High Molecular Weight Polypeptides Research Articles

Effects of intracellular structural associations on degradation of algal chloropigments in natural oxic and anoxic seawaters

To understand the effects of intracellular structural associations on degradation of algal chloropigments, we conducted a series of microcosm experiments by incubating Emiliania huxleyi cells (a marine haptophyte) in natural oxic and anoxic seawaters collected from a stratified water column in the Cariaco Basin. The incubated cell detritus were sequentially treated with two buffer solutions to separate pigment components into soluble and insoluble fractions. By using non-denaturing gel electrophoresis, several chlorophyll-complexes, free chlorophyll, and another unknown chlorophyll-containing component were further separated from the soluble fraction. The chlorophyll-complexes included those bound with high molecular weight core-proteins (CP-I and CP43+CP47) and low molecular weight polypeptides (LHC-I and LHC-II) in the cellular photosystems PS-I and PS-II. Overall pigment recovery from these fractions and gel bands was well equivalent to the total amount from direct acetone extraction of the cells. We followed the time-dependent concentration changes of chlorophyll-a (Chl-a), phaeophorbide-a (Ppb-a), and phaeophytin-a (Ppt-a) in all fractions and complexes to estimate the degradation rate constants of chloropigments in natural oxic and anoxic seawaters. Our experimental results demonstrated that the intracellular structural associations had important influences on degradation of chloropigments under different redox conditions. In general, total Chl-a degraded faster (∼4X) in oxic seawater than in anoxic seawater. However, the rate differences between oxic and anoxic conditions varied among the fractions and complexes. Degradation rate constants of Chl-a in soluble fraction were much higher (>10X) than those in insoluble fraction under both oxic and anoxic conditions. Chl-a bound with the complexes in PS-II appeared to be more reactive (∼2X) than that in PS-I under oxic conditions but the difference in degradation rate constants between two photosystems became indistinguishable under anoxic conditions. Variations of Ppb-a in different fractions and complexes during incubation showed different patterns, implying that cellular Chl-a could degrade through two different pathways: (1) internal degradation into Ppb-a within insoluble pool and polypeptide complexes; and (2) release first from protein complexes and followed by external degradation.

My Contributions to Science and Society

My Contributions to Science and Society

Conserved C-termini of Spidroins are secreted by the major ampullate glands and retained in the silk thread.

The C-termini of Spidroins produced in the major and minor ampullate glands of spiders are highly conserved. Despite this conservation, no corresponding peptides have been identified in the spinning dopes or the silk filaments so far. To prove their presence or absence, polyclonal antibodies derived against fusion proteins containing the conserved C-terminal regions of both Spidroin 1 and 2 from the spider Nephila clavipes were generated. The antibodies reacted with high molecular weight polypeptides of the corresponding gland extracts and solubilized major ampullate filament and in addition to filament cross-sections. This demonstrates the existence of C-terminal specific peptides in the spinning dope and the mature Spidroins. Both the fusion proteins as well as the proteins contained within the gland lumen showed a reduction in their size under reducing conditions indicating the presence of disulfide bonds. Their high conservation and the biochemical data suggest crucial roles the C-termini play in the formation and/or structure of the corresponding silk filaments.

Typical changes of erythrocytes in chronic inflammation.

Structural and metabolic status and function of erythrocytes were studied in patients with chronic bronchitis, chronic inflammatory diseases of the upper airways, and chronic colitis. Complex study of the peripheral component of the erythron showed that disorders in erythrocyte morphology and function (decreased erythrocyte dry weight, low concentration of sulfhydryl groups and lipoproteins, decreased content of high molecular weight polypeptides in parallel with increased content of low molecular weight proteins, increased number of transformed cells, and enhanced reversible aggregation) were nonspecific and did not depend on the location of the inflammatory process.

Solubilization of dynein from Tetrahymena ssp. axonemes using phosphate analogues

One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp. ( T. pyriformis or T. thermophila) in the presence of ATP. This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein. Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins. These results suggest that the solubilized protein is an outer arm dynein and also that a dynein–ADP–phosphate complex decreases its affinity with the adjacent microtubules within axonemes. Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue. The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.

