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PubMed1992

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants.

Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

Amino Acid Sequence↗
PubMed1986

Cell-cycle regulation of center initiation in Dictyostelium discoideum.

The center-initiating behavior of Dictyostelium discoideum amoebae in various cell-cycle phases was investigated. Small populations of synchronized AX-2 cells were seeded 1 in 1000 into cultures of a nonsignaling mutant (NP160) incapable of initiating centers. The ability of the wild-type AX-2 cells to initiate centers among mutant amoebae displayed cell-cycle regulation. Approximately 50% of a population of S-phase cells initiated centers while only 7.5% of a population of late G2-phase cells resulted in center formation. The timing of center formation also varied with cycle position. Synchronous cultures containing only AX-2 S-phase amoebae (no NP160) displayed the initial signs of aggregation after 4.5 hr of starvation and streaming into the aggregate was complete after 6 hr. In contrast, cultures of late G2-phase amoebae initiated aggregation centers after 5.5 hr of starvation and did not complete streaming until 7.5 hr. In addition, the number of aggregates formed by these synchronous cultures of AX-2 cells also varied with cycle position. In general, these results suggest a cell-cycle modulation of the autonomous signaling responsible for center initiation.

Cell Aggregation↗
PubMed1995

Cell-to-cell contact modulates the expression of the beta 1 family of integrins in primary cultures of thyroid cells.

The expression of the beta 1 family of integrins was studied in normal thyroid tissue cultures and monolayer cell cultures. The expression of the various subunits was measured by flow cytofluorometry with specific monoclonal antibodies and by Northern analysis. In monolayer cell cultures but not in tissue cultures, the expression of the alpha 3 subunit on the cell membrane progressively increased soon after plating, reaching a 30-fold higher intensity. The alpha 2 subunit, not detectable in native follicular cells, was expressed de novo and reached a remarkable high level. Up-regulation of alpha 2 and alpha 3 in monolayer cell cultures was serum-independent and preceded the expression of proliferating cell nuclear antigen, [3H]thymidine incorporation, and cell replication. Northern analysis demonstrated an increased level of beta 1 integrin mRNA. The increase of alpha 2 and alpha 3 was readily reversible since the expression of these molecules returned to a lower level when cultures reached a high cell density. Down-regulation did not occur until cell cultures were confluent. When cells from high cell density and low integrin expression were harvested and sparsely seeded in culture, up-regulation of integrins was observed again, while rapid reaggregation of isolated cells inhibited this phenomenon. Altogether these data suggest that cell-to-cell contact may regulate the expression of beta 1 integrins in thyroid primary cultures.

Antigens, CD↗
PubMed1976

Correction of essential fatty acid deficiency in newborn infants by cutaneous application of sunflower-seed oil.

Two newborn infants receiving long-term, fat-free parenteral nutrition developed essential fatty acid (EFA) deficiency. Biochemical evidence of EFA deficiency was documented in plasma, red blood cells, and adipose tissue and included a decrease in arachidonic and linoleic acids, an increase in 5,8,11-eicosatrienoic acid, palmitoleic and oleic acids and a trienoic/tetraenoic ratio of more than 0.4. Cutaneous application of sunflower-seed oil, a source rich in the essential fat linoleic acid, rapidly reversed the clinical and biochemical manifestations of deficiency in plasma.

Adipose Tissue↗
PubMed1990

Leukocyte inhibitory factor activates human neutrophils and macrophages to release leukotriene B4 and thromboxanes.

Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products. Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation. Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalanine (FMLPs) mediated responses. Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2). Resuspended at a final concentration of greater than 95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2. Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min. Adherent macrophages were used for the cytokine stimulation study. Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP. Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants. Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production. This was compared with IL 6 and GM-CSF, which had no effects. In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions. The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced. The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcimycin↗
PubMed2005

Improving small-diameter vascular grafts: from the application of an endothelial cell lining to the construction of a tissue-engineered blood vessel.

