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PubMed1999

Chondrocyte aggregation and reorganization into three-dimensional scaffolds.

Articular cartilage has a very limited self-repairing capacity; thus, chondral lesions normally result in chronic degeneration and, eventually, osteoarthritis development. Currently, tissue engineering offers a new tool for the clinical treatment of osteochondral defects. The present investigation aimed to develop an in vitro engineered cartilage using a new class of semisynthetic scaffolds. Two nonwoven meshes of hyaluronan esters (Hyaff(R) derivatives) were seeded with sternal chick embryo chondrocytes cultured for up to 21 days, after which time they were assessed for both the cellular growth profile and histological features. Avian chondrocytes easily adhered and proliferated onto hyaluronan-based scaffolds, demonstrating a significant preference for the fully esterified benzylic form. Histochemical staining revealed the presence of a neosynthesized glycosaminoglycan-rich extracellular matrix, and immunohistochemistry confirmed the deposition of collagen type II. Moreover, ultrastructural observations supported evidence that chondrocytes grown onto a hyaluronan-derived three-dimensional scaffold maintained their unique phenotype and organization in a cartilage-like extracellular matrix. These findings support the further pursuit of a transplantable engineered cartilage using human chondrocytes for the regeneration of chondral lesions.

Animals↗
PubMed2005

Behaviour of bone marrow osteoblast-like cells on mineral trioxide aggregate: morphology and expression of type I collagen and bone-related protein mRNAs.

AIM: To investigate the in vitro behaviour of rat bone marrow cells (RBM) on mineral trioxide aggregate (MTA) (ProRoot, MTA Root Canal Repair Material; Dentsply Tulsa, Tulsa, OK, USA) compared with intermediate restorative materials (IRM) (Dentsply Caulk, Milford, DE, USA). METHODOLOGY: RBM were obtained from rat femur and were primary cultured and then subcultured. Cells were then seeded on three dishes of each material, and cultured for 3 days, after which they were evaluated morphologically using scanning (SEM) and transmission (TEM) electron microscopy. Furthermore, the calcium released from hydrated material, the cell proliferation ratio and alkaline phosphatase (ALP) activity were analysed, and the expression of type I collagen and bone-related protein mRNAs were evaluated. The data were averaged and analysed via one-way analysis of variance (anova) and were then compared by the Scheffe's test. RESULTS: SEM showed that RBM attached to MTA and had a flattened appearance without nuclear protrusions and microspikes. TEM showed that the cells attached in the same manner as the control group, but gaps larger than 2 microm were frequently seen. The calcium released from hydrated MTA was about 130 ppm after 3 days of immersion in saline. The ALP activity was similar to the control group. Cell proliferation and expression of type I collagen mRNA was significantly lower, while the expression of osteopontin mRNA was significantly higher than the control group at the third day of culture. In IRM groups, a few rounded cells were observed on the material but no living cells were seen. CONCLUSIONS: MTA is a material of low toxicity which does not inhibit cell growth, but does suppress the differentiation of osteoblast-like cells.

Alkaline Phosphatase↗
PubMed1995

Soluble factors from the olfactory bulb attract olfactory Schwann cells.

Olfactory Schwann cells (OSCs) extend processes that ensheathe bundles of olfactory axons as they course from the olfactory epithelium to the olfactory bulb (OB). Results of morphological and immunohistochemical studies have led to speculation that OSCs may be involved in guiding the olfactory axons to their target tissue. In this study we have explored this possibility by investigating the relationship between OSCs and the OB. Olfactory Schwann cells labelled with 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were injected into the nasal region of E14 rat embryos and entire embryos were cultured for 24 hr. It was found in some embryos, that the OSCs had migrated toward the presumptive OB. Cocultures of neonatal OB explants on OSC monolayers showed that the OSCs were attracted to the OB and formed a ring-like aggregate around the explant after 48 hr culture. This attraction was absent when a piece of cerebrum was used in place of the OB. When medium conditioned by neonatal OBs was placed in the lower compartment of the chemotaxis chamber, OSCs seeded in the upper compartment migrated through the pores of the nucleopore filter to reach the underside which was in contact with the conditioned medium. After 6 hr of incubation, scanning electron microscopy was performed on the underside of the nucleopore filters. Cell counts of OSCs showed that the cell density was significantly higher when medium conditioned by OBs was used instead of unconditioned medium or medium conditioned by cerebrum. The results of these experiments show that the OSCs migrate toward the OB under the influence of soluble factor(s) secreted by the target tissue.