Structure and formation of self-assembled monolayers of helical poly ( γ-benzyl l-glutamate) by surface plasmon resonance and infrared spectroscopy

The self-assembly of a rigid, rod-like, α-helical polypeptide was investigated on gold substrates. A disulfide labeled poly( γ-benzyl- l-glutamate) (PBLG) was prepared by a novel synthetic route wherein the polymerization of the N-carboxyanhydride was initiated with an amine terminated disulfide. Two different SSPBLG materials with molecular weight of 11 and 28 kDa were prepared and characterized using dielectric spectroscopy. The kinetics of self-assembly of the two polypeptides was studied using surface plasmon resonance (SPR) and the organization of the self-assembled monolayers (SAMs) was characterized by polarization-modulation infrared absorption spectroscopy (PM-IRRAS). SPR revealed that self-assembly of the high molecular weight polypeptides on gold substrates proceeds rapidly on a time-scale of minutes and is comparable to the time of assembly of simple n-alkanethiols. The increase in length of the polypeptide led to an increase in the dipole moment from 156 to 363 D. The enhanced electrostatic interactions in the SAMs were manifested as an increase in the average tilt of the helix axis from the normal to the surface and monolayers with smaller thickness.

Self-Assembly of a Rodlike Polypeptide on Solid Surfaces: Role of Solvent, Molecular Weight, and Time of Assembly

The interplay between preparative conditions and molecular characteristics during the self-assembly of a rigid, rodlike, α-helical polypeptide was investigated. Poly(γ-benzyl-l-glutamate) was prepared by a novel synthetic route wherein the polymerization of the N-carboxyanhydride was initiated with an amine terminated disulfide, and the SSPBLG was characterized using light scattering, viscometry, and size-exclusion chromatography. Self-assembly of SSPBLG on gold substrates was found to proceed rapidly and high molecular weight (∼27 kDa) polypeptides formed self-assembled monolayers (SAMs) on a time scale of minutes. Characterization of the SAMs by polarization modulation infrared absorption spectroscopy (PM-IRRAS) was used to explore the effects of the molecular weight of the polypeptide, the time of assembly, and the solvent. An increase in the length of the polypeptide from 66 to 156 Å caused an increase in the average tilt of the helix axis from the normal to the surface. The use of a nonpolar solvent (benzene), where antiparallel aggregation of helices is known to occur, did not prove to be an effective strategy for improving orientation of the longer polypeptides. The time of assembly was found to play a key role, and long times of assembly led to a decrease in surface coverage and increase in tilt angle. This effect was more pronounced for the short polypeptide than the long polypeptide. Self-assembly of a debenzylated SSPBLG showed that physisorption due to hydrophobic interactions was important in the preparation of SAMs formed from long polypeptides. Reduction of physisorption by use of the debenzylated SSPBLG led to an enhancement in surface coverage and decrease in the tilt angle of the higher molecular weight SSPBLG.

Cold stress induced high molecular weight membrane polypeptides are responsible for cold tolerance in Rhizobium DDSS69

Cold stress induces a lag phase in the growth cycle of Rhizobium DDSS69. Two cold sensitive mutants of DDSS69 were generated through Tn5 tagged mutagenesis. These mutants do not grow below 15 degrees C but show a growth curve comparable with the wild type grown at 5 degrees C. There is a rapid induction of two high molecular weight membrane polypeptides of 135 and 119 kDa within 15 min of exposure to 5 degrees C in DDSS69. PAGE membrane protein profiles of stressed and non-stressed cells reveal differential regulation of genes. At 15 degrees C both mutants lack the high molecular weight polypeptides, suggesting a role in alleviation of cold stress.

Characterization of an axonemal dynein heavy chain expressed early in airway epithelial ciliogenesis.

The most conspicuous evidence of airway epithelial maturation and vitality is the presence of motile cilia. In an effort to generate genetic and antigenic markers of airway maturation, injury, and repair, we characterized airway epithelial expression of a gene identified by two human expressed sequence tags that encoded peptides with sequence similarity to an invertebrate ciliary dynein heavy chain (DHC). Molecular analyses showed that the gene has a very large RNA transcript that encodes a very high molecular weight polypeptide with biochemical properties that are characteristic of a dynein heavy chain. Expression of the gene transcript correlated with the presence of ciliated cells in tissues, and immunohistochemical localization of the gene product confirmed its presence in the cilia of mature airway epithelium. In epithelium undergoing ciliogenesis ex vivo, expression of the gene transcript preceded ciliation of the epithelium and the gene product was present in the cytoplasm and at the apical border of nonciliated cells. These data suggested that the gene encodes an axonemal DHC that is expressed early during ciliogenesis, before the appearance of cilia.

