Novel vitamin K-dependent pathways regulating cell survival

Historically, the vitamin K₁-dependent proteins have been associated primarily with blood coagulation and secondarily with bone formation. Recent identification of K₁-dependent proteins as specific ligands for the receptor tyrosine kinases (RTKs) that can stimulate cell replication and transformation and participate in cell survival highlighted a previously unrecognized and potentially important role for vitamin K₁ in cell signaling. Growing evidence suggests that most normal and tumor cells possess an active K₁-dependent -carboxylation mechanism necessary for the production of -carboxyglutamic acid (Gla)-containing proteins. Gla residues in proteins facilitate calcium-dependent protein/phospholipid interaction. Recent studies demonstrating the potentially positive effects of a vitamin K-dependent receptor:ligand system on cell growth and survival in general and the effects of the overexpression of these RTKs on malignant cell survival provide a new perspective on the role of vitamin K₁, its dependent protein ligands, and their receptors. These cumulative observations also provide an explanation for the rigidly controlled K₁ levels in the mammalian fetus and the minimal hepatic stores in the adult.     

Compression, compaction, and disintegration properties of low crystallinity celluloses produced using different agitation rates during their regeneration from phosphoric acid solutions

The tabletting characteristics of low crystallinity celluloses (LCPC)-LCPC-700, LCPC-2000, and LCPC-4000-prepared using agitation rates of 700, 2000, and 4000 rpm, respectively, during their regeneration from phosphoric acid, were evaluated and compared with those of Avicel PH-102 and Avicel PH-302. The mean deformation pressure values calculated from the linear region of the Athy-Heckel curves indicated LCPC-4000 to be the most ductile material. The area under the Athy-Heckel curve for LCPC-4000 was 330 MPa, whereas LCPC-700 and LCPC-2000 showed a corresponding value similar to that of Avicel PH-102 and Avicel PH-302 (192–232 MPa). The tensile strength of LCPC and Avicel compacts increased linearly with increasing applied pressures. A comparison of the area under the tensile strength-compression pressure curves indicated that LCPC-4000 formed the strongest tablets. The strengths of LCPC-700 and LCPC-2000 compacts, in contrast, were slightly lower than that of Avicel PH-302 and Avicel PH-102, respectively. The compacts of both LCPC-4000 and Avicel PH-102 were intact in water for 6 hours, whereas LCPC-2000 and Avicel PH-302 compacts disintegrated in 4 minutes and 2 minutes, respectively. In conclusion, LCPC-4000 was the most ductile material and exhibited the highest compression and compaction characteristics. The corresponding properties of LCPC-700 and LCPC-2000, in contrast, were comparable to that of Avicel PH-102 or Avicel PH-302.     

Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.     

Selection of Tn5::lacZ mutants isogenic to wild type Azospirillum brasilense strains capable of growing at sub-optimal temperature

Azospirillum brasilense strains, CDJA and A40, capable of growing at sub-optimal temperature were tagged with stable chromogenic marker Tn5-lacZ. Mutants were screened for plant growth promoting activities at 20, 22, 25, 30 and 37 °C. Mutants MC48 and MA3 were found to fix nitrogen upto 85% and produced indole acetic acid (IAA) and siderophore in isogenic manner to their respective wild type strains, CDJA and A40, at sub-optimal temperatures. Co-inoculation of mutants with their respective parent (1:1 ratio) to the wheat revealed that colonization potential of the mutants was affected greatly. Tn5-lacZ tagged mutants MC48 and MA3 were found isogenic to their respective wild type Azospirillum strain, with regards to plant growth promoting activities and root colonization ability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos')

 St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N′-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 μmol·l^–1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders. Received: 19 March 1999 / Revision received: 5 July 1999 · Accepted: 17 August 1999     

Genetic fidelity of organized meristem-derived micropropagated plants: A critical reappraisal