Animals↗
PubMed2001

Integrin alpha3beta1 engagement disrupts intercellular adhesion.

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.

Antibodies, Monoclonal↗
PubMed1981

Molecular properties of the enzymic phytohemagglutinin of mung bean.

Mung bean seeds possess a tetrameric galactose-binding protein that displays two types of activities: (a) a hemagglutinin activity, and (b) an alpha-galactosidase activity. This protein can be reversibly converted by pH changes from a tetrameric form, which possesses both enzymic and hemagglutinin activities, to a monomeric form which possesses enzymic activity only. This observation suggests that the enzymic phytohemagglutinin is an aggregated form of a monomeric alpha-galactosidase. The tetrameric alpha-galactosidase has a pH optimum of about pH 7.0, while the monomeric form displays a pH optimum of 5.6. Circular dichroism difference spectra and inhibition studies suggest that aggregation induces conformational changes in the subunits sufficient to alter their enzymatic properties. The possibility of in vivo changes in subunit equilibria, when combined with the accompanying alterations in activity, provides a new concept worthy of consideration with respect to the physiological role of phytohemagglutinins.

Calcium-Binding Proteins↗
PubMed2000

Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth.

Sialoproteins such as bone sialoprotein (BSP) and dentin sialoprotein (DSP) accumulate at the mineralization fronts in bone and dentin, respectively, suggesting they have some function in the mineralization process. BSP, a highly phosphorylated protein rich in polyglutamate repeats, is an effective nucleator of hydroxyapatite (HA) formation in vitro. The present study examines the effect of DSP, a low phosphorylated but related sialoprotein, on the formation and growth of HA. In vitro, in a gelatin gel diffusion system, DSP at low concentrations (<25 microg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50-100 microg/ml) it slightly inhibited accumulation. Fewer mineral crystals were formed in the presence of high concentrations of DSP but they tended to aggregate (making them appear larger by electron microscopic analysis) than those formed in DSP-free gels. X-ray diffraction line broadening analysis failed to show significant changes in c-axis crystal dimensions with increasing DSP concentration. When HA-seed crystals were coated with DSP before inclusion in the gelatin gel there was a reduction in mineral accumulation relative to HA-seeds which had not been coated with DSP, but the extent of inhibition was significantly less than that seen in this system with other mineralized tissue matrix sialoproteins, such as osteopontin or BSP. The low affinity of DSP for well-characterized seed crystals and the limited effect of this protein on HA formation and growth suggest that the role of DSP in dentin is not primarily that of a mineralization regulator.

Animals↗
PubMed1987

Plasma membrane glycoproteins covalently bound to silica beads as a model for molecular studies of cell-cell interactions in culture.

In previous studies, we have shown that plasma membrane glycoproteins are of major importance in the density-dependent regulation of growth of normal diploid fibroblasts. Due to the hydrophobic portions of these molecules, functional studies in cell culture are often difficult to perform and to interpret. Specifically, the addition of these molecules in soluble form to cell culture, after depletion of detergents needed for their solubilization, leads to aggregation and internalization. Therefore, we developed a method for the covalent immobilization of the solubilized plasma membrane proteins to derivatized silica beads for further investigations on the molecular nature of the active molecules. The addition of immobilized plasma membrane glycoproteins to sparsely seeded human fibroblasts resulted in cellular reactions similar to those found in confluent cell cultures (strongly reduced cell proliferation; high collagen type III synthesis). The method consists in the derivatization of silica beads (Lichrosphere Si 500, 10 microns) with isothiocyanatopropyltriethoxysilane. Amino-groups react with the SCN group under physiological conditions, resulting in a stable linkage of amino-group bearing molecules with the silica beads. Due to the easy handling of the silica beads (e.g. washing by short centrifugation steps), the mild coupling conditions, and the stable bondings this system is highly suited for functional studies of molecules involved in cell-cell interactions.

Cell Division↗
PubMed2004

In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc).

