Growth and biomass production in potato grown in the hot tropics as influenced by paclobutrazol

Two similar field trials were carried out during 2003 in a hot tropical region of eastern Ethiopia to investigate the effect of leaf and soil applied paclobutrazol on the growth, dry matter production and assimilate partitioning in potato. A month after planting paclobutrazol was applied as a foliar spray or soil drench at rates of 0, 2, 3, and 4 kg a.i. paclobutrazol ha^–1. Plants were sampled during treatment application and subsequently 2, 4, 6 and 8 weeks after treatment application. The data was analyzed using standard growth analyses techniques. None of the growth parameters studied was affected by the method of paclobutrazol application. Paclobutrazol decreased leaf area index, crop growth rate, and total biomass production, and increased specific leaf weight, tuber growth rate, net assimilation rate, and partitioning coefficient of potato. At all harvesting stages, paclobutrazol reduced the partitioning of assimilate to the leaves, stems, and roots and stolons and increased allocation to the tubers. Although paclobutrazol decreased the total biomass production it improved tuber yield by partitioning more assimilates to the tubers. Paclobutrazol improved the productivity of potato under tropical conditions by redirecting assimilate allocation to the tubers.     

Fluorescence studies of chicken liver fatty acid synthase. Segmental flexibility and distance measurements

The 4'-phosphopantetheine of chicken liver fatty acid synthase was specifically labeled with the fluorescent substrate analog coenzyme A 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoate at low salt concentrations. A serine at the active site of the thioesterase was specifically labeled with the fluorescent compounds 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminopentylmethylphosphono fluoridate and/or pyrenebutyl methylphosphonofluoridate. Dynamic anisotropy measurements indicate the thioesterase has considerable segmental flexibility, whereas the fluorescent labeled 4'-phosphopantetheine does not display detectable local or segmental flexibility. Fluorescence resonance energy transfer measurements indicate that the distance between the fluorescent label at the end of the 4'-phosphopantetheine and NADPH bound to the beta-ketoacyl reductase or enoyl reductase site on the same polypeptide chain is essentially the same, approximately 38 A. The two types of reductases were distinguished by specifically blocking enoyl reductase with pyridoxal 5'-phosphate. No significant energy transfer occurs between sites on different polypeptide chains so that the distances must be greater than 55 A. The distance between the serine on the thioesterase and the 4'-phosphopantetheine on the same polypeptide is 48 A; again no interpolypeptide chain energy transfer was observed. The distance between the serines of the two thioesterases within a fatty acid synthase molecule is greater than 56 A. The monomeric enzyme obtained at 1 degree C does not have beta-ketoacyl synthase and reductase activities. Also fluorescent titrations indicate NADPH is not bound to beta-ketoacyl reductase in monomeric enzyme. The addition of potassium phosphate to the monomers at 1 degree C rapidly dimerizes the enzyme and restores the beta-ketoacyl reductase activity. The beta-ketoacyl synthase activity is slowly restored when the dimer is raised to room temperature. The results obtained suggest that relatively large conformational changes may be part of the catalytic cycle.     

Strain-specific ureolytic microbial calcium carbonate precipitation

During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.     

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is unlocked by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.     

Flexibility, diversity, and cooperativity: pillars of enzyme catalysis

This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, nuclear magnetic resonance, and single-molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model.     

Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.

BACKGROUND: Chronic diabetes causes structural changes in the retinal capillaries of nearly all patients with a disease duration of more than 15 years. Acellular occluded vessels cause hypoxia, which stimulates sight-threatening abnormal angiogenesis in 50% of all type I diabetic patients. The mechanism by which diabetes produces acellular retinal capillaries is unknown. MATERIALS AND METHODS: In this study, evidence of programmed cell death (PCD) was sought in the retinas of early diabetic rats, and the effect of nerve growth factor (NGF) on PCD and capillary morphology was evaluated. RESULTS: Diabetes induced PCD primarily in retinal ganglion cells (RGC) and Muller cells. This was associated with a transdifferentiation of Muller cells into an injury-associated glial fibrillary acidic protein (GFAP)-expressing phenotype, and an up-regulation of the low-affinity NGF receptor p75NGFR on both RGC and Muller cells. NGF treatment of diabetic rats prevented both early PCD in RGC and Muller cells, and the development of pericyte loss and acellular occluded capillaries. CONCLUSIONS: These data provide new insight into the mechanism of diabetic retinal vascular damage, and suggest that NGF or other neurotrophic factors may have potential as therapeutic agents for the prevention of human diabetic retinopathy.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase

The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 microM. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)