Compared responses of poplar cuttings and in vitro raised shoots to short-term chilling treatments.

In woody plants, chilling stress occurs during the early spring growth and can have important economic consequences. The aim of this study was to compare the reliability of two different experimental systems, 3-month-old softwood cuttings and in vitro-grown shoots, to study chilling effects in a poplar clone (Populus tremula×P. tremuloides cv. Muhs1). Different parameters were recorded: lignin content, sucrose concentration and protein and fresh weight variation, during a 2-week treatment at 10 °C. Two families of polypeptides of high molecular weight (110 and 116 kDa) were shown to accumulate in response to chilling in both cuttings and microshoots. For some of the parameters studied, i.e. appearance of some groups of polypeptides and reduction of fresh weight gain, both in vitro and ex vitro systems were suitable and produced similar results. In contrast, for some other observations, i.e. on sucrose concentration and lignin content, the systems led to different conclusions. While sucrose and lignin contents were shown to increase in cuttings submitted to chilling, no variation in lignin and only a small temporary peak of sucrose could be observed in microshoots kept under chilling. These parameters seem not to be suitable for studying the response of poplar in the in vitro system in such a short-term study.

Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM.

Major histocompatibility complex (MHC) class II molecules bind and present to CD4(+) T cells peptides derived from endocytosed antigens. Class II molecules associate in the endoplasmic reticulum with invariant chain (Ii), which (i) mediates the delivery of the class II-Ii complexes into the endocytic compartments where the antigenic peptides are generated; and (ii) blocks the peptide-binding site of the class II molecules until they reach their destination. Once there, Ii must be removed to allow peptide binding. The bulk of Ii-class II complexes reach late endocytic compartments where Ii is eliminated in a reaction in which the cysteine protease cathepsin S and the accessory molecule H-2DM play an essential role. Here, we here show that Ii is also eliminated in early endosomal compartments without the intervention of cysteine proteases or H-2DM. The Ii-free class II molecules generated by this alternative mechanism first bind high molecular weight polypeptides and then mature into peptide-loaded complexes.

CHARACTERIZATION OF HYDROPSYCHE SLOSSONAE (TRICHOPTERA: HYDROPSYCHIDAE) CAPTURE NET POLYPEPTIDES

AbstractThe aim of this study was to characterize polypeptide components of the capture net spun by trichopteran larvae Hydropsyche slossonae (Banks) (Trichoptera: Hydropsychidae). Thirty-one polypeptide bands were identified by SDS – polyacrylamide gel electrophoresis (SDS–PAGE) from extracted net material, with molecular weights ranging from 8500 to 179 000. Comparison with published data on Bombyx mori (L.) (Lepidoptera: Bombycidae) silk, treated under similar denaturing conditions, shows that six low molecular weight polypeptides ranging between 8500 and 18 800 in the silk of H. slossonae are absent from that of B. mori; furthermore, two high molecular weight polypeptides (210 000 and 220 000) detected in the silk of B. mori are not present in that of H. slossonae. Differences between both groups are probably related to their mode of living and to the specific use of silk (in air versus under water). Our findings are consistent with the current trend in the literature that silk spun by aquatic and terrestrial insects, as well as those spun by different species, is apparently made of different biopolymers according to the protein constituents. Hence, the polypeptide characterization of silk, combined with sequence data and (or) antibodies cross-reactivity data, could represent a potential tool for taxonomic classification improvement of aquatic insects. These results could eventually be used to characterize hydropsychid capture net anomalies induced by environmental pollution.

Protein and amino acid composition of silks from the cob weaver, Latrodectus hesperus (black widow)

The silks from the cob weaving spider, Latrodectus hesperus (black widow), have been examined with the goal of expanding our understanding of the relationship between the protein structure and mechanical performance of these unique biomaterials. The scaffolding, dragline and inner egg case silks each appear to be distinct fibers based on mole percent amino acid composition and polypeptide composition. Further, we find that the amino acid composition of dragline and egg case silk are similar to the analogous silks produced by orb weaving spiders, while scaffolding silk may represent a novel silk. The black widow silks are comprised of multiple high molecular weight polypeptides, however, the egg case and scaffolding silks also contain some smaller polypeptides.