The "protein only" hypothesis postulates that the infectious agent of prion diseases, PrP(Sc), is composed of the prion protein (PrP) converted into an amyloid-specific conformation. However, cell-free conversion of the full-length PrP into the amyloid conformation has not been achieved. In an effort to understand the mechanism of PrP(Sc) formation, we developed a cell-free conversion system using recombinant mouse full-length PrP with an intact disulfide bond (rPrP). We demonstrate that rPrP will convert into the beta-sheet-rich oligomeric form at highly acidic pH (<5.5) and at high concentrations, while at slightly acidic or neutral pH (>5.5) it assembles into the amyloid form. As judged from electron microscopy, the amyloid form had a ribbon-like assembly composed of two non-twisted filaments. In contrast to the formation of the beta-oligomer, the conversion to the amyloid occurred at concentrations close to physiological and displayed key features of an autocatalytic process. Moreover, using a shortened rPrP consisting of 106 residues (rPrP 106, deletions: Delta23-88 and Delta141-176), we showed that the in vitro conversion mimicked a transmission barrier observed in vivo. Furthermore, the amyloid form displayed a remarkable resistance to proteinase K (PK) and produced a PK-resistant core identical with that of PrP(Sc). Fourier transform infrared spectroscopy analyses showed that the beta-sheet-rich core of the amyloid form remained intact upon PK-digestion and accounted for the extremely high thermal stability. Electron and real-time fluorescent microscopy revealed that proteolytic digestion induces either aggregation of the amyloid ribbons into large clumps or further assembly into fibrils composed of several ribbons. Fibrils composed of ribbons were very fragile and had a tendency to fragment into short pieces. Remarkably, the amyloid form treated with PK preserved high seeding activity. Our work supports the protein only hypothesis of prion propagation and demonstrates that formation of the amyloid form that recapitulates key physical properties of PrP(Sc) can be achieved in vitro in the absence of cellular factors or a PrP(Sc) template.

Amyloid↗
PubMed2003

Cowpea mosaic virus-based chimaeras. Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses.

Expression of foreign peptides on the surface of cowpea mosaic virus particles leads to the creation of chimaeras with a variety of phenotypes and yields. Two factors were shown to be particularly significant in determining the properties of a given chimaera: the length of the inserted sequence and its isoelectric point. The deleterious effect of high isoelectric point on the ability of chimeras to produce a systemic infection occurs irrespective of the site of insertion of the peptide. Ultrastructural analysis of tissue infected with chimaeras with different phenotypes showed that all produced particles with a tendency to aggregate, irrespective of the size or isoelectric point of the insert. Host range and transmission studies revealed that the expression of a foreign peptide did not (1) alter the virus host range, (2) increase the rate of transmission by beetles or through seed, or (3) change the insect vector specificity. These findings have implications for both the utility and the biosafety of Cowpea mosaic virus-based chimaeras.

Amino Acid Sequence↗
PubMed1994

Changes in regional pulmonary blood flow during lobar bronchial occlusion in man.

1. Acute hypoxic pulmonary vasoconstriction is important in the restoration of ventilation-perfusion balance in the presence of regional alveolar hypoventilation. However, the magnitude and time course of this response in man has not been adequately characterized in regions smaller than an entire lung. We have studied the effectiveness of hypoxic vasoconstriction in diverting blood from hypoxic lobes in normal supine subjects, and have documented the redistribution of pulmonary blood flow under these conditions. 2. Lobar hypoxia was induced for 80-300 s by placing occluding balloon-tipped catheters in lobar bronchi during fibreoptic bronchoscopy in 10 normal subjects. Respiratory gas partial pressures within occluded lobes were measured with a mass spectrometer. The percentage reduction in blood flow to the hypoxic lobes was assessed after injection of 99mTc-labelled albumin by gamma-scintigraphy, and compared with a control scan performed 1 week later. A computer program was used to analyse changes in regional pulmonary perfusion. 3. During lobar bronchial occlusion respiratory gas partial pressures rapidly approached reported values for mixed venous partial pressures. After a mean time of occlusion of 3.5 min lobar blood flow was reduced by 47 +/- 5%. During occlusions pulmonary blood flow was not evenly redistributed, but was preferentially redistributed to more cranial lung regions. 4. We conclude that acute hypoxic pulmonary vasoconstriction in occluded lobes is more effective at rapidly diverting pulmonary blood flow away from hypoxic lung regions than has previously been reported in man during unilateral hypoxia of an entire lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase.