One of the main reasons why vascular reconstruction with synthetic small-diameter grafts has limited success is the absence of endothelial cells. To improve the outcome of nonvenous vascular bypass surgery, cell seeding of vascular grafts and other tissue-engineering techniques were developed. In this article, an overview is given of the artificial blood vessel as an alternative for venous vascular bypass surgery.

Blood Coagulation↗
PubMed2004

Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs?

High levels of Silver (Ag), Barium (Ba) and Strontium (Sr) and low levels of copper (Cu) have been measured in the antlers, soils and pastures of the deer that are thriving in the chronic wasting disease (CWD) cluster zones in North America in relation to the areas where CWD and other transmissible spongiform encephalopathies (TSEs) have not been reported. The elevations of Ag, Ba and Sr were thought to originate from both natural geochemical and artificial pollutant sources--stemming from the common practise of aerial spraying with 'cloud seeding' Ag or Ba crystal nuclei for rain making in these drought prone areas of North America, the atmospheric spraying with Ba based aerosols for enhancing/refracting radar and radio signal communications as well as the spreading of waste Ba drilling mud from the local oil/gas well industry across pastureland. These metals have subsequently bioconcentrated up the foodchain and into the mammals who are dependent upon the local Cu deficient ecosystems. A dual eco-prerequisite theory is proposed on the aetiology of TSEs which is based upon an Ag, Ba, Sr or Mn replacement binding at the vacant Cu/Zn domains on the cellular prion protein (PrP)/sulphated proteoglycan molecules which impairs the capacities of the brain to protect itself against incoming shockbursts of sound and light energy. Ag/Ba/Sr chelation of free sulphur within the biosystem inhibits the viable synthesis of the sulphur dependent proteoglycans, which results in the overall collapse of the Cu mediated conduction of electric signals along the PrP-proteoglycan signalling pathways; ultimately disrupting GABA type inhibitory currents at the synapses/end plates of the auditory/circadian regulated circuitry, as well as disrupting proteoglycan co-regulation of the growth factor signalling systems which maintain the structural integrity of the nervous system. The resulting Ag, Ba, Sr or Mn based compounds seed piezoelectric crystals which incorporate PrP and ferritin into their structure. These ferrimagnetically ordered crystals multireplicate and choke up the PrP-proteoglycan conduits of electrical conduction throughout the CNS. The second stage of pathogenesis comes into play when the pressure energy from incoming shock bursts of low frequency acoustic waves from low fly jets, explosions, earthquakes, etc. (a key eco-characteristic of TSE cluster environments) are absorbed by the rogue 'piezoelectric' crystals, which duly convert the mechanical pressure energy into an electrical energy which accumulates in the crystal-PrP-ferritin aggregates (the fibrils) until a point of 'saturation polarization' is reached. Magnetic fields are generated on the crystal surface, which initiate chain reactions of deleterious free radical mediated spongiform neurodegeneration in surrounding tissues. Since Ag, Ba, Sr or Mn based piezoelectric crystals are heat resistant and carry a magnetic field inducing pathogenic capacity, it is proposed that these ferroelectric crystal pollutants represent the transmissible, pathogenic agents that initiate TSE.

Acoustic Stimulation↗
PubMed2002

Ecology of southern ocean pack ice.

