches, olfactory epithelium, olfactory pit, spinal cord, photoreceptor and retina (ganglion cell layer), it should be noted that the expression of nell3 is special, was only detected at 96 hpf in the brain and spinal cord of zebrafish. Overall, our results indicate that nell2a and nell2b genes are expressed in the nervous system and eyes of zebrafish embryo, while nell3 is expressed in different regions in the nervous system. The phylogenetic analysis also shows that nell3 sequences are significantly different from nell2a and nell2b. This study provides new evidence to better understand the role of nel in zebrafish embryo development.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Mitochondrial thioredoxin-2 from disk abalone (Haliotis discus discus): molecular characterization, tissue expression and DNA protection activity of its recombinant protein

Thioredoxin-2 is a mitochondria-specific member of the thioredoxin (TRx) super-family that plays an important role as a component of the mitochondrial antioxidant system. The gene coding mitochondrial TRx-2 was isolated from the disk abalone (Haliotis discus discus) cDNA library, denoted as AbTRx-2. It contains 1214-bp full length with 519-bp open reading frame, encoding 173 amino acids. AbTRx-2 showed characteristic TRx active site at ⁹⁶WCGPC¹⁰⁰ and mitochondrial targeting peptide at the N-terminal amino acid sequence. The deduced amino acid comparison showed that AbTRx-2 shares 43 and 42% identity with Xenopus laevis and human TRx-2, respectively. Purified recombinant AbTRx-2 fusion protein was shown to catalyze insulin reduction and protect supercoiled plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. Constitutive AbTRx-2 mRNA was detected in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes, in a tissue specific manner. The AbTRx-2 mRNA was up-regulated in gill and digestive tract tissues initially at 3 h post-injection of H₂O₂ and maintained higher level at 6 h. Our results suggest that abalone TRx-2 may play an important role in regulating oxidative stress in mitochondria by catalyzing protein disulfide reduction, scavenging of ROS, and minimizing the DNA damage.     

Purification and characterization of mycoferritin from Aspergillus parasiticus (255)

As intracellular iron storage molecules, only hydroxymate type siderophores have been reported in ascomycetes and basidiomycetes. This is the first report documenting the presence of mycoferritin in ascomycetes. The fungus, Aspergillus parasiticus (255), is capable of producing mycoferritin only upon induction with iron in yeast extract sucrose (YES) medium. The same has been purified from Aspergillus sps by application of conventional biochemical techniques. The molecular mass, yield, iron and carbohydrate contents of the HPLC purified protein were 460kDa, 0.012mg/g of wet mycelia, 1.6% and 6.0%, respectively. The iron content was much lower than Mortierella alpina mycoferritin (17%). Native PAGE revealed the presence of trimeric and monomeric forms of ferritin. Subunit analysis by SDS-PAGE showed a single protein subunit of approximately 20kDa suggesting structural simplicity of the apoferritin shell. Variation in amino acid composition was noted upon comparison with ferritins of other species. Interestingly, no phenylalanine could be detected in the mycoferritin of Aspergillus sps. The acidic amino acid content was 1.5-1.6 fold higher than mammalian and fish ferritins. The spectral characteristics (UV/VIS and fluorescence) of mycoferritin were akin to equine spleen ferritin. However, circular dichroic spectra revealed a lower degree of helicity.     

Expression analysis of asthma candidate genes during human and murine lung development

Background

Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.

Objective

To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.

Methods

Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.

Results

In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.

Conclusions

Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.     

Health and survival of red abalone, Haliotis rufescens, under varying temperature, food supply, and exposure to the agent of withering syndrome

Withering syndrome (WS) is a disease of wild and cultured abalone caused by a Rickettsiales-like prokaryote (WS-RLP). This study compared the pathologic changes that occur during the progression of WS in red abalone to those caused by environmental stresses consisting of elevated temperature and food limitation and determined the impact of these stressors on WS prevalence and intensity. Farmed red abalone were administered a feed-based oxytetracycline therapeutic treatment to assure WS-RLP-free status prior to initiation of the experiment. Groups were then held in each of eight combinations of exposed vs. unexposed to WS-RLP, elevated vs. ambient temperature, and high vs. low food supply, for 447 days. Mortality was associated with starvation and disease but not elevated temperature alone. Elevated temperature significantly affected WS-RLP transmission: only 1.7% of WS-RLP- exposed abalone held at ambient temperature (12.3 degrees C) became infected compared to at least 72% of those held at elevated temperature (18.7 degrees C). Among exposed abalone at elevated temperature, fed animals exhibited greater infection prevalence but not greater infection intensity or digestive gland changes than starved animals, suggesting that abalone acquire infections by ingesting contaminated food. Food, temperature, WS-RLP exposure, and most of their interactions had significant effects on body condition and foot atrophy. Immunohistochemical detection of cell proliferation and apoptosis revealed no differences between normal digestive gland and that infected with WS-RLP. Body mass shrinkage, foot atrophy, elevated mortality, and decreased foot and digestive gland glycogen were observed in both WS-affected and starved, unexposed abalone, with the WS-RLP-exposed, starved group held at elevated temperature faring worst. Among exposed and unexposed animals, food supply but not temperature affected body mass and growth. These data demonstrate that the high morbidity and mortality exhibited by WS-RLP-infected abalone is a consequence of disease and not direct thermal stress. Drug residue analysis indicated oxytetracycline concentrations of up to 600 ppm in the digestive gland at 38 days post-treatment, an unusual degree of tissue retention of this therapeutant.     

Myogenesis in primary cell cultures from Drosophila melanogaster: protein synthesis and actin heterogeneity during development.

