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At least 163 records · Page 9Linked to original sources
PubMed1987

Cottonseed malate synthase : biogenesis in maturing and germinated seeds.

The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and imbibition, then increases again following germination. The research reported herein is a comparative study of the synthesis and acquisition of MS into glyoxysomes as they occur in maturing and germinated seeds. Rate-zonal centrifugation of cotyledon extracts revealed that the 5 Svedberg unit (S) cytosolic form of MS was the only form present at 42 days postanthesis (DPA) when activity was first detectable. At later stages (48 DPA, 0 day, 26 hours, and 48 hours), both the 5S and glyoxysomal 20S forms were present, with the 20S form becoming much more prevalent. Western blot analyses revealed that no other form(s) of MS were present in the phosphate-buffered gradients, and that 5S and 20S forms had the same subunit molecular weight in maturing and germinated seeds. Comparisons of radiospecific activity of MS immunoprecipitates following in vivo labeling with [(35)S]methionine for varying time intervals provided strong evidence for a 5S-precursor to 20S-product relationship during both seed maturation and seedling growth. Comparisons of MS labeled in vivo and in vitro in wheat germ and rabbit reticulocyte lysates programmed with poly(A)(+)RNA (from maturing and germinated seeds) revealed no detectable differences in subunit molecular weights. These results reinforced our other data indicating that MS was synthesized in the cytosol and acquired by glyoxysomes in both maturing and germinated cotton seeds without involvement of an intervening aggregate pool in the endoplasmic reticulum, or via processing of a cleavable precursor molecule. MS was translated from poly(A)(+)RNA extracted from 28 DPA cotton seeds. This was nearly 2 weeks before MS activity or protein was detected in vivo. This finding invites further study on the regulation of RNA transcripts during maturation.

Journal Article↗
PubMed1982

Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro.

This study correlates endogenous levels of cAMP and cGMP with their immunohistochemical localization during chondrogenic differentiation of C57B1/6J mouse limb mesenchyme in vivo and in vitro. A transient decrease in cGMP but not cAMP was found from days 12 to 13 in vivo correlating with early stages of chondrogenesis in the developing limb. Intracellular levels of both cAMP and cGMP in high density limb mesenchyme cultures increased 25% after 24 hr in culture when aggregate and nodule formation was detectable. When cells were seeded at different initial plating densities to delay the onset of aggregate and nodule formation, increased levels of intracellular cAMP correlated temporally with the appearance of nodules. Both cyclic AMP and cGMP were immunohistochemically localized in perichondrial cells and chondrocytes in vivo and in vitro. Therefore, (1) cAMP levels correlated temporally with the appearance of chondrogenic cells and (2) cAMP and cGMP were immunohistochemically localized to chondrogenic cells. These data indicate that fluctuations of both cAMP and cGMP levels may be involved in limb cartilage differentiation. Although increases in both nucleotides were found to correlate with the onset of chondrogenesis in vitro, in vivo data suggest that the amount of cAMP relative to cGMP rather than the absolute amount of an individual cyclic nucleotide may be more significant in modulating differentiation.

Animals↗
PubMed2006

Mathematical modelling of engineered tissue growth using a multiphase porous flow mixture theory.

This paper outlines the framework of a porous flow mixture theory for the mathematical modelling of in vitro tissue growth, and gives an application of this theory to an aspect of tissue engineering. The problem is formulated as a set of partial differential equations governing the space and time dependence of the amounts of each component of the tissue (phase), together with the physical stresses in each component. The theory requires constitutive relations to specify the material properties of each phase, and also requires relations to specify the stresses developed due to mechanical interactions, both within each phase and between different phases. An application of the theory is given to the study of the mobility and aggregation of a population of cells seeded into an artificial polymeric scaffold. Stability analysis techniques show that the interplay of the forces between the tissue constituents results in two different regimes: either the cells form aggregates or disperse through the scaffold.

Algorithms↗
PubMed1987

Effects of defined medium, fetal bovine serum, and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: inference for in vitro assays.

We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors.

Animals↗
PubMed1998

Identification of structural domains in inter-alpha-trypsin involved in calcium oxalate crystallization.

