Tubulin in sea urchin embryonic cilia: characterization of the membrane-periaxonemal matrix

1991 ◽  
Vol 100 (3) ◽  
pp. 521-531 ◽  
Author(s):  
R.E. Stephens
Keyword(s):  
Membrane Protein ◽  
Sea Urchin ◽  
Posttranslational Modifications ◽  
Calcium Binding ◽  
Specific Activity ◽  
Cross Reactivity ◽  
Alpha Tubulin ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Matrix Fraction

When the membranes of isolated embryonic cilia from three species of sea urchin are detergent-solubilized, the major proteins found are two equi-molar polypeptides comigrating with tubulin subunits. Cross-reactivity with a variety of tubulin antibodies confirms their identity. Calmodulin and other calcium-binding proteins are prominent minor constituents of the extract. Removal of the solubilizing detergent by adsorption to polystyrene beads, followed by a freeze-thaw cycle, produces membrane leaflets and vesicles of uniform bouyant density. Such reconstituted membranes incorporate most of the tubulin and minor proteins but not calmodulin. Equivalent cross-reactivity with antibodies to acetylated or detyrosinated alpha-tubulin indicates that the tubulin derived from the membrane-periaxonemal matrix and axoneme are indistinguishable from each other in terms of these posttranslational modifications but are distinct from the mainly unmodified tubulin of the embryonic cytoplasm. Pulse labeling with [3H]palmitate does not label either tubulin subunit but acylation does occur on a 190 × 10(3) Mr membrane protein. Its specific activity is essentially the same whether label is applied to embryos with existing or regenerating cilia, suggesting rapid ciliary membrane protein exchange or physical turnover. Using pulse-chase labeling with [3H]leucine during steady-state ciliary growth or induced regeneration in both normal and zinc-animalized embryos, the specific activity of the membrane-periaxonemal matrix-derived tubulin is initially higher than that of the axoneme but the degree of labeling equalizes in successive regenerations, consistent with derivation from a common pool. Many heavily labeled axonemal architectural proteins, such as tektin-A, are not reflected in the membrane-periaxonemal matrix fraction, suggesting that this fraction is not simply a pool of unassembled axonemal precursors.

Tubulin in sea urchin embryonic cilia: post-translational modifications during regeneration

1992 ◽  
Vol 101 (4) ◽  
pp. 837-845 ◽  
Author(s):  
R.E. Stephens
Keyword(s):  
Monoclonal Antibody ◽  
Sea Urchin ◽  
Growth Phase ◽  
Linear Growth ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Alpha Tubulin ◽  
Ciliary Membrane ◽  
Membrane Matrix ◽  
Matrix Fraction

Tubulin is the major protein found in the membrane/periaxonemal matrix fraction of mature sea urchin embryonic cilia but its distribution and possible function during ciliary assembly are unknown. Hypertonic salt may be used to deciliate the embryos, allowing synchronous regrowth of cilia and subsequent deciliation of the regenerating embryos at various times. During the earliest stages of regeneration, the amounts of tubulin in the axoneme and membrane/matrix fractions are nearly equal, but the proportion of tubulin in the axoneme fraction increases coincident with the quasi-linear growth phase while the membrane/matrix tubulin remains constant. Antibodies to tyrosinated and detyrosinated alpha-tubulin show that both the membrane/matrix and axonemal tubulin fractions are primarily unmodified (i.e. tyrosinated) at the earliest stages of regeneration but are progressively and equally detyrosinated coincident with regeneration, approaching a final level of 50% C-terminal Glu. A monoclonal antibody to acetylated alpha-tubulin reveals that both tubulin fractions are equally and maximally acetylated at relatively early stages of regeneration. In contrast, three-times-repolymerized tubulin from either unfertilized eggs or midgastrula embryos is primarily tyrosinated (greater than 97%) and not detectably acetylated. These data suggest that membrane/matrix tubulin is a precursor to axonemal tubulin and that acetylation and detyrosination may be involved in partitioning tubulin among cytoplasmic, ciliary membrane/matrix, and 9 + 2 compartments.

