Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.     

Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation

Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization.     

Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures

Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15–16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 μM) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 ± 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 ± 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD₂ ? PGF_2α > TXB₂ ? PGE₂) and astroglial cells (PGD₂ > TXB₂ ? PGF_2α ? PGE₂ ? 6-ketoPGF_1α). Preincubation with arachidonic acid (1 μg/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4–6-fold by addition of the calcium ionophore A23187 (1 μM). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.     

Mepiquat chloride (PIX)-induced changes in photosynthesis and growth of cotton

Mepiquat chloride (N, N-dimethylpiperidinium chloride), well known as PIX, is a potential systemic plant growth regulator. The effects of PIX on plant height, stem elongation, leaf area, net photosynthetic rates, chlorophyll content, sucrose and starch levels, and RuBP carboxylase activity in cotton (Gossypium hirsutum L. cv. DES 119) plants were measured. PIX was sprayed (0, 7.65, 15.3, 30.6 or 61.2 g active ingredient ha^–1) on the plants at first square (25 days after emergence) and measurements were made at frequent intervals. Plant height was clearly reduced by PIX. The total length of vegetative branches and fruiting branches was 40% and 50% less than the control. Total leaf area in PIX treated plants was 16% less than the control. Net photosynthetic rates were 25% less in PIX-treated leaves. PIX treated leaves had more chlorophyll content. The activity of RuBP carboxylase was decreased in PIX treated plants. Starch accumulation was noticed in PIX treated leaves while sucrose content was not changed. The data reported here suggest that reduced growth responses induced by PIX results in partial loss of photosynthetic capacity in cotton at least up to 20 days after application of the growth regulator.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Effect of salinity on phosphate accumulation and injury in soybean

Many soybean [Glycine max (L.) Merr.] genotypes that are grown in solution cultures are highly sensitive to the combination of both salinity and inorganic phosphate (Pi) in the substrate. This effect has been observed on numerous occasions on plants grown in a saline medium that contained a substantial amount of Ca (i.e., CaCl₂/NaCl=0.5 on a molar basis). Because Ca is important in regulating ion transport and membrane permeability, solution culture experiments were designed to examine the effects of various concentrations of Pi and ratios of CaCl₂/NaCl (0 to 0.5 on a molar basis) at a constant osmotic potential (−0.34 MPa) on this adverse interaction. Four soybean cultivars (‘Lee’, ‘Lee 74’ ‘Clark’ and ‘Clark 63’) were tested. No adverse salinity x Pi interaction was found on Lee at any ratio and leaf P and Cl were maintained below 300 and 200 mmol kg⁻¹ dry wt, respectively. Clark, Clark 63 and Lee 74 soybean plants, on the other hand, were severely injured by solution salinity (−0.34 MPa osmotic potential) when substrate Pi was ≥0.12 mM. Reduced substrate Ca did not intensify the salinity x Pi interaction. On the contrary, the onset of injury was hastened and more severe with increased CaCl₂/NaCl ratios in isotonic solutions. Shoot and root growth rates decreased as injury increased. Leaf P concentrations from these cultivars grown in saline solutions with 0.12 mM Pi were excessive (>600 mmol kg⁻¹ dry wt) compared with concentrations commonly found in soybean leaf tissue yet they were independent of the severity of injury. Since leaf Cl increased wiht increased CaCl₂/NaCl ratio, we suspect that the severity of foliar injury was related to the combined effects of excessive P and Cl within the tissue. Lee 74, the only injured cultivar examined that excluded Cl from its leaves, was less sensitive than either Clark cultivar and its injury was characteristically different. Other ion interactions were reported that may have played a role in injury susceptibility.     

INFLUENCE OF FLUCTUATING PHOSPHATE SUPPLY ON THE REGULATION OF PHOSPHATE UPTAKE BY THE BLUE-GREEN ALGA ANACYSTZS NIDULANS1

The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of ³²P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range .