Stress Induced Polypeptides in Lathyrus sativus

Effect of stress during seed germination showed both qualitative and quantitative changes in respect to polypeptide pattern. In general, the number of polypeptides decreased with induced stress. Polypeptides of 46, 40, 35 and 28 kD were present even at sublethal dehydration state of the plant thereby indicating the possibility of their role in overcoming severe stress. This has also been confirmed by RNA dot-blot analysis using drought responsive DNA probe which showed an abundance of stress inducible transcripts on salinity, mannitol and ABA treatments. Salinity induced greater reduction in the expression of high molecular weight polypeptides than other stress treatments. ABA alongwith mannitol/salt induced higher synthesis of a 46 kD polypeptide.

α-Helix Nucleation Constant in Copolypeptides of Alanine and Ornithine or Lysine

The α-helix is one of a small number of fundamental structural motifs of the peptide backbone that are abundant in native proteins. The forces responsible for its stability have attracted a great deal of theoretical and experimental attention. Helix formation can naturally be considered in terms of two processes, initial nucleation of a helix from a sequence of disordered residues and propagation or growth of helical structure from such a nucleus. Data from a variety of short peptide and polypeptide models have revealed more details about the propagation of helix structure than about helix nucleation. To investigate helix nucleation, we have synthesized two series of high molecular weight polypeptides containing differing ratios of alanine and one of the basic side chains, ornithine or lysine; poly l-alanine is insoluble in water. The CD signals of these copolymers have been analyzed by a program that evaluates the helix content in terms of the experimental chain-length distribution and composition, with ...

Preliminary characterization of a structural defect in homozygous sickled cell alpha spectrin demonstrated by a rabbit autoantibody.

We have identified a rabbit autoantibody that strongly reacts with the core membrane skeleton of control red blood cells, and does not react with low- or high-density sickle cell core skeletons upon indirect immunofluorescence. Western blot analysis of red blood cell membrane proteins, utilizing this autoantibody, indicated no reactivity to any protein when SDS-PAGE was conducted in the presence of the reducing agent, dithiothreitol. However when SDS-PAGE was performed on control red blood cell membrane proteins separated in the absence of dithiothreitol, the autoantibody specifically reacted with a high molecular weight polypeptide (apparent Mr approximately equal to 310 kD) representing a DTT sensitive form of control alpha spectrin, which we refer to as alpha' spectrin. There was no staining of high density or low density sickle cell alpha or alpha' spectrin. This autoantibody should be an excellent tool for the fine mapping of structural change(s) in control vs. sickle cell alpha spectrin, and determination of whether the structural alteration effects spectrin dimer-tetramer interconversion and/or the spectrin-actin interaction. The modification in alpha spectrin, detected by this antibody, is very specific for homozygous SS alpha spectrin because sickle cell beta+ thalassemic alpha spectrin and sickle cell trait alpha spectrin react intensely with the autoantibody.

Dynein heavy chain-related polypeptides are associated with organelles in pollen tubes of Nicotiana tabacum

It is known that pollen tubes contain two high molecular weight polypeptides which share some biochemical and immunological properties with dynein heavy chains. This paper reports data on the subcellular localization of the two dynein heavy chain-related polypeptides during pollen tube growth. Immunofluoresence studies using a purified antibody (Dy-1) raised against a synthetic peptide reproducting the P-loop conserved sequence of dynein heavy chains showed spot-like structures, with a characteristic distribution pattern that depended on the tube length. Biochemical evidence confirmed the presence of dynein heavy chain-related bands in the pollen tube membrane fraction. The association of proteins carrying dynein heavy chain-related polypeptides to cell membranes was affected by detergent (Triton×100), whereas other stripping agents, like NaCl and Na2CO3, did not significantly influence the interaction of dynein heavy chain-related doublet with their cytoplasmic targets. These data suggest that dynein heavy chain-related polypeptides associate with membranous organelles within the vegetative cell of Nicotiana tabacum pollen tubes, implying their involvement in the cytoplasmic distribution of these organelles.

