Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.     

Inhibition of farnesylation blocks growth but not differentiation in FRTL-5 thyroid cells.

The cholesterol lowering drug lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, blocks DNA synthesis and proliferation of thyrotropin (TSH) primed FRTL-5 rat thyroid cells. The blockade can be completely prevented and/or reversed by mevalonate and largely prevented and/or reversed by farnesol whereas cholesterol and/or other non-sterol mevalonate derivatives such as ubiquinone, dolichol or isopentenyladenosine are ineffective. TSH-dependent augmentation of cyclic-AMP and cAMP dependent differentiated functions, such as iodide uptake, are unaffected by lovastatin. 3H-Thymidine incorporation into DNA is also decreased by alpha-hydroxyfarnesyl-phosphonic acid, an inhibitor of protein farnesylation which mimicks the effect of lovastatin since it also leaves unaffected TSH stimulated iodide uptake. It is suggested that the HMG-CoA reductase inhibitor lovastatin affects cell proliferation mainly through inhibition of protein farnesylation which results in altered function proteins relevant for proliferation control, notably p21ras and/or other small GTPases.     

Pharmakogenetik der oralen Antikoagulation mit Cumarinen

The recent identification of VKORC1 has made important contributions to our understanding of the vitamin K cycle. The VKORC1 enzyme was shown to be the molecular target of coumarin drugs. Mutations and polymorphisms in coding and noncoding regions of the VKORC1 gene have been shown to cause both a partial to total coumarin resistance and coumarin sensitivity. Availability of molecular diagnostics (VKORC1, CYP2C9) and drug monitoring by HCPLC (determination of coumarin, vitamin K, and vitamin K epoxide levels) is helpful for detecting hereditary and acquired factors influencing coumarin therapy. In the future, these tools may be instrumental in designing individualized oral anticoagulation therapy regimens.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Immunosuppressive activity of T cell clones generated from human T cells stimulated with autologous TPHA cells

T cell clones were generated from human T cells stimulated with autologous phytohemagglutinin (PHA)-activated T (TPHA) cells. Characterization of three T cell clones originated from donor SF and one from donor JM showed that they proliferated when stimulated with autologous TPHA cells, non-T cells, and peripheral blood mononuclear cells, but did not proliferate when stimulated with allogeneic TPHA cells, non-T cells, and mononuclear cells, with autologous and allogeneic resting T cells, and with PHA. These results in conjunction with the blocking of the proliferation by anti-histocompatibility leukocyte antigen class II monoclonal antibodies indicate that these class II antigens are involved in the proliferation of T cell clones stimulated with autologous lymphoid cells. The four T cell clones are cytotoxic neither to autologous lymphoid cells nor to a panel of cultured human cell lines. The four T cell clones display immunosuppressive activity, since they inhibit the proliferation of autologous and allogeneic cells stimulated with antigens and mitogens and the secretion of immunoglobulin by B cells stimulated with pokeweed mitogen in presence of T cells. Furthermore, the four T cell clones display differential inhibitory activity on the proliferation of cultured human cell lines. The immunosuppressive activity is species-specific, since the T cell clones do not inhibit the proliferation of murine cells. The suppression is mediated by a factor(s) with an apparent m.w. of 13,000 to 16,000. The suppressor activity is labile at alkaline pH and is lost following incubation with pronase (100 U/ml) for 30 min at 37 degrees C.     

Effect of dolichyl monophosphate on the permeability properties of lipid membranes

The effect of dolichyl monophosphate on the permeability properties of dimyristoylphosphatidylcholine bilayers to alkaline cations, Ca²⁺ and glucose has been determined by stop-flow spectrophotometry. The results show that, in con trast to free dolichol effects, the monophosphate derivative increased the permeability following a decreasing order of the permeating particle size. Phase diagrams indicate that dolichyl monophosphate is fully incorporated into the phosphatidylcholine bilayer around 0.75% weight/weight ratio. For these ratios, the permeation of ions is higher in the gel than in the liquid crystalline state.     

Purification and properties of the enzyme 6-phosphogluconate dehydrogenase from Dicentrarchus labrax L. liver

The enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ oxidoreductase, decarboxylating EC 1.1.1.44) from bass liver has been purified to over 95% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to about 100,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed to be a dimeric protein. The effect of pH and kinetic properties were studied.     

Effects of seed-irradiation on meiosis in M1 plants of Lactuca and related plants

Meiosis was studied in control and seed-irradiated materials of Lactuca sativa, cultivars Tom, Cos and Webbs, L. serriola from the wild, and Cichorium intybus. In meiosis of the M₁ plants from irradiated seed, observations of univalent and multivalent formation, bridge formation at Anaphase I and II, micronucleus formation and persistence, and pollen fertility, were carried out, and compared with controls. Quantitative estimations of bridge formation were made in L. sativa Tom and Cos and in L. serriola, at anaphase I only. It was found that the frequency of anaphase I bridges and reduction of pollen fertility in the M₁ and also M₂ generation, were different for each variety or species; these could be sequenced radiation-resistant to sensitive in the order Tom, Cos and L. serriola.     

Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.