Muscle cell cultures from Drosophila melanogaster were obtained by plating dissociated gastrula stage embryo cells on protamine-treated culture dishes. They myogenic cells in these cultures fuse to form multinucleated pulsating cells by 15 hr after plating. An analysis of protein synthesis during myogenesis in these cultures, as measured by the incorporation of 35S-methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis, showed profound changes in the pattern of protein synthesis. This analysis enabled us to identify three distinct classes of proteins. Class A proteins, the most abundant, are synthesized continuously throughout myogenesis, class B proteins are those proteins whose synthesis is initiated during myogenesis and continued throughout development; class C proteins are those synthesized at specific times during development. In addition, three forms of actin have been identified in these cultures. Actin I, which shows increased synthesis concomitant with the myogenic development in these cultures, is apparently a muscle-specific form of actin. Actin II, the predominant "cytoplasmic" form of actin in the nonmuscle Schneider cell line 2, is also the major form in the gastrula cultures before differentiation begins. Synthesis of this actin continues in the myogenic cultures. Actin III is a rapidly turning over form of actin which does not accumulate in either the Schneider cells or the myogenic cultures.     

A miRacle in plant development: role of microRNAs in cell differentiation and patterning

MicroRNAs (miRNAs) are endogenous small regulatory RNAs, which control gene expression in eukaryotes. In plants they repress mRNA targets containing a highly complementary site, either by cleavage or translational repression. Studies of individual miRNA/target interactions highlight the involvement of the miRNA-based regulations in a broad range of developmental programs, throughout plant lifecycle. MicroRNAs can have distinct regulatory functions on their targets: some determine their spatial accumulation, some have a buffering role that ensures the robustness of their expression pattern, and finally others establish the temporal expression of targeted genes.     

Ganglioside changes in the chicken optic lobes and cerebrum during embryonic development

Summary The developmental profiles of 15 different gangliosides of the optic lobes and cerebrum of the chicken were followed from the 6 th day of incubation to hatching and correlated to morphological development. Five of these gangliosides appearing in both structures between the sixth and tenth day, have not been reported previously in higher vertebrates. Three chromatographed on TLC-plates similarly to G_T3, G_T2, and G_T1c gangliosides, which have been demonstrated in fish brain. One fraction moved just below G_Q1b and is suggested, to contain G_Q1c. These novel gangliosides, which are possibly related to a recently proposed separate and probably phylogenetically older biosynthetic pathway, contained up to 20% of total ganglioside sialic acid. The fifth novel fraction, containing up to 16% of total ganglioside-sialic acid, moved below the penta-sialoganglioside G_P1 and is suggested to contain hexa-sialogangliosides.There were two main changes in ganglioside synthesis, which were identical in both structures.The first occurred from the sixth to the eleventh day, parallel to decreased proliferation, maximal cell migration and neuroblast differentiation, G_D3 and G_D2 decreased rapidly in favour of G_Q1b, G_P1, and to the novel fractions, described above.The second occurred from the eleventh to the eighteenth day, parallel to increased growth and arborization of dendrites and axons as well as functional establishment of synaptic contacts, there was a sharp rise in the amount of G_D1b, G_T1b, and G_D1a. Concomitantly the novel gangliosides decreased. At hatching G_D1a was the predominant ganglioside. G_M3, G_M2, and G_M1 were always minor fractions, each accounting for less than 4% of total ganglioside-sialic acid. G_M4 was never detected, indicating neglegible myelinisation until hatching.Abbreviations NeuAc N-Acetylneuraminic acid - G_M4 I³NeuAc-GalCer - G_M3 II³NeuAc-LacCer - G_M2 II³NeuAc-G_gOse₃Cer - G_M1 II³NeuAc-G_gOse₄Cer - G_D3 II³(NeuAc)₂LacCer - G_D1a IV³NeuAc, II³NeuAc G_gOse₄Cer - G_T3 II³ (NeuAc)₃LacCer - G_D2 II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₃Cer - G_D1b II³(NeuAc)₂G_gOse₄Cer - G_T2 II³(NeuAc)₃G_gOse₃Cer - G_T1b IV³NeuAc, II³(NeuAc)₂G_gOse₄Cer - G_T1c II³(NeuAc)₃G_gOse₄Cer - G_Q1b IV³(NeuAc)₂ II³(NeuAc)₂G_gOse₄Cer - G_Q1c IV³NeuAc, II³(NeuAc)₃G_gOse₄Cer - G_P1 IV³(NeuAc)₂, II³(NeuAc)₃G_gOse₄Cer - G_H(?) IV₃(NeuAc)₃, II³(NeuAc)₃G_gOse₄Cer     

Macromolecular structure of the organic framework of nacre in Haliotis rufescens: implications for growth and mechanical behavior

We have performed a macromolecular structural analysis of the interlamellar and intertabular parts of the organic framework of the nacreous part of the shell of Haliotis rufescens, including the identification of structural chitin. Using histochemical optical microscopy we have mapped the locations of carboxylates and sulfates of proteins and chitin on the surfaces and within the core of the interlamellar layers and the intertabular matrix that together form the external organic matrix of composite nacre. This extends the earlier work of Nudelmann et al. [Nudelman, F., Gotliv, B.A., Addadi, L. and Weiner, S. 2006. Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonite tablet in nacre. J. Struct. Biol. 153, 176–187] and Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H. 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Omori, M., Watabe, N. (Eds.) The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Toyko] on Nautilus pompilius. Our mapping identifies distinct regions, defined by the macromolecular groups, including what is proposed to be the sites of CaCO₃ nucleation and that play a key role in nacre growth. Using AFM scanning probe microscopy we have identified a fibrous core within the framework that we associate with chitin. The structural picture that is evolved is then used to develop a simple structural model for the organic framework which is shown to be consistent with mechanical property measurements. The role of the intracrystalline matrix within the nacre tablets in mediating nacre’s mechanical response is noted within the framework of our model.