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PubMed2000

A novel treatment for lupus nephritis: lignan precursor derived from flax.

BACKGROUND: Flaxseed has renoprotective effects in animal and human lupus nephritis. We have recently extracted the lignan precursor (secoisolariresinol diglucoside) (SDG) to determine if this more palatable derivative of flaxseed would exert renoprotection similar to the whole flaxseed in the aggressive MRL/lpr lupus mouse model. METHODS: 131 MRL/lpr mice were randomly assigned to saline gavage, 600, 1,200 and 4,800 microg lignan gavage groups. At 7 weeks, 6 animals underwent platelet aggregating factor (PAF) lethal challenge and 40 were studied with urine collection to determine the levels of secoisolariresinol, enterodiol and enterolactone in the gavaged animals. A baseline study of 10 saline gavaged animals took place at 6 weeks. 25 animals in the saline gavage, 600 and 1200 microg lignan groups were studied at 14 and 22 weeks for GFR, spleen lymphocyte S-phase and organ weight studies. RESULTS: Metabolic studies indicated that secoisolariresinol is the major metabolite absorbed and the lowest lignan dose provides a lengthening in survival for the PAF lethal challenge. Body weight, fluid and water intake studies demonstrated that the lignan was well tolerated. Changes in proteinuria, GFR and renal size showed a time- and dose-dependent protection for the lignan precursor. Cervical lymph node size and spleen lymphocyte cells in the S-phase demonstrated modest dose-dependent reductions in the lignan gavaged groups. CONCLUSION: SDG was converted in the gut to secoisolariresinol, which was absorbed and well tolerated by the MRL/lpr mice. Renoprotection was evidenced, in a dose-dependent fashion, by a significant delay in the onset of proteinuria with preservation in GFR and renal size. This study suggests that SDG may have a therapeutic role in lupus nephritis.

4-Butyrolactone↗
PubMed1993

Kinetics and thermodynamics of the mechanism of interaction of sodium phytate with alpha-globulin.

The precipitation mechanism of alpha-globulin in the presence of myo-inositol hexaphosphate (sodium phytate) was studied in detail. The maximum interaction was found at pH 2.3 where the protein was in a dissociated state having an 8.3S aggregate and a 1.5S monomer. This interaction was predominantly dependent upon the sodium phytate to protein ratio. Velocity sedimentation studies indicated polymer formation due to preferential progressive binding of ligand to polymer, whose size and concentration increased with an increase in sodium phytate concentration. The polymer formation was shown to be ligand mediated and exists independently in solution along with the monomer. The binding isotherm by equilibrium dialysis confirmed differential binding of sodium phytate to the polymer and the monomer as indicated by two sets of binding sites, one having 7 +/- 2 sites of a K value 1.3 x 10(-4) mol-1 and the other having 56 +/- 3 sites with a K value of 2.8 x 10(-3) mol-1. Binding resulted in perturbation of chromophores of protein due to charge effects. The kinetics of the polymer formation was shown to be a pseudo-first-order reaction having two steps. The initial fast reaction involving conformational changes has rate constants of k1 = 52.4 x 10(-3) s-1 and k' = 67.5 x 10(-3) s-1, followed by a slow reaction step of rate constants k2 = 4.3 x 10(-3) s-1 and k'2 = 2.9 x 10(-3) s-1 at sodium phytate concentrations of 1 x 10(-4) M and 5 x 10(-4) M, respectively.

Alpha-Globulins↗
PubMed2000

Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking.

Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented.

Models, Biological↗
PubMed1991

Advances in pharmacological studies of silymarin.

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.6% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-O-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular system have been studied in this university since 1980. P. O silymarin 800 mg/kg/d or silybin 600 mg/kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured "on top" and "inside" different extracellular matrix substitutes.

An essentially pure population of bile ductular epithelial cells isolated from bile duct-ligated rats was placed in primary culture by plating the cells either "on top" of or "inside" different extracellular matrix substitutes, including basement membrane Matrigel, type I collagen gel, and agarose gel. Plating efficiencies of greater than 60% were obtained when the cells were seeded in the presence of 1.0% fetal calf serum on top of Matrigel and collagen gel, but there was very little if any cell attachment to the agarose surface. In contrast, the cells could be maintained equally well and at very similar densities when they were cultured inside the various gel substances, including agarose. Regardless of substratum condition, bile ductular cells at 10 days in primary culture expressed specific activities of the marker enzymes gamma-glutamyl transpeptidase and leucine aminopeptidase which were significantly higher than those shown by freshly isolated cells. On the other hand, alkaline phosphatase activity of the cells became undetectable by day 3 of culture when they were cultured on top of either Matrigel or collagen gel but was retained at approximately 50% of its original level in cells cultured for 10 days within Matrigel or agarose gel. Treatments with dexamethasone or hydrocortisone (i.e., 10(-6) M) inhibited the increase in gamma-glutamyl transpeptidase activity of the cultured cells but did not affect the other enzyme changes. Subcultures of the bile ductular epithelial cells were developed by passing the cells in the presence of 10% fetal calf serum on surfaces coated with either type I collagen or Matrigel. In either case, cells subjected to at least 4-6 passages (up to 100 days of culture) were still characterized by a high gamma-glutamyl transpeptidase activity. Preliminary results obtained with cells plated at very low density within Matrigel also indicated the development of cell growths that appeared to be organized in the form of distinct acinar-like structures.

Animals↗
PubMedDate not supplied

Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system.

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.

Cartilage, Articular↗