The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Regenerating Sciatic Nerve Does Not Utilize Circulating Cholesterol

The rapid accumulation of myelin in the peripheral nervous system during the early postnatal period requires large amounts of cholesterol, a major myelin lipid. All of the cholesterol accumulating in the developing rat sciatic nerve is synthesized locally within the nerve, rather than being derived from the supply in lipoproteins in the systemic circulation (Jurevics and Morell, J. Lipid Res. 5:112–120; 1994). Since this lack of utilization of circulating cholesterol may relate to exclusion by the blood-nerve barrier, we examined the sources of cholesterol needed for regeneration following nerve injury, when the blood-nerve barrier is breached. One sciatic nerve was crushed or transected, and at various times later, the rate of cholesterol accumulation was compared with the rate of local in vivo synthesis of cholesterol within the nerve, utilizing intraperitoneally injected ³H₂O as precursor. The accumulation of additional cholesterol in nerve during regeneration and remyelination could all be accounted for by that locally synthesized within the nerve. There was also an increase in cholesterol esters in injured nerve segments; in crushed nerves, these levels decreased during regeneration and remyelination, consistent with reutilization of cholesterol originally salvaged by phagocytic macrophages and Schwann cells. Thus, regeneration and remyelination following injury in sciatic nerve utilizes both salvaged cholesterol and cholesterol synthesized locally within the nerve, but not cholesterol from the circulation.     

Prevention of aneuploidy by S-adenosyl-methionine in human cells treated with sodium arsenite

Alterations in methyl group's metabolism affect availability of S-adenosyl-L-methionine (SAM); these modifications can be originated by enzyme polymorphisms, nutritional deficiencies, and exposure to chemical agents. There are several types of chemicals that interfere with methyl groups, among them is arsenic. It deserves special attention because it modifies a number of cell functions that influence the development of diseases such as cancer. Since part of arsenic's toxicity is influenced by changes on SAM availability, in a previous study we investigated whether exogenous addition of SAM to cells treated with sodium arsenite (NaAsO(2)) has an effect on its genotoxicity. Results demonstrated that SAM reduces the frequency of cells presenting micronuclei (MN) and tubulin-cytoskeleton defects after treatment with NaAsO(2). MN are fragments of the cell nucleus that may contain whole chromosomes or chromosome fragments depending on whether they derive either from the aneugenic or from the clastogenic action of chemicals. Therefore one question generated by these results was whether SAM reduced only the frequency MN resulting from aneugenic damage. To answer this question, in the present work we used an all-centromere DNA probe to distinguish the type of MN reduced by SAM after treatment with NaAsO(2) and vinblastine. In addition, the capacity of SAM to reduce clastogenicity was also evaluated. Results show that SAM decreases the frequency of cells with MN containing whole chromosomes in cultures treated either with NaAsO(2) or with vinblastine; however, induction of double-strand breaks by NaAsO(2) was not prevented by SAM.     

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells

Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin™ is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin™ could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin™ were generated. Immunoblot experiments showed that Solulin™ accumulated to maximum levels of 115 and 27 μg g⁻¹ plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin™ was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin™ from the BY2 culture supernatant. The sequence was identical to that of Solulin™ produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.     

PCR Detection of Coxiella burnetii from Different Clinical Specimens, Especially Bovine Milk, on the Basis of DNA Preparation with a Silica Matrix

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.