Summary The commercial multiplication of a large number of diverse plant species represents one of the major success stories of urilizing tissue culture technology profitably. Micropropagation has now become a multibillion dollar industry, practised all over the world. Of the various methods used to micropropagate plants, somatic embryogenesis and enhanced axillary branching have become the principal methods of multiplication. Long-term benefits of this enterprise, however, lie in the production of clonally uniform plants. The concept of genetic uniformity among micropropagated plants derived through organized meristems was exploded by several convincing reports of the incidence of somaclonal variation at morphological, cytological (chromosome number and structure), cytochemical (genome size), biochemical (proteins and isozymes), and molecular (nuclear and organellar genomes) levels. Somaclonal variation is not limited to any particular group of plants; it has been reported, for example, in ornamentals, plantation crops, vegetable and food crops, forest species and fruit trees. The upsurge of these reports, facilitated to a large extent by the technical developments made in molecular biology, is a matter of great concern for any micropropagation system. The economic consequences of somaclonal variation can be enormous in forest trees and woody plants, as they have long life cycles. Therefore, somaclonal variation has to be dispensed with if large-scale micropropagation of diverse plant species is to become not only successful but also accepted by end-users. In the light of the various factors (genotype, ploidy level, in vitro culture age, explant and culture type, etc.) that lead to somaclonal variation of divergent genetic changes at the cellular and molecular levels, genetic analysis of micropropagated plants using a multidisciplinary approach, especially at the DNA sequence level, initially and at various cultural stages, is essential. The results obtained at early multiplication stages from these tests could help in modifying the protocol/s for obtaining genetically true-to-type plants, and ultimate usage by entrepneneurs without any ambiguity.     

Generation of human class I major histocompatibility complex activating factor in serum free medium and its partial characterization

Human peripheral blood mononuclear cells (PBMCs) activated with Con-A release a soluble factor which augments the expression of class I major histocompatibility complex (MHC) antigens by a variety of tumour cells. Previous attempts to purify this factor called MHC-activating factor (AF) (MHC-AF) made us realize that the presence of large numbers and quantities of irrelevant fetal calf serum proteins in the culture supernatants of the activated human PBMCs, interfered with the purification procedure. It was therefore necessary to standardize the use of a serum free culture medium to generate human MHC-AF. In the present communication we have tried several types of culture media and have identified DCCM-2 as the most suitable culture medium to generate human MHC-AF. MHC-AF generated in DCCM-2 medium appears to be a protein molecule resistant to pH 2 treatment but sensitive to heat treatment (56°C × 45 min) and treatment with proteolytic enzymes trypsin and chymotrypsin.     

Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling

Lipoprotein lipase (LPL) hydrolyzes triglyceride in plasma lipoprotein primarily while bound to vascular endothelial cells. LPL metabolism by cultured endothelial cells was studied. Purified radioiodinated bovine LPL bound to porcine aortic endothelial cells at 4 degrees C with an association constant of 0.18 x 10(7) m-1. Analysis of the time course of LPL dissociation from endothelial cells at 4 degrees C yielded a dissociation rate constant of 3.9 x 10(-6)s-1. After 1 h at 37 degrees C, 28% of the LPL initially bound to the cell surface was no longer releasable by heparin or trypsin treatments, suggesting that LPL was internalized by the cells. Addition of heparin to the medium or pretreatment of the cells with heparinase markedly reduced the amount of LPL internalized, establishing a requirement for cell surface heparan sulfate proteoglycans in the process. When cells containing internalized LPL were incubated at 37 degrees C, a time-dependent increase in the amount of LPL in the medium and a corresponding decrease in LPL associated with the cells was found. This suggested that internalized LPL was released back into the medium. The catalytic activity, molecular size, and heparin-binding characteristics of the released LPL was similar to native LPL. Addition of either heparin, heparinase, or excess unlabeled LPL to prevent the rebinding of released 125I-LPL to the cell surface increased the amount of 125I-LPL present in the medium, suggesting that there is a process of recycling of 125I-LPL bound to the cell surface. Studies examining the effect of pH on dissociation of LPL from its binding site showed less dissociation of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5. These results suggest that even at acidic pH as in endocytotic vesicles, LPL remains bound to proteoglycans and this may facilitate the recycling of internalized LPL molecules.