Around Antarctica the annual five-fold growth and decay of sea ice is the most prominent physical process and has a profound impact on marine life there. In winter the pack ice canopy extends to cover almost 20 million square kilometres--some 8% of the southern hemisphere and an area larger than the Antarctic continent itself (13.2 million square kilometres)--and is one of the largest, most dynamic ecosystems on earth. Biological activity is associated with all physical components of the sea-ice system: the sea-ice surface; the internal sea-ice matrix and brine channel system; the underside of sea ice and the waters in the vicinity of sea ice that are modified by the presence of sea ice. Microbial and microalgal communities proliferate on and within sea ice and are grazed by a wide range of proto- and macrozooplankton that inhabit the sea ice in large concentrations. Grazing organisms also exploit biogenic material released from the sea ice at ice break-up or melt. Although rates of primary production in the underlying water column are often low because of shading by sea-ice cover, sea ice itself forms a substratum that provides standing stocks of bacteria, algae and grazers significantly higher than those in ice-free areas. Decay of sea ice in summer releases particulate and dissolved organic matter to the water column, playing a major role in biogeochemical cycling as well as seeding water column phytoplankton blooms. Numerous zooplankton species graze sea-ice algae, benefiting additionally because the overlying sea-ice ceiling provides a refuge from surface predators. Sea ice is an important nursery habitat for Antarctic krill, the pivotal species in the Southern Ocean marine ecosystem. Some deep-water fish migrate to shallow depths beneath sea ice to exploit the elevated concentrations of some zooplankton there. The increased secondary production associated with pack ice and the sea-ice edge is exploited by many higher predators, with seals, seabirds and whales aggregating there. As a result, much of the Southern Ocean pelagic whaling was concentrated at the edge of the marginal ice zone. The extent and duration of sea ice fluctuate periodically under the influence of global climatic phenomena including the El Niño Southern Oscillation. Life cycles of some associated species may reflect this periodicity. With evidence for climatic warming in some regions of Antarctica, there is concern that ecosystem change may be induced by changes in sea-ice extent. The relative abundance of krill and salps appears to change interannually with sea-ice extent, and in warm years, when salps proliferate, krill are scarce and dependent predators suffer severely. Further research on the Southern Ocean sea-ice system is required, not only to further our basic understanding of the ecology, but also to provide ecosystem managers with the information necessary for the development of strategies in response to short- and medium-term environmental changes in Antarctica. Technological advances are delivering new sampling platforms such as autonomous underwater vehicles that are improving vastly our ability to sample the Antarctic under sea-ice environment. Data from such platforms will enhance greatly our understanding of the globally important Southern Ocean sea-ice ecosystem.

Animals↗
PubMed1998

Analysis of thermal stability of soya globulins using monoclonal antibodies.

The epitopes of two monoclonal antibodies (Mabs), one raised to soya glycinin (IFRN 0025) and one to beta-conglycinin (IFRN 0089), have been defined. The epitope of 0025 corresponds to residues 86-104 of the acidic polypeptide of glycinin A1aB1b and lies at the C terminus of the proteolytic intermediate known as glycinin-T, whilst that of 0089 corresponds to residues 78-84 in the acidic extension present in the alpha' subunit of beta-conglycinin. As the Mabs bind to synthetic peptides corresponding to the epitope regions both epitopes can be considered as being continuous in nature. The regions recognised correspond to surface loops, which are probably flexible in nature. Both Mabs were used to investigate thermally induced conformational changes in soya globulins. Thermally treated glycinin was recognised more strongly than native protein, possibly due to disruption of the disulphide bond joining the acidic and basic polypeptides. Disruption could increase epitope accessibility, as could the conformational changes associated with denaturation. The binding of anti-beta-conglycinin Mab 0089 was unaffected by heating, suggesting that its epitope remains on the surface of the aggregates formed on heating. This study demonstrates that Mabs with defined specificities can be sensitive probes for monitoring local conformational changes within a protein molecule during thermal denaturation.

Amino Acid Sequence↗
PubMed2006

Adhesion and growth of human umbilical vein endothelial cells on collagen-treated PU/PEGDA IPNs.