The urinary glycoprotein that inhibits calcium oxalate (CaOx) crystallization in vitro shows a structural similarity to urinary trypsin inhibitor (UTI; recently termed bikunin), the light chain of inter-alpha-trypsin inhibitor (I alpha I). The functional domains of I alpha I involved in its inhibitory activity of CaOx crystallization have been investigated using isolated intact domains of I alpha I produced from controlled proteolytic digests of the protein. The fragments investigated include the heavy chains of I alpha I, UTI, chondroitinase AC-treated UTI, and the carboxyl-terminal domain of UTI (termed HI-8). The effects of I alpha I and its fragments on the inhibitory activity of CaOx crystallization were evaluated in vitro using CaOx crystal aggregation and growth assays, and seeded crystal generation assay as well as using crystal matrix protein generation assay. UTI, but not the heavy chains of I alpha I, had a discernible effect on CaOx crystallization inhibitory activity. Less requirement of the carbohydrate moiety of UTI is implicated by the observation that chondroitinase AC-treated UTI fragment was also found to inhibit CaOx crystallization with almost the same activity as UTI. HI-8 also efficiently inhibited CaOx crystallization, while I alpha I showed a weak inhibitory activity. The results are almost consistent with a seed crystal generation assay and a crystal adsorption inhibition assay, in which I alpha I or its derivatives inhibits prothrombin fragment 1 (F1) adsorption to CaOx crystals. In conclusion, these results suggest that the part of the I alpha I protein responsible for inhibition of CaOx crystallization is the carboxyl-terminal domain of UTI.

Alpha-Globulins↗
PubMed1999

Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.

Amino Acid Sequence↗
PubMed2000

The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein.

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.

Amyloid↗
PubMed2007

A novel three-dimensional system to study interactions between endothelial cells and neural cells of the developing central nervous system.

BACKGROUND: During angiogenesis in the developing central nervous system (CNS), endothelial cells (EC) detach from blood vessels growing on the brain surface, and migrate into the expanding brain parenchyma. Brain angiogenesis is regulated by growth factors and extracellular matrix (ECM) proteins secreted by cells of the developing CNS. In addition, recent evidence suggests that EC play an important role in establishing the neural stem cell (NSC) niche. Therefore, two-way communication between EC and neural cells is of fundamental importance in the developing CNS. To study the interactions between brain EC and neural cells of the developing CNS, a novel three-dimensional (3-D) murine co-culture system was developed. Fluorescent-labelled brain EC were seeded onto neurospheres; floating cellular aggregates that contain NSC/neural precursor cells (NPC) and smaller numbers of differentiated cells. Using this system, brain EC attachment, survival and migration into neurospheres was evaluated and the role of integrins in mediating the early adhesive events addressed. RESULTS: Brain EC attached, survived and migrated deep into neurospheres over a 5-day period. Neurospheres express the ECM proteins fibronectin and laminin, and brain EC adhesion to neurospheres was inhibited by RGD peptides and antibodies specific for the beta1, but not the alpha6 integrin subunit. CONCLUSION: A novel 3-D co-culture system for analysing the interactions between EC and neural cells of the developing CNS is presented. This system could be used to investigate the reciprocal influence of EC and NSC/NPC; to examine how NSC/NPC influence cerebral angiogenesis, and conversely, to examine how EC regulate the maintenance and differentiation of NSC/NPC. Using this system it is demonstrated that EC attachment to neurospheres is mediated by the fibronectin receptor, alpha5beta1 integrin.

Animals↗
PubMed1998

Recruitment and the role of nuclear localization in polyglutamine-mediated aggregation.

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat-containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.

Animals↗
PubMed1995

Dietary mustard, rape seed oils and selenium exert distinct effects on serum Se, lipids, peroxidation products and platelet aggregability.

