Effect of the extracts from Glycyrrhiza uralensis Fisch on the growth characteristics of human cell lines: Anti-tumor and immune activation activities

Immune modulating activity of ethanol extracts from Glycyrrhiza uralensis Fisch was investigated by conserving growth characteristics of several human cell lines. All of the samples did not show severe cytotoxicity on normal human liver cell line, WRL-68, showing less than 25% inhibition of cell growth. The crude extract and its fractionized samples (F1 and F3) inhibited the growth of human hepatoma, Hep3B, down to ca. 70% of normal cell growth in adding 1.0 g l^-1 of fraction F3. The result of anticancer experiments was well matched to the results of antimutagenicity using Chinese Hamster Lung cell lines(CHL V79). In adding 1.0 g l^-1 of fraction F1, the growth of human B cell was enhanced, up to 60% of control growth. The secretion of two kinds of cytokines, Interleukin-6 and Tumor Necrosis Factor-α from human B cells was also enhanced in adding the crude extract, but not the standards such as Glycyrrhizin (GL) or 18,β-glycyrrhetinic acid (GM). It was found that both of the apoptosis and differentiation were more accelerated in supplementing the crude extract and fraction F1 than in adding the standards. A spot was found only in the crude extract and fractions, not standards by Thin Layer Chromatography(TLC) analysis. It tells that there must be another unknown component in crude and/or fraction F1 as a possible candidate of immune modulators. This component seems to be a derivative of a monomer, GM since its R_f was close to the monomer. It was also interesting that glycyrrhizin, a major component in G. uralensis Fisch was biologically activated by first being hydrolyzed by an enzyme. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ethnobotanical study and conservation status of trees in the district Sargodha,Punjab, Pakistan

Sargodha district is one of the least studied regions of Pakistan regarding its ethnobotanical values. This paper is the first report related to the documentation and conservation status of the tree species in the Sargodha district, and their folk ethnobotanical uses. An interview base survey was conducted in the study area in 2010-2013. The ethnobotanical data revealed the use of 100 tree species (6 gymnosperms, 94 angiosperms) belonging to 77 genera (6 gymnosperms, 71 angiosperms) and 39 families (4 gymnosperms, 35 angiosperms), with the Fabaceae ranking first with 19 tree species, followed by the Moraceae (12 species). Tree species like Aegle marmelos, Butea monosperma, Diospyrus malabarica, Gmelina arborea, Kigelia africana, Manilkara hexandra, Manilkara zapota, Mimusops elengi, Nyctanthes arbor-tristis, Putranjiva roxburghii, Terminalia arjuna and Terminalia bellerica are not only unique in their medicinal value but also interesting because of their unusual occurrence here. Thevetia peruviana, Cassia fistula, Celtis australis, Delonix regia, Diospyrus malabarica, Grevillea robusta, Haplophragma adenophylum, Jacaranda mimosifolia, Lagerstroemia speciosa, Plumeria rubra, Pterospermum acerifolium, Roystonea regia, Taxodium distichum and Tectona grandis are included among the worth looking ornamental tree species. Capparis decidua, Dalbergia sissoo, Tamarix aphylla, Tamarix dioica, Prosopis cineraria and Ziziphus mauritiana are the most commonly used timber species. Other common ethnobotanical utilization of these trees includes either sheltering or fuel or agricultural uses. Lack of awareness about the potential uses of these species, and particularly ignorance of the concerned authorities, have led to a decline in the population of this precious tree flora. Documentation of this tree flora, and as-sociated indigenous knowledge, can be used as a basis for developing management plans for conservation and sustainable use of this flora in the study area. A well-organized management is critical to restore and conserve this endangered natural resource in the District Sargodha, Pakistan. The immense medicinal and timber value of these tree species make it necessary to promote their conservation to simultaneously alleviate the poverty and improve the socio-economic status of the study area.     

Monitoring of lung tumour cell growth in artificial membranes

Morbidity of many tumour types is associated with invasion of tumour cells through the basement membrane and subsequent metastasis to vital organs. Tumour invasion is frequently detected late on as many patients present with advanced disease. The method of detecting invasion is through conventional histological staining techniques, which are time consuming and require processing of the sample. This can affect interpretation of the results. In this study, a new imaging technique, optical coherence tomography (OCT), was used to monitor lung tumour cell growth in two artificial membranes composed of either collagen type I or Matrigel. In parallel, standard histological section analysis was performed to validate the accuracy of the monitoring by OCT. Cross-sectional images from OCT revealed that lung tumour cells infiltrated only when low cell seeding density (5 x 10(5)) and low collagen concentration (1.5 mg/ml) were combined. The cells could be easily differentiated from the artificial membranes and appeared as either a brighter layer on the top of the membrane or brighter foci embedded within the darker membrane. These cell-membrane morphologies matched remarkably to the standard histological section images. Our results suggest that OCT has a great potential to become a useful tool for fast and robust imaging of cell growth in vivo and as a potential assessment of cell invasion.     