Growth performance and gut function of piglets weaned at four weeks of age and fed protease-treated soya-bean meal

Two experiments were carried out to investigate the effects of the protease-treatment of soya-bean meal (SBM) on its composition and nutritive value when fed to piglets weaned at 4 weeks of age in cereal-based diets. In experiment 1, two proteases, active at either alkaline (protease P1) or acid pH (protease P2), were compared with alkali or acid treatment alone. In experiment 2, treatment with protease P2 at acid pH was compared with untreated-SBM, acid-treated SBM, a soya protein concentrate or skim milk powder+fishmeal. Protease treatment increased SBM alpha amino nitrogen concentrations and reduced in vitro SBM antigenicity. Protease P1 reduced in vitro antigenicity to a greater extent than protease P2. However, protease P1 hydrolysed SBM polypeptides non-specifically whereas protease P2 preferentially hydrolysed a high molecular weight polypeptide. In experiment 1, piglets (12 diet −1) fed alkali-treated SBM gained more weight than piglets fed acid-treated SBM in the first 7 days after weaning. Protease P1 did not improve live-weight gain compared with alkali-treatment whereas protease P2 did improve live-weight gain compared with acid treatment. In experiment 2, piglets (24 diet −1) fed protease P2-treated SBM or skim milk powder+fishmeal gained significantly more weight over the first 7 days post-weaning than piglets fed untreated SBM. In neither experiment were there significant differences between diets in piglet serum anti-SBM specific antibodies. There were no consistent changes in small intestine enzyme specific activities in response to the diets fed. Histopathological examination of the small intestine of piglets in experiment 2 revealed no evidence of enteric disease. Neither were there any differences between diets in villus height or crypt depth.

Production and characterization of monoclonal antibodies against mink leukocytes

Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32–40 kDa polypeptides and the platelet reactive antibody 17 kDa, 22 kDa plus two high molecular weight (> 100 kDa) polypeptides. Immunohistological studies of the mAbs were performed in order to localize the cellular distribution of the detected antigens in various organs. The T lymphocyte reactive antibody detected an antigen, which was widely distributed in the T cell area of lymph nodes and spleen and in the thymic medulla. We conclude that this antibody is an anti-CD3 mAb and suggest that the platelet reactive antibody reacts to the CD41 CD61 integrin molecule. In addition to our own mAbs, more than 100 mAbs against leukocytes of human and various animal species have been analysed for cross-reactivity to mink leukocytes. We found eight to cross-react with mink. Of particular importance was an anticanine CD11a mAb, an antihuman CD79a mAb and an antihuman bcl-2 mAb.

ISCOM vaccine against contagious bovine pleuropneumonia (CBPP) 1. Biochemical and immunological characterization

A better vaccine than the existing ones against contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) would improve the chances for eradication of CBPP. In such an effort, immunostimulating complexes (ISCOMS) have been prepared from the whole detergent-solubilized cells of MmmSC and characterized biochemically and immunologically. The most efficient detergent for solubilization of the mycoplasma was MEGA-10 which yielded a high recovery of proteins in the ISCOMS. The ISCOMS showed the typical cage-like structure by EM and sedimented as 19S by sucrose gradient centrifugation. The protein pattern of the ISCOMS, analyzed in SDS-PAGE, revealed a great number of bands distributed along the gel as high and low molecular weight polypeptides. The Western blot developed with a serum from a CBPP infected animal detected a reduced number of polypeptides. In samples from whole mycoplasma cells and in ISCOMS, lectin blots revealed more than 20 carbohydrate structures. The ISCOMS induced a strong primary antibody response in mice measured by ELISA and the boost resulted in a 6-fold increase of the serum antibody response. The IgG response was distributed into various IgG subclasses with high IgG1, IgG2a and IgG2b titres while the IgG3 response was low. In cattle the ISCOM vaccine induced strong primary and long lasting secondary antibody responses of similar magnitudes as those of naturally infected animals as recorded by ELISA which persisted more than a year. IgG response was equally distributed in IgG1 and IgG2 subclasses. Also a cell-mediated immune response measured by proliferation assay was induced by low dose of ISCOMS. In the growth inhibition test, sera from vaccinated cattle readily inhibited colony growth already after the first immunization.