Evaluation of Fourier-transform infrared spectroscopy for data capture in predictive microbiology

Chemical changes in the medium, induced by the fermentative species Lactobacillus plantarum and Lactobacillus brevis and by the enzymatic action of a proteolytic, spoilage species, Yarrowia lipolytica, were analysed using Fourier-transform i.r. spectroscopy (FTIR). Changes in the absorbance data over time could be modelled using one of the more current predictive, mathematical models of microbial growth, such as the Gompertz equation. Moreover, a linear correlation between FTIR data (expressed as absorbance of some selected peaks) and viability data (expressed as log₁₀ c.f.u./g or ml) was observed during the fermentation process, both for L. plantarum and L. brevis.     

Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.     

Secondary structure of β-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from Hα, Cα, Cβ and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

Summary Nearly complete backbone ¹H, ¹⁵N and ¹³C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although ¹⁵N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins. Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D ¹H–¹⁵N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and ³J_HN data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed.     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound ⁴⁵Ca²⁺ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca²⁺ concentration. Crosslinking experiments with BS³ showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg²⁺-ATPase activity of myosin in the presence of Ca²⁺.     

Effect of daily photon receipt on the susceptibility of dwarf bean (Phaseolus vulgaris L.) leaves to photoinhibition of photosynthesis

Bean (Phaseolus vulgaris L.) plants were grown at two light periods of 8 and 13 h with a similar photon flux density (PFD) giving a daily photon receipt (DPR) of 17.9 and 38.2 mol · m–2, respectively. Shoot growth and leaf area development were followed at regular intervals and diurnal whole-plant photosynthesis measured. Single mature trifoