Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.     

Antisense expression and overexpression of biotin carboxylase in tobacco leaves.

The plastid acetyl-coenzyme A carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis and in most plants is present as a heteromeric complex of at least four different protein subunits: the biotin carboxylase (BC), the biotin carboxyl carrier protein, and the alpha and beta subunits of the carboxyltransferase. To gain insight into the subunit organization of this heteromeric enzyme complex and to further evaluate the role of ACCase in regulating fatty acid synthesis, BC expression was altered in transgenic plants. Tobacco (Nicotiana tabacum) was transformed with antisense-expression and overexpression tobacco BC constructs, which resulted in the generation of plants with BC levels ranging from 20 to 500% of wild-type levels. Tobacco plants containing elevated or moderate decreases in leaf BC were phenotypically indistinguishable from wild-type plants. However, plants with less than 25% of wild-type BC levels showed severely retarded growth when grown under low-light conditions and a 26% lower leaf fatty acid content than wild-type plants. A comparison of leaf BC and biotin carboxyl carrier protein levels in plants with elevated and decreased BC expression revealed that these two subunits of the plastid ACCase are not maintained in a strict stoichiometric ratio.     

Chlamydomonas reinhardtii Phosphoribulokinase : Sequence, Purification, and Kinetics

The sequence and kinetic properties of phosphoribulokinase purified from Chlamydomonas reinhardtii were determined and compared with the spinach (Spinacea oleracea) enzyme. Chlamydomonas phosphoribulokinase was purified to apparent homogeneity, with a specific activity of 410 micromoles per minute per milligram. Polyclonal antibodies to the purified protein were used to isolate a Chlamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. The transit peptide cleavage site was determined by Edman analysis of the mature protein. The precursor protein consists of a 31 amino acid transit peptide and a 344 amino acid mature polypeptide. The mature polypeptide has a calculated molecular weight of 38.5 kilodaltons and a pl of 5.75. The V_max of the purified enzyme was 465 micromoles per minute per milligram, with apparent K_m values of 62 micromolar ATP and 56 micromolar ribulose 5-phosphate. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Southern blot analysis of Chlamydomonas genomic DNA indicated the presence of a single phosphoribulokinase gene. Comparison of the mature proteins from Chlamydomonas and spinach revealed 86 amino acid differences in primary structure (25% of the total) without a major difference in kinetic properties. The transit peptides of the spinach and Chlamydomonas proteins possessed little sequence homology.     

Field Evaluation of Alternative Earthen Final Covers

Five methods to assess percolation rate from alternative earthen final covers (AEFCs) are described in the context of the precision with which the percolation rate can be estimated: trend analysis, tracer methods, water balance method, Darcy's Law calculations, and lysimetry. Trend evaluation of water content data is the least precise method because it cannot be used alone to assess the percolation rate. The precision of percolation rates estimated using tracer methods depends on the tracer concentration, percolation rate, and the sensitivity of the chemical extraction and analysis methods. Percolation rates determined using the water balance method have a precision of approximately 100 mm/yr in humid climates and 50 mm/yr in semiarid and drier climates, which is too large to demonstrate that an AEFC is meeting typical equivalency criterion (30 mm/yr or less). In most cases, the precision will be much poorer. Percolation rates computed using Darcy's Law with measured profiles of water content and matric suction typically have a precision that is about two orders of magnitude (or more) greater than the computed percolation rate. The Darcy's Law method can only be used for performance assessment if the estimated percolation rate is much smaller than the equivalency criterion and preferential flow is not present. Lysimetry provides the most precise estimates of percolation rate, but the precision depends on the method used to measure the collected water. The lysimeter used in the Alternative Cover Assessment Program (ACAP), which is described in this paper, can be used to estimate percolation rates with a precision between 0.00004 to 0.5 mm/yr, depending on the measurement method and the flow rates.     

