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PubMedDate not supplied

Development of cell-seeded patellar tendon allografts for anterior cruciate ligament reconstruction.

Patellar tendon (PT) allografts for anterior cruciate ligament (ACL) reconstruction are potentially immunogenic and incorporate slowly compared with autografts. Our tissue-engineering approach to improve allograft efficacy is to (1) remove intrinsic cells from the graft to reduce antigenicity and then (2) seed the graft with extrinsic cells to improve ligamentization. To remove cells, tendons were soaked in 1% extraction solutions of tri(n-butyl)phosphate (TBP) or sodium dodecyl sulfate (SDS) for various time periods (24-72 h) and rinsed exhaustively. After treatment, we measured tendon cellularity, crimp structure, and mechanical properties. Treatment with either SDS or TBP removed approximately 70-90% of the intrinsic PT cells. Mechanical properties of treated PTs were similar to those of controls, despite changes in appearance. TBP- and SDS-treated PTs were then seeded with fibroblasts and cultured for up to 2 weeks in vitro. Fibroblast proliferation was retarded on SDS-treated PTs; in contrast, TBP-treated PTs supported cell proliferation similar to that of untreated controls. Extrinsic fibroblasts were successfully cultured on the TBP-treated PTs in vitro, creating viable tissue-engineered grafts potentially useful for ACL reconstruction. These modified allografts have the potential to be developed into mechanically functional delivery vehicles for cells, gene therapy vectors, or other biological agents.

Animals↗
PubMed2001

Brassicaceae express multiple isoforms of biotin carboxyl carrier protein in a tissue-specific manner.

Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity.

Acetyl-CoA Carboxylase↗
PubMed1984

Molecular cloning and analysis of a gene coding for the Bowman-Birk protease inhibitor in soybean.

We have constructed cDNA clones from size-selected mRNA of developing soybean seeds to identify genes encoding the Bowman-Birk protease inhibitor. Recombinant clones containing sequences coding for the Bowman-Birk inhibitor were identified by mRNA hybrid selected translation and immunoprecipitation. The nucleotide sequence of the insert of plasmid pB38 corresponds to the mRNA for the Bowman-Birk inhibitor, although it is missing a portion of the coding sequence at the 5' end. Northern hybridization analysis of total RNA isolated from developing soybean seeds showed that the mRNA for the inhibitor is approximately 450 nucleotides long and that it accumulates early in embryogeny. Southern hybridization analysis of restriction enzyme-digested soybean nuclear DNA indicated that the gene encoding the Bowman-Birk inhibitor is not highly reiterated in the genome. We have isolated a gene encoding the Bowman-Birk inhibitor from a soybean genomic library constructed in Charon 4A. DNA sequence analysis of the genomic clone reveals that it is similar, although not identical, to the cDNA clone and that it contains no intervening sequences.

Amino Acid Sequence↗
PubMed2003

In vitro distribution and characterization of membrane-associated PLD and PI-PLC in Brassica napus.

Two types of phospholipid degrading enzyme, phospholipase D (PLD; EC 3.1.4.4) and phosphatidyl- inositol-specific phospholipase C (PIP(2)-PLC; PI-PLC 3.1.4.11) were studied during the development of seeds and plants of Brassica napus. PLD exhibits two types of activity; polyphosphoinositide-requiring (PIP(2)-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLDalpha). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP(2)-dependent PLD activity shows the opposite trend when compared to PLDalpha, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLDalpha. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca(2+), pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLDalpha with G-proteins under in vitro conditions was not confirmed.

Brassica napus↗
PubMed1999

[Seed germinative characteristics and embryo development of Schisandra chinensis (Turcz.) Bail].

OBJECTIVE: To detect the germinative character of S. chinensis seeds. METHOD: Using low temperature and gibberellin treatment. RESULT: The breach rate of seeds was increased by both low temperature and gibberellin, but the breach on different seeds appeared untidy, which showing a character. The embryo development is normal before breaching and increased in volume rapidly after breaching. CONCLUSION: The bypoplasia of embryos is not the primary reason for long dormancy of the seeds.

Plants, Medicinal↗
PubMed2003

Muscle-derived stem cells seeded into acellular scaffolds develop calcium-dependent contractile activity that is modulated by nicotinic receptors.