OBJECTIVE: A degenerative cardiomyopathy (DCM) has been observed among mustard oil (MUST) users in China who eat 150 ml/week. Though the condition may be a selenium (Se) deficiency, MUST, rich in erucic acid (22:1), may bear a cardiotoxic or pro-oxidant substrate(s). The purpose of this feeding study was to compare the effects of feeding MUST, high erucic acid rape seed oil (HEAR), low erucic acid rape seed oil (LEAR) and corn oil, with or without Se addenda, on serum cholesterol, triglycerides, thiobarbituric acid reactive substances (TBARS), alpha-tocopherol, platelet aggregation and hepatic glutathione peroxidase (GSHPXase). Heart muscle pathology was evaluated. METHODS: Eighty male, weanling Wistar rats were divided into four dietary groups and fed AIN76A diet for 8 weeks with one of four oils: 1) MUST, 2) HEAR, 3) LEAR, or CORN oil. Half of each group received 0.43 mg/kg Se supplement. A purified diet was fed for 8 weeks. RESULTS: Neither Se nor oil type affected growth, though serum Se rose with dietary Se addendum (p < 0.01), and hepatic GSHPXase rose for each oil (p < 0.01). Se deprivation led to elevated serum cholesterol, except for the CORN oil group (p < 0.01). Only with LEAR did HDL cholesterol decrease. Serum triglycerides decreased with MUST and LEAR feedings (p < 0.05). In Se deficiency, HEAR led to elevated TBARS (p < 0.01), though not MUST, which contained twice as much alpha-tocopherol. Though supplementary Se protected the HEAR-fed animals from secondary peroxidation as TBARS (p < 0.01), the MUST group was not so protected. Ration Se also spared serum alpha-tocopherol for each diet group except the MUST/Se group (p < 0.05). MUST oil feeding resulted in increased platelet aggregation; MUST and HEAR resulted in increased platelet ATP release compared with CORN oil. CONCLUSION: Oxidative mechanisms may be involved in the DCM involving both Se limitation and pro-oxidant stress related to the usual intake of MUST. Human studies have been undertaken to test this thesis.

Adenosine Triphosphate↗
PubMed1989

[Inhibitory effect of sodium pentosan polysulfate on the formation, growth and aggregation of calcium oxalate in vitro].

The inhibitory effects of sodium pentosan polysulfate (SPP) on the formation, growth and aggregation of calcium oxalate (CaOx) crystals were estimated by means of a Coulter counter TA II by the seed crystal method, whole urine method and infrared spectroscopic method. In the seeded crystal system, the inhibitory activities on aggregation (Ia), growth (Ig) and size (Is) were calculated from the change of the number, volume and diameter of crystals. At above 0.5 microgram/ml, SPP inhibited the aggregation and growth of added CaOx monohydrate (COM) and dihydrate (COD) crystals. In the whole urine system, after determining the metastable limit, the formation and growth of CaOx crystals precipitated in response to a load of sodium oxalate was measured. SPP had a strong inhibitory effect on the formation and growth in the concentration of over 5 micrograms/ml. In infrared spectroscopic study, the ratio of COD/COM formed in whole urine increased with the concentration of SPP. SPP therefore might have prevented the recurrence of stone formation.

Agglutination↗
PubMed2002

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts.

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.

Escherichia coli O157↗
PubMed2004

Differential Behavior Between Isolated and Aggregated Rabbit Auricular Chondrocytes on Plastic Surfaces.

A knowledge of the behavior of chondrocytes in culture is relevant for tissue engineering. Chondrocytes dedifferentiate to a fibroblast-like phenotype on plastic surfaces. Dedifferentiation is reversible if these cells are then cultured in suspension. In this report a description is given of how when chondrocyte aggregates formed in suspension are next seeded on plastic, most of them attach as round or polygonal cells. This morphological differentiation, with synthesis of type II collagen, is stable for long culture periods. This simple method can be of use as a model for studies of chondrocyte behavior on plastic. The results indicate that in addition to culture conditions, such as cell isolation method or cell density, chondrocyte behavior on plastic depends on the presence of aggregates.

Journal Article↗
PubMed2004

Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro.