scholarly journals Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Katharina N. Schwaiger ◽  
Monika Cserjan-Puschmann ◽  
Gerald Striedner ◽  
Bernd Nidetzky
Keyword(s):  
Specific Activity ◽  
Dry Mass ◽  
High Yield ◽  
Sucrose Phosphorylase ◽  
Bifidobacterium Adolescentis ◽  
Enzymatic Production ◽  
Intracellular Enzyme ◽  
Whole Cell ◽  
Freeze Thaw ◽  
Thaw Cycle

Abstract Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

scholarly journals Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species

2020 ◽  
Vol 22 (1) ◽  
pp. 32
Author(s):  
Shaymaviswanathan Karnaneedi ◽  
Roger Huerlimann ◽  
Elecia B. Johnston ◽  
Roni Nugraha ◽  
Thimo Ruethers ◽  
...  
Keyword(s):  
De Novo ◽  
Penaeus Monodon ◽  
Clinical Importance ◽  
Major Allergen ◽  
Cross Reactivity ◽  
Multiple Sequence ◽  
Alpha Tubulin ◽  
Shrimp Species ◽  
Allergen Sources ◽  
Shellfish Allergy

Shellfish allergy affects 2% of the world’s population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.

scholarly journals Sustainable Development of Chitosan/Calotropis procera-Based Hydrogels to Stimulate Formation of Granulation Tissue and Angiogenesis in Wound Healing Applications

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3284
Author(s):  
Muhammad Zahid ◽  
Maria Lodhi ◽  
Zulfiqar Ahmad Rehan ◽  
Hamna Tayyab ◽  
Talha Javed ◽  
...  
Keyword(s):  
Wound Healing ◽  
Blood Vessels ◽  
Granulation Tissue ◽  
Chorioallantoic Membrane ◽  
Calotropis Procera ◽  
Connective Tissues ◽  
Blood Capillaries ◽  
Chick Chorioallantoic Membrane ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle

The formation of new scaffolds to enhance healing magnitude is necessarily required in biomedical applications. Granulation tissue formation is a crucial stage of wound healing in which granulation tissue grows on the surface of a wound by the formation of connective tissue and blood vessels. In the present study, porous hydrogels were synthesized using chitosan incorporating latex of the Calotropis procera plant by using a freeze–thaw cycle to stimulate the formation of granulation tissue and angiogenesis in wound healing applications. Structural analysis through Fourier transform infrared (FTIR) spectroscopy confirmed the interaction between chitosan and Calotropis procera. Latex extract containing hydrogel showed slightly higher absorption than the control during water absorption analysis. Thermogravimetric analysis showed high thermal stability of the 60:40 combination of chitosan (CS) and Calotropis procera as compared to all other treatments and controls. A fabricated scaffold application on a chick chorioallantoic membrane (CAM) showed that all hydrogels containing latex extract resulted in a significant formation of blood vessels and regeneration of cells. Overall, the formation of connective tissues and blood capillaries and healing magnitude decreased in ascending order of concentration of extract.

scholarly journals Effect of freeze–thaw cycle on physical and mechanical properties and damage characteristics of sandstone

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Longxiao Chen ◽  
Kesheng Li ◽  
Guilei Song ◽  
Deng Zhang ◽  
Chuanxiao Liu
Keyword(s):  
Mechanical Properties ◽  
Shear Failure ◽  
Triaxial Compression ◽  
Physical And Mechanical Properties ◽  
Freeze Thaw ◽  
Damage Characteristics ◽  
Large Pore ◽  
Natural Rock ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle

AbstractRock deterioration under freeze–thaw cycles is a concern for in-service tunnel in cold regions. Previous studies focused on the change of rock mechanical properties under unidirectional stress, but the natural rock mass is under three dimensional stresses. This paper investigates influences of the number of freeze–thaw cycle on sandstone under low confining pressure. Twelve sandstone samples were tested subjected to triaxial compression. Additionally, the damage characteristics of sandstone internal microstructure were obtained by using acoustic emission (AE) and mercury intrusion porosimetry. Results indicated that the mechanical properties of sandstone were significantly reduced by freeze–thaw effect. Sandstone’ peak strength and elastic modulus were 7.28–37.96% and 6.38–40.87% less than for the control, respectively. The proportion of super-large pore and large pore in sandstone increased by 19.53–81.19%. We attributed the reduced sandstone’ mechanical properties to the degenerated sandstone microstructure, which, in turn, was associated with increased sandstone macropores. The macroscopic failure pattern of sandstone changed from splitting failure to shear failure with an increasing of freeze–thaw cycles. Moreover, the activity of AE signal increased at each stage, and the cumulative ringing count also showed upward trend with the increase of freeze–thaw number.