For ideal non-thrombogenicity under normal physiologic conditions, we propose endothelialization. Endothelialization means that synthetic biomaterials are seeded by endothelial cells to mimic natural blood vessels. In our study, we synthesized amphiphilic polyurethane (PU)/poly(ethyleneglycol)diacrylate (PEGDA) interpenetrating polymer networks (IPNs) with different levels of surface energy to investigate the effect of adhesion and the growth of human umbilical vein endothelial cells (HUVECs). Collagen with cell-binding molecules was adsorbed on the surface of PU/PEGDA IPNs to enhance the adhesion of HUVECs. The morphology of collagens adsorbed on the IPN surfaces depends highly on the surface energy of the IPNs. As the surface becomes hydrophilic, there is greater aggregation of the adsorbed collagens on the IPN surface. The HUVECs successfully adhere to the collagen-immobilized IPN surface. The morphology of the endothelial cells (ECs) that adhere to IPN 2k-C and IPN 2k after 1 day and after 3 days incubation shows that ECs were successfully spread. The adhesion and the proliferation of ECs increase on non-treated IPN surfaces as the hydrophobicity of the IPNs increases. The surface energy of IPN 2k-C is suitable for the adhesion and proliferation of ECs. Therefore, platelet adhesion is significantly reduced on the EC-hybridized surface of IPNs.

Biopolymers↗
PubMed1988

IL-1-induced adhesion of polymorphonuclear leukocytes to cultured human endothelial cells. Role of platelet-activating factor.

In early studies we found that IL-1 stimulated endothelial cells (EC) to produce platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Inasmuch as this phospholipid has a wide range of biologic activities, including polymorphonuclear leukocytes (PMN) aggregation and chemotaxis, we investigated whether EC-associated PAF could contribute to IL-1-induced PMN adhesion to EC. When four selective PAF antagonists were added to IL-1-stimulated EC during the PMN adhesion assay, adhesion was reduced in a concentration-related way. Similarly, pre-treatment of PMN with PAF before the adhesion assay to induce desensitization to this phospholipid reduced PMN adhesion to IL-1-treated EC. However, comparing the time course and the concentration response curve of IL-1-induced EC adhesivity and PAF synthesis, we found that increased EC adhesivity to PMN required a shorter incubation time and lower concentration of IL-1 to become apparent than PAF production. When acetyl-coenzyme A was added to EC cultures at a concentration that raised PAF synthesis by 60%, no significant increase in PMN adhesion was observed. In addition, after 9 to 10 doublings, the EC ability to synthesize PAF decreased by 85 to 90%, whereas IL-1-induced EC adhesivity to PMN was only slightly diminished. When IL-1-alpha and -beta were tested on EC, we observed that both were equally active in promoting PMN adhesion to EC but only the alpha-form was able to stimulate PAF production. When PMN were seeded on IL-1-treated EC, increased amounts of PAF were detected even when EC were fixed; in addition, the inhibitory effect of a PAF antagonist was evident also in these conditions. Overall these results indicate that IL-1-induced PAF production by EC does not significantly contribute to PMN adhesion to them. We hypothesize that the observed inhibitory effect of PAF antagonists and PAF desensitization of PMN might be directed at PAF produced by PMN themselves during adhesion to IL-1-treated EC.

Cell Adhesion↗
PubMed1989

The effects of citrate on hydroxyapatite induced calcium oxalate crystallization and on the formation of calcium phosphate crystals.

The addition of different amounts of hydroxyapatite crystals (HAP) to a solution, metastably supersaturated with respect to calcium oxalate (CaOx) resulted in heterogenous crystallization at seed concentrations exceeding 0.2 mmol/l. The induction period varied between 1 and more than 8 h with the shortest period for a seed concentration of 2 mmol/l. Addition to the system of 1 and 2% of whole urine and citrate in concentrations corresponding to approximately 1% of that found in normal urine inhibited the crystallization for as long as 4 h. In a system supersaturated with respect to calcium phosphate (CaP) the total number of crystals was markedly reduced by citrate concentrations exceeding 0.5 mmol/l. The fractions of medium sized and large crystals were sharply reduced and small crystals predominated at higher citrate concentrations. This might indicate effects of citrate on both crystal growth and crystal aggregation. We conclude that increased citrate concentrations during treatment with alkali leads to a significant inhibition of CaOx growth on HAP as well as to a prevention of the formation of large CaP crystals from solutions supersaturated with respect to CaP.

Calcium Oxalate↗
PubMed2005

Low level laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seeded on a three-dimensional biomatrix.