Gastrin-Releasing Peptide Receptor Knockdown Induces Senescence in Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, characterized by excessive cell proliferation, resistance to apoptosis, and invasiveness. Due to resistance to currently available treatment options, the prognosis for patients with GBM is very dismal. The activation of gastrin-releasing peptide receptors (GRPR) stimulates GBM cell proliferation, whereas GRPR antagonists induce antiproliferative effects in in vitro and in vivo experimental models of GBM. However, the role of GRPR in regulating other aspects of GBM cell function related to tumor progression remains poorly understood, and previous studies have not used RNA interference techniques as tools to examine GRPR function in GBM. Here, we found that stable GRPR knockdown by a lentiviral vector using a short hairpin interfering RNA sequence in human A172 GBM cells resulted in increased cell size and altered cell cycle dynamics consistent with cell senescence. These changes were accompanied by increases in the content of p53, p21, and p16, activation of epidermal growth factor receptors (EGFR), and a reduction in p38 content. These results increase our understanding of GRPR regulation of GBM cells and further support that GRPR may be a relevant therapeutic target in GBM.     

Dexamethasone reverses the memory impairment induced by antagonism of hippocampal gastrin-releasing peptide receptors

Storage of emotionally influenced memory is regulated by activation of glucocorticoid receptors (GRs) as well as of gastrin-releasing peptide receptors (GRPRs) in the dorsal hippocampus. In the present study, male Wistar rats were given a bilateral infusion of saline or the GRPR antagonist (D-Tpi6, Leu13 psi[CH2NH]-Leu14) bombesin (6-14) (RC-3095) (1.0 microg/side) into the dorsal hippocampus 10 min before training on an inhibitory avoidance task, followed by an immediate post-training i.p. injection of vehicle or the GR agonist dexamethasone (0.3 mg/kg). A retention test trial, carried out 24 h after training, indicated that intrahippocampal infusion of RC-3095 impaired inhibitory avoidance retention. Post-training administration of dexamethasone induced an enhancement of retention regardless of whether the animals had received saline or RC-3095 into the hippocampus before training. The findings indicate that hippocampal GRPR blockade does not prevent memory enhancement induced by dexamethasone. Together with previous results, these findings suggest that endogenous activation of GRPRs in the hippocampus modulates the consolidation of emotional memory, but is not a critical receptor system mediating memory formation.     

Non-associative learning and anxiety in rats treated with a single systemic administration of the gastrin-releasing peptide receptor antagonist RC-3095

The gastrin-releasing peptide receptor (GRPR) has been implicated in the modulation of emotionally-motivated memory. In the present study, we investigated the role of the GRPR on non-emotional, non-associative memory, and anxiety. Adult male Wistar rats were given a systemic injection of the GRPR antagonist [D-Tpi⁶, Leu¹³ psi(CH₂NH)-Leu¹⁴] bombesin (6–14) (RC-3095) (0.2, 1.0 or 5.0 mg/kg) 30 min before exposure to an open field or an elevated plus maze. Habituation to the open field was tested in a retention trial carried out 24 h after the first exposure to the open field. Rats given RC-3095 at the doses of 1.0 or 5.0 mg/kg showed impaired habituation. Animals treated with 5.0 mg/kg of RC-3095 spent significantly more time in the closed arms of the elevated plus maze. No effects of RC-3095 on locomotion or exploratory behavior were observed. The results implicate the GRPR in the regulation of non-emotional, non-associative memory as well as in anxiety.     

Intrahippocampal infusion of the bombesin/gastrin-releasing peptide antagonist RC-3095 impairs inhibitory avoidance retention

Bombesin (BN)-like peptides regulate cell proliferation and cancer growth as well as neuroendocrine and neural functions. We evaluated the effects of the BN/gastrin-releasing peptide (GRP) antagonist RC-3095 on memory formation. Male Wistar rats were given a bilateral infusion of saline or RC-3095 (0.2, 1.0 or 5.0 microg) into the dorsal hippocampus either immediately or 2 h after training in an inhibitory avoidance (IA) task. Retention test trials were carried out 1.5 h (short-term memory) and 24 h (long-term memory) after training. RC-3095 impaired both short- and long-term retention only when given immediately after training. The results suggest that the hippocampal BN/GRP receptor system modulates IA memory formation.     

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.