Rice (Oryza sativa) accumulates prolamins and glutelins as storage proteins. The latter storage protein is synthesized on the endoplasmic reticulum (ER) as a 57-kD proglutelin precursor, which is then processed into acidic and basic subunits in the protein storage vacuole. Three esp2 mutants, CM1787, EM44, and EM747, contain larger amounts of the 57-kD polypeptide and corresponding lower levels of acidic and basic glutelin subunits than normal. Electron microscopic observation revealed that esp2 contained normal-appearing glutelin-containing protein bodies (PB-II), but lacked the normal prolamin-containing PB (PB-I). Instead, numerous small ER-derived PBs of uniform size (0.5 microm in diameter) and low electron density were readily observed. Immunoblot analysis of purified subcellular fractions and immunocytochemistry at the electron microscopy level showed that these new PBs contained the 57-kD proglutelin precursor and prolamin polypeptides. The 57-kD proglutelin was extracted with 1% (v/v) lactic acid solution only after removal of cysteine-rich prolamin polypeptides, suggesting that these proteins form glutelin-prolamin aggregates via interchain disulfide bonds within the ER lumen. The endosperm of esp2 mutants contains the lumenal chaperones, binding protein and calnexin, but lacks protein disulfide isomerase (PDI) at the protein and RNA levels. The transcript of PDI was expressed in the seed only during the early stage of seed development in the wild type. These results suggest that PDI plays an essential role in the segregation of proglutelin and prolamin polypeptides within the ER lumen.

Blotting, Northern↗
PubMed2004

Heterogeneous genetic structure in a Fagus crenata population in an old-growth beech forest revealed by microsatellite markers.

The within-population genetic structure of Fagus crenata in a 4-ha plot (200 x 200 m) of an old-growth beech forest was analysed using microsatellite markers. To assess the genetic structure, Moran's I spatial autocorrelation coefficient was calculated. Correlograms of Moran's I showed significant positive values less than 0.100 for short-distance classes, indicating weak genetic structure. The genetic structure within the population is created by limited seed dispersal, and is probably weakened by overlapping seed shadow, secondary seed dispersal, extensive pollen flow and the thinning process. Genetic structure was detected in a western subplot of 50 x 200 m with immature soils and almost no dwarf bamboos (Sasa spp.), where small and intermediate-sized individuals were distributed in aggregations with high density because of successful regeneration. By contrast, genetic structure was not found in an eastern subplot of the same size with mature soils and Sasa cover, where successful regeneration was prevented, and the density of the small and intermediate-sized individuals was low. Moreover, genetic structure of individuals in a small-size class (diameter at breast height < 12 cm) was more obvious than in a large-size class (diameter at breast height >/= 12 cm). The apparent genetic structure detected in the 4-ha plot was therefore probably the result of the structure in the western portion of the plot and in small and intermediate-sized individuals that successfully regenerated under the favourable environment. The heterogeneity in genetic structure presumably reflects variation in the density that should be affected by differences in regeneration dynamics associated with heterogeneity in environmental conditions.

Demography↗
PubMed1992

Effect of polyethylene glycol-400 at low concentrations on long-term growth of muscle phosphoglucomutase crystals from concentrated salt solutions.

Although rabbit muscle phosphoglucomutase occasionally deposits tetragonal crystals from solutions of ammonium sulfate at about 47% of saturation, low concentrations of polyethylene glycol-400 (PEG), 1 to 4.5% w/v, must be included to sustain crystal growth. A comparison of long-term growth rates for macroscopic crystals in the presence and absence of added PEG suggests that at high salt concentration this cosolute exerts its primary effect on disordered protein aggregates, either in the external medium or at the surface of the crystal, and thereby allows the growth of much larger crystals. Since the observed effects may arise from a PEG-induced increase in the "solubility" of the aggregate that exceeds the induced increase in solubility of the crystalline phase under these conditions, the physical basis for a cosolute-induced increase in solubility in the presence of a precipitant is considered. The applicability of such a rationale to the present system is supported by an assessment of the relative effects of polyethylene glycol and beta-octylglucoside on amorphous, salt-induced precipitates of phosphoglucomutase. PEG also produces what appears to be a differential effect on nucleation efficiency and crystal growth rate. Thus, seed crystals cannot be enlarged at a significant rate at high salt concentration without producing showers of extraneous nucleation centers when the concentration of added PEG is 3% or less. But PEG concentrations of 4.5% essentially eliminate the showering problem, ostensibly by increasing the supersaturation required for nucleation to a greater extent than that required for crystal growth. The same type of effect is observed during de novo growth. Again a solubility-based mechanism is posed. Hysteretic effects related to properties of amorphous aggregates of the protein also are described.

Ammonium Sulfate↗
PubMed1979

Methods of study of the invasion of malignant C3H-mouse fibroblasts into embryonic chick heart in vitro.