OBJECTIVES: To explore the contractile activity and physiologic properties of muscle-derived stem cells (MDSCs) incorporated into small intestinal submucosa (SIS) scaffolds. METHODS: MDSCs were harvested from mice hind leg muscles using the preplate technique and stably transfected with a plasmid to express the LacZ reporter gene. Fifty different preparations of SIS cultured with MDSCs (MDSC/SIS) or SIS alone were incubated at 37 degrees C for 1, 4, and 8 weeks and also were mounted in a bath to measure the isometric contractions. RESULTS: LacZ and Masson-trichrome staining revealed MDSCs could migrate into and distribute throughout the SIS and form myotubes. In MDSC/SIS, spontaneous contractile activities were noted in the 4-week (five of six specimens) and 8-week (eight of eight specimens) cultures, but not in 1-week cultures (n = 11). All SIS control groups after 1 (n = 11), 4 (n = 6), and 8 (n = 8) weeks of incubation did not show any activity. In most of the 4-week, and all of the 8-week, MDSC/SIS cultures, the frequency and amplitude of spontaneous contractile activities were decreased by succinylcholine 10 microM and 20 microM. Electrical field stimulation, carbachol, and KCl did not alter the frequency, amplitude, or pattern of spontaneous contractile activities in MDSC/SIS. Spontaneous contractile activities were blocked by Ca(32+)-free Krebs solution with ethyleneglycoltetraacetic acid 200 microM and distilled water. CONCLUSIONS: MDSCs could be incorporated into SIS-forming myotubes capable of contracting. The contractile activity of this three-dimensional construct is Ca(2+) dependent and is modulated by nicotinic receptors. MDSC seeding of an acellular matrix may become a functional sling to reengineer the deficient sphincter or as contractile bladder augmentation.

Animals↗
PubMed1990

The inhibitory effect of neonatal rat smooth muscle cells and elastic extracellular matrix on development of the newly seeded cell population in culture.

Neonatal smooth muscle cells were seeded in standard plastic Falcon flasks, on top of another 2-month-old culture of the same cell population or on top of an acellular matrix prepared by removal of these cells. The effect of both complete and acellular layers on the production of elastin, collagen and total extracellular matrix (EM) proteins as well as on cell division was measured. Compared with the standard population grown on plastic, the complete cell layer almost completely prevented the newly seeded cells from dividing. The acellular matrix did not affect cell doubling but caused a distinct decrease in the production of EM components.

Animals↗
PubMed1977

Delayed conversion of squalene to sterols during development of Pinus pinea seeds.

During germination of seeds of the gymnosperm, Pinus pinea, radioactivity from [2-14C]-mevalonate proceeded principally through the anaerobic reactions leading to squalene in the first 24 hr in both the haploid endosperm and the diploid embryo, and only with succeeding time (3-9 days) in both cases was label transferred to sterols in oxygen-requiring steps. The rates of turnover must be real and independent in the two tissues, since no consequential interchange of labelled lipids occurred between the endosperm and the embryo. Similar delayed conversion of squalene to sterols has been observed previously during germination of seeds of the angiosperm, Pisum sativum.

Female↗
PubMed1996

In vivo endoproteolytically cleaved phaseolin is stable and accumulates in developing Phaseolus lunatus L. seeds.

Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present - in residual amounts - in the nature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.

Amino Acid Sequence↗
PubMed2000

Control of plant growth and development through manipulation of cell-cycle genes.

The plant embryo is a relatively simple structure consisting of a primordial shoot and root, whose development is frozen in the form of a seed. Most development of the mature plant takes place post-embryonically, and is the consequence of cell division and organogenesis in small regions known as meristems, which originate in the embryonic shoot and root apices. Significant recent progress has been made in understanding the mechanisms that control the plant cell cycle at a molecular level, and the first attempts have been made to control plant growth through modulation of cell-cycle genes. These results suggest that there is significant potential to control plant growth and architecture through manipulation of cell division rates. However, a full realisation of the promise of such strategies will probably require a much greater understanding of cell division control and how its upstream regulation is co-ordinated by spatial relationships between cells and by environmental signals.

Cell Cycle↗
PubMed1984

Intracellular transport and posttranslational cleavage of oat globulin precursors.

The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The alpha and beta globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The alpha and beta subunits appeared during the same chase period and assembled into a 12S oligomer.The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the alpha and beta subunits forming a 12S oligomer.

Journal Article↗
PubMed1996

A long-term culture system for the expansion of hematopoietic stem cells from embryonic yolk sac with the capacity to seed erythroid and lymphoid development in vitro and to reconstitute the lymphoid compartment in severe combined immunodeficient mice.

We have established a long-term culture system under which hematopoietic stem cells derived from the embryonic yolk sac may be maintained for long periods. Evidence for the persistence of stem cells in this culture is provided by three experimental observations. First, erythroid progenitors, as evidenced by morphological, functional, and phenotypical analysis, may be generated from these yolk sac cultures after more than 7 months in culture. The yolk sac derived erythroid progenitors are distinct from those of bone marrow derived cells both qualitatively and quantitatively. This is evidenced by the high colony plating efficiency, large colony size, different growth factor requirements, increased sensitivity to Epo and other cytokines, as well as significant and prolonged expansion capability, suggesting that the yolk sac derived progenitors are both more proliferative and more primitive than their bone marrow derived analogs. Second, under different conditions, lymphoid progenitors may also be derived from these long term yolk sac cultures in the presence of the appropriate cytokines. Third, preliminary data suggest the engraftment of these yolk sac cells and reconstitution of at least some compartments of the hematopoietic system of host animals. This long-term culture system will provide a useful model for the study of early embryonic hematopoiesis, and the cells derived from this culture system may also have the potential of serving as donor cells for hematopoietic cell transplantation.

Animals↗