Mesenchymal stem cells (MSCs) seeded on three-dimensional (3D) coralline (Porites lutea) biomatrices were irradiated with low-level laser irradiation (LLLI). The consequent phenotype modulation and development of MSCs towards ossified tissue was studied in this combined 3D biomatrix/LLLI system and in a control group, which was similarly grown, but was not treated by LLLI. The irradiated and non irradiated MSC were tested at 1-7, 10, 14, 21, 28 days of culturing via analysis of cellular distribution on matrices (trypan blue), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregates formation (oil red O), alkaline phosphatase (ALP) activity, scanning electron microscopy (SEM) and electron dispersive spectrometry (EDS). The results obtained from the irradiated samples showed enhanced tissue formation, appearance of phosphorous peaks and calcium and phosphate incorporation to newly formed tissue. Moreover, in irradiated samples ALP activity was significantly enhanced in early stages and notably reduced in late stages of culturing. These findings of cell and tissue parameters up to 28 days of culture revealed higher ossification levels in irradiated samples compared with the control group. We suggest that both the surface properties of the 3D crystalline biomatrices and the LLLI have biostimulatory effects on the conversion of MSCs into bone-forming cells and on the induction of ex-vivo ossification.

Alkaline Phosphatase↗
PubMed1981

Gap junction assembly and endocytosis correlated with patterns of growth in a cultured adrenocortical tumor cell (SW-13).

After seeding at subconfluent densities, human adrenocortical adenocarcinoma cells (SW-13) proliferate to form a typical epithelial monolayer upon the culture vessel substratum. Single cells of the confluent monolayer are spontaneously released into the medium, however, where they may remain isolated or reaggregate with other floating cells. In this study, we examined the growth patterns of these cells in culture and analyzed alterations of their most prominent intercellular contact specialization, the gap junction, as cellular relationship changed during growth and development of the cell culture. We report here details of gap junction assembly during cellular aggregation and correlate the apparent endocytosis of gap junctions with the spontaneous release of single cells from the monolayer into the medium.

Adenocarcinoma↗
PubMed2002

Physicochemical and structural properties of oat globulin polymers formed by a microbial transglutaminase.

Oat globulin was polymerized by a microbial transglutaminase (TG), and some physicochemical and functional properties of polymers were studied. Reversed-phase HPLC revealed that the number of epsilon-(gamma-glutamyl) lysine isopeptide bonds formed after 4 h of enzyme incubation was 2.21 micromol/g of protein. SDS-PAGE showed that the oat globulin acidic polypeptides (AP) were more susceptible to polymerization than the basic polypeptides (BP), and the reactivities of both AP and BP were enhanced by the addition of other substrate proteins. Differential scanning calorimetry showed that both the denaturation temperature and denaturation enthalpy were decreased after TG treatment. Fourier transform infrared spectroscopy revealed marked increases in the intensity of two intermolecular beta-sheet bands associated with aggregate formation but little conformational changes in the polymerized protein. TG incubation led to progressive changes in flow properties of oat globulin dispersions, indicating enhanced pseudoplasticity and increased viscosity and yield stress.

Avena↗
PubMed1998

The roles of the N-linked glycans and extension regions of soybean beta-conglycinin in folding, assembly and structural features.

Beta-conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, alpha, alpha' and beta. All subunits are N-glycosylated and alpha and alpha' contain extension regions in addition to the core regions common to all subunits. Non-glycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of alpha and alpha' were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of beta-conglycinin. Density-gradient centrifugation, gel-filtration and differential scanning calorimetry profiles of the recombinant proteins and the native beta-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of beta-conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subunits with those of deletion mutants, only the alpha and alpha' subunits were soluble at lower ionic strength (mu < 0.25) at around the pH value of the endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.

Amino Acid Sequence↗
PubMed1988

Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.

As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.

Catalase↗
PubMed2006

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner.

Amyloid fibrils are typically rigid, unbranched structures with diameters of approximately 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.

Amyloid↗