Non-linear creep damage model of sandstone under freeze-thaw cycle

2021 ◽  
Vol 28 (3) ◽  
pp. 954-967
Author(s):  
Jie-lin Li ◽  
Long-yin Zhu ◽  
Ke-ping Zhou ◽  
Hui Chen ◽  
Le Gao ◽  
...  
Keyword(s):  
Damage Model ◽  
Creep Damage ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Linear Creep ◽  
Non Linear ◽  
Freeze Thaw Cycle ◽  
Creep Damage Model

scholarly journals Strength and Microscopic Damage Mechanism of Yellow Sandstone with Holes under Freezing and Thawing

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Huren Rong ◽  
Jingyu Gu ◽  
Miren Rong ◽  
Hong Liu ◽  
Jiayao Zhang ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Pore Size ◽  
Power Function ◽  
Damage Mechanism ◽  
Uniaxial Compression Test ◽  
Freezing And Thawing ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Microscopic Damage

In order to study the damage characteristics of the yellow sandstone containing pores under the freeze-thaw cycle, the uniaxial compression test of saturated water-stained yellow sandstones with different freeze-thaw cycles was carried out by rock servo press, the microstructure was qualitatively analyzed by Zeiss 508 stereo microscope, and the microdamage mechanism was quantitatively studied by using specific surface area and pore size analyzer. The mechanism of weakening mechanical properties of single-hole yellow sandstone was expounded from the perspective of microstructure. The results show the following. (1) The number of freeze-thaw cycles and single-pore diameter have significant effects on the strength and elastic modulus of the yellow sandstone; the more the freeze-thaw cycles and the larger the pore size, the lower the strength of the yellow sandstone. (2) The damage modes of the yellow sandstone containing pores under the freeze-thaw cycle are divided into five types, and the yellow sandstone with pores is divided into two areas: the periphery of the hole and the distance from the hole; as the number of freeze-thaw cycles increases, different regions show different microscopic damage patterns. (3) The damage degree of yellow sandstone is different with freeze-thaw cycle and pore size. Freeze-thaw not only affects the mechanical properties of yellow sandstone but also accelerates the damage process of pores. (4) The damage of the yellow sandstone by freeze-thaw is logarithmic function, and the damage of the yellow sandstone is a power function. The damage equation of the yellow sandstone with pores under the freezing and thawing is a log-power function nonlinear change law and presents a good correlation.

Studies the Properties of Polyaspartic Polyurea Coated Concrete under Coaction of Salt Fog and Freeze-Thaw

2012 ◽  
Vol 455-456 ◽  
pp. 781-785
Author(s):  
Ping Lu ◽  
Xin Mao Li ◽  
Xue Qiang Ma ◽  
Wei Bo Huang
Keyword(s):  
Elastic Modulus ◽  
Frost Resistance ◽  
Dynamic Elastic Modulus ◽  
Freeze Thaw ◽  
Decrease Rate ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Salt Fog

. This paper mainly studied the properties of PAE polyurea coated concrete under coactions of salt fog and freeze-thaw. After exposed salt fog conditions for 200d, T3, B2, F2 and TM four coated concrete relative dynamic elastic modulus have small changes, but different coated concrete variation amplitude is different. T3 coated concrete after 100 times of freeze-thaw cycle the relative dynamic elastic modulus began to drop, 200 times freeze-thaw cycle ends, relative dynamic elastic modulus variation is the largest, decrease rate is 95%, TM concrete during 200 times freeze-thaw cycle, relative dynamic elastic modulus almost no change, B2 concrete and F2 concrete the extent of change between coating T3 and TM. After 300 times the freeze-thaw cycle coated concrete didn't appear freeze-thaw damage phenomenon. Four kinds of coating concrete relative dynamic elastic modulus variation by large to small order: T3 coated concrete > B2 coated concrete >F2 coated concrete > TM coated concrete, concrete with the same 200d rule. Frost resistance order, by contrast, TM coated concrete > B2 coated concrete > F2 coated concrete > T3 coated concrete.

Sign in / Sign up

Export Citation Format

Share Document