A conserved seven amino acid stretch important for murine mitochondrial glycerol-3-phosphate acyltransferase activity. Significance of arginine 318 in catalysis

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. We previously cloned the cDNA sequence to murine mitochondrial GPAT (Yet, S-F., Lee, S., Hahm, Y. T., and Sul, H.S. (1993) Biochemistry 32, 9486-9491). We expressed the protein in insect cells which was targeted to mitochondria, purified, and reconstituted mitochondrial GPAT activity using phospholipids (Yet, S.-F., Moon, Y., and Sul, H. S. (1995) Biochemistry 34, 7303-7310). Deletion of the seven amino acids from mitochondrial GPAT, (312)IFLEGTR(318), which is highly conserved among acyltransferases in glycerolipid biosynthesis, drastically reduced mitochondrial GPAT activity. Treatment of mitochondrial GPAT with arginine-modifying agents, phenylglyoxal and cyclohexanedione, inactivated the enzyme. Two highly conserved arginine residues, Arg-318, in the seven amino stretch, and Arg-278, were identified. Substitution of Arg-318 with either alanine, histidine, or lysine reduced the mitochondrial GPAT activity by over 90%. On the other hand, although substitution of Arg-278 with alanine and histidine decreased mitochondrial GPAT activity by 90%, replacement with lysine reduced activity by only 25%. A substitution of the nonconserved Arg-279 with either alanine, histidine, or lysine did not alter mitochondrial GPAT activity. Moreover, R278K mitochondrial GPAT still showed sensitivity to arginine-modifying agents, as in the case of wild-type mitochondrial GPAT. These results suggest that Arg-318 may be critical for mitochondrial GPAT activity, whereas Arg-278 can be replaced by a basic amino acid. Examination of the other conserved residues in the seven amino acid stretch revealed that Phe-313 and Glu-315 are also important, but conservative substitutions can partially maintain activity; substitution with alanine reduced activity by 83 and 72%, respectively, whereas substituting Phe-313 with tyrosine and Glu-315 with glutamine had even lesser effect. In addition, there was no change in fatty acyl-CoA selectivity. Kinetic analysis of the R318K and R318A mitochondrial GPAT showed an 89 and 95%, respectively, decrease in catalytic efficiency but no major change in substrate binding as indicated by the K(m) values for palmitoyl-CoA and glycerol 3-phosphate. These studies indicate importance of the conserved seven amino acid stretch for mitochondrial GPAT activity and the significance of Arg-318 for catalysis.     

Mice lacking paternally expressed Pref-1/Dlk1 display growth retardation and accelerated adiposity

Preadipocyte factor 1 (Pref-1/Dlk1) inhibits in vitro adipocyte differentiation and has been recently reported to be a paternally expressed imprinted gene at human chromosome 14q32. Studies on human chromosome 14 deletions and maternal uniparental disomy (mUPD) 14 suggest that misexpression of a yet-to-be-identified imprinted gene or genes present on chromosome 14 causes congenital disorders. We generated Pref-1 knockout mice to assess the role of Pref-1 in growth and in vivo adipogenesis and to determine the contribution of Pref-1 in mUPD. Pref-1-null mice display growth retardation, obesity, blepharophimosis, skeletal malformation, and increased serum lipid metabolites. Furthermore, the phenotypes observed in Pref-1-null mice are present in heterozygotes that harbor a paternally inherited, but not in those with a maternally inherited pref-1-null allele. Our results demonstrate that Pref-1 is indeed paternally expressed and is important for normal development and for homeostasis of adipose tissue mass. We also suggest that Pref-1 is responsible for most of the symptoms observed in mouse mUPD12 and human mUPD14. Pref-1-null mice may be a model for obesity and other pathologies of human mUPD14.     

Differential expression of proteins in rat plasma exposed to benzene

Benzene, a ubiquitous environmental contaminant, is an important solvent in the chemical industry and is also known as a constituent of petroleum. It has been reported that benzene is associated with hematotoxicity including leukemia in humans and cancer in laboratory animals. To study protein expression alterations in rat plasma exposed to benzene, rats were exposed to levels of 1, 10, 100 ppm benzine for 6 h/day and 5 d/week for 2 or 6 weeks. Two-dimensional gel electrophoresis of rat plasma was carried out, and approximately 1000 protein spots were detected on the gels. The 11 spots which showed significantly different expression were selected and identified with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. Analyzing the targeted 11 spots, there was no correlation between the 2 and 6 weeks benzene-inhaled groups on up-regulated proteins (zinc finger protein, and tristetraprolin) and on down-regulated proteins (cAMP-regulated guanine nucleotide exchange factor II, protein kinase and unknown protein). The overexpressed proteins (inhibitor of kappaB-like protein, GTP-binding protein rab14, T-cell receptor alpha chain, and somatostatin transactivating factor-1) were detected only in groups inhaling benzene for 6 weeks. Among them the expression level of T-cell receptor alpha chain was confirmed by Western blot.