Prions are infectious protein conformations that are generally ordered protein aggregates. In the absence of prions, newly synthesized molecules of these same proteins usually maintain a conventional soluble conformation. However, prions occasionally arise even without a homologous prion template. The conformational switch that results in the de novo appearance of yeast prions with glutamine/aspargine (Q/N)-rich prion domains (e.g., [PSI+]), is promoted by heterologous prions with a similar domain (e.g., [RNQ+], also known as [PIN+]), or by overexpression of proteins with prion-like Q-, N-, or Q/N-rich domains. This finding led to the hypothesis that aggregates of heterologous proteins provide an imperfect template on which the new prion is seeded. Indeed, we show that newly forming Sup35 and preexisting Rnq1 aggregates always colocalize when [PSI+] appearance is facilitated by the [RNQ+] prion, and that Rnq1 fibers enhance the in vitro formation of fibers by the prion domain of Sup35 (NM). The proteins do not however form mixed, interdigitated aggregates. We also demonstrate that aggregating variants of the polyQ-containing domain of huntingtin promote the de novo conversion of Sup35 into [PSI+]; whereas nonaggregating variants of huntingtin and aggregates of non-polyQ amyloidogenic proteins, transthyretin, alpha-synuclein, and synphilin do not. Furthermore, transthyretin and alpha-synuclein amyloids do not facilitate NM aggregation in vitro, even though in [PSI+] cells NM and transthyretin aggregates also occasionally colocalize. Our data, especially the in vitro reproduction of the highly specific heterologous seeding effect, provide strong support for the hypothesis of cross-seeding in the spontaneous initiation of prion states.

Alleles↗
PubMed2000

Occurrence of resveratrol in edible peanuts.

Resveratrol has been associated with reduced cardiovascular disease and reduced cancer risk. This phytoalexin has been reported in a number of plant species, including grapes, and may be one of the compounds responsible for the health benefits of red wine. Analytical methods for measuring resveratrol in wine and peanuts were adapted to isolate, identify, and quantify resveratrol in several cultivars of peanuts. Aqueous ethanol (80% v/v) extracts from peanuts without seed coats were purified over alumina/silica gel columns and analyzed by reversed phase HPLC using a C-18 column. Peanuts from each market type, Virginia, runner, and Spanish, produced in four different locations contained from 0.03 to 0.14 microg of resveratrol/g. Seed coats from runner and Virginia types contained approximately 0.65 microg/g of seed coat, which is equivalent to <0.04 microg/seed. Quantitative analysis of 15 cultivars representing 3 peanut market types, which had been cold stored for up to 3 years, indicated a range of 0.02-1.79 microg/g of peanut compared to 0.6-8.0 microg/mL in red wines.

Arachis↗
PubMed1981

Urinary macromolecular substances as natural inhibitors of calcium oxalate crystal aggregation.

We studied the inhibitory effect of urinary macromolecular substances (UMM), more than 10,000 mol. wt., on aggregation of calcium oxalate crystals in a crystal-seed system and a non-crystal-seed system. In both assay systems, UMM from non-stone-farmers showed a marked inhibitory activity on crystal aggregation, whereas UMM from recurrent stone-formers showed a weak inhibitory activity. The distinction between the two types of UMM was greater in the non-crystal-seed system. The macromolecular inhibitors seemed to be composed of proteins or protein-complexes inasmuch as approximately 70 to 90 per cent of the macromolecular inhibitory activity was destroyed by protein digestion with pronase. Urinary acid glycosaminoglycans, which have been considered possible inhibitors, played a small role in the inhibition of crystal aggregation.

Adult↗
PubMedDate not supplied

The reaction front hypothesis in solid-state digestion: estimation of minimum size of viable seed body.

An alternative mechanism for the anaerobic digestion of a bed of solids was recently proposed. A multizoned reaction front might form around a suitable body of seed material, then advance through the bed, within the solid phase. Such a solid-phase mechanism might coexist with liquid-phase digestion but one or the other of these two parallel mechanisms might generally be dominant. It is envisaged that solid-phase digestion would be favored by the presence of viable seed bodies: single particles of a suitable seed material above a minimum size or equivalent aggregates. A key determinant of the feasibility of this mechanism is expected to be the minimum viable size for a seed body (dmin). This would depend on the thickness of the reaction zones, some of which must initially be accommodated within the seed body. In this article, we present some theoretical estimates of dmin, which indicate a magnitude of 7-700 mm. Such values suggest that solid-phase digestion might be the norm in semi-dry waste digesters. Such digestion might be rare in unseeded landfills but it would appear likely that it could easily be initiated by suitable seeding.

Bacteria, Anaerobic↗