Methods of study of tumour invasiveness in vitro were investigated using the interactions between malignant virally transformed C3H mouse fibroblasts (MO4) with fragments from embryonic chick heart. Invasion of MO4 cells into the heart tissue could be demonstrated in all three-dimensional cultures. On the contrary, seeding of MO4 cells on top of a monolayer of heart cells did not mimic invasion. Precultured heart fragments were more suitable for the study of the early phase of invasion than freshly cut ones because they presented healthy well-delineated borders. Aggregates of MO4 cells proved more suitable than suspensions or monolayer fragments because they were amenable to quantitation, were not traumatized or treated with enzymes during harvest. They allowed precise control of the initial site of contact with the heart tissue. The nature of the culture medium predominantly affected the growth of the MO4 population. This factor influenced the pattern of invasion quantitatively, but did not alter the invasive capacity of the MO4 cells itself. In cultures on adhesive substrates the interaction between the MO4 cells and the heart tissue was complicated by the fact that both also interacted with the aritficial substrate. Shaker cultures appeared better than comparable static cultures because they allowed better aeration and so delayed central necrosis.

Animals↗
PubMed2006

Tuning amyloidogenic conformations through cosolvents and hydrostatic pressure: when the soft matter becomes even softer.

Compact packing, burial of hydrophobic side-chains, and low free energy levels of folded conformations contribute to stability of native proteins. Essentially, the same factors are implicated in an even higher stability of mature amyloid fibrils. Although both native insulin and insulin amyloid are resistant to high pressure and influence of cosolvents, intermediate aggregation-prone conformations are susceptible to either condition. Consequently, insulin fibrillation may be tuned under hydrostatic pressure or-- through cosolvents and cosolutes-- by preferential exclusion or binding. Paradoxically, under high pressure, which generally disfavors aggregation of insulin, an alternative "low-volume" aggregation pathway, which leads to unique circular amyloid is permitted. Likewise, cosolvents are capable of preventing, or altering amyloidogenesis of insulin. As a result of cosolvent-induced perturbation, distinct conformational variants of fibrils are formed. Such variants, when used as templates for seeding daughter generations, reproduce initial folding patterns regardless of environmental biases. By the close analogy, this suggests that the "prion strains" phenomenon may mirror a generic, common feature in amyloids. The susceptibility of amyloidogenic conformations to pressure and cosolvents is likely to arise from their "frustration", as unfolding results in less-densely packed side-chains, void volumes, and exposure of hydrophobic groups. The effects of cosolvents and pressure are discussed in the context of studies on other amyloidogenic protein models, amyloid polymorphism, and "strains".

Amyloid↗
PubMed2005

Molecular properties of food allergens.

Plant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators.

Allergens↗
PubMed1979

Changes in immunoferritin labeling of Rickettsia tsutsugamushi after serial cultivation in 60Co-irradiated BHK cells.

The immunolabeling characteristics of Rickettsia tsutsugamushi (Gilliam strain) were examined by using a purified immunoglobulin G fraction of antibody to R. tsutsugamushi raised in rabbits. Formalin-fixed rickettsiae were reacted with this antibody and then with ferritin-conjugated goat anti-rabbit Fc antibody. R. tsutsugamushi cultivated in yolk sacs was used to raise antibody for this study. When rickettsiae in BHK-21 cells infected from yolk sac seed material were immunoferritin labeled, the binding of ferritin was found to be dense and uniform on the outer surface of the rickettsiae in disrupted host cells. Immunolabeling of purified suspensions of extracellular rickettsiae resulted in the uniform ferritin labeling of the microorganism. Aggregation of these rickettsiae by antibody appeared to depend upon the purity of the pellets. Immunoferritin labeling examined at high magnification revealed ferritin very close to the outer dense leaflet of the outer membrane. On some rickettsiae or on focal sites of others, the labelin; was several ferritin particles thick, suggesting the presence of a thick coating. The immunoferritin labeling of R. tsutsugamushi during successive serial passages in BHK-21 cells revealed decreased labeling with each passage, and by the 10th passage there was no detectable labeling. However, these rickettsiae inoculated back into yolk sacs regained their immunoferritin labeling. R. tsutsugamushi passed back into yolk sacs after four serial propagations in BHK-21 cells regained their labeling on the first passage in yolk sacs. However, rickettsiae from the 20th serial passage in BHK-21 cells required five passages in yolk sacs to reestablish their previous labeling affinity. Rickettsiae which did not label after 20 passages in BHK cells regained some of their labeling characteristics when sonicated. Antibody against rickettsiae cultivated in BHK-21 cells continued labeling rickettsiae even after 9 serial passages in BHK-21 cells.

Animals↗
PubMed1986

Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds.

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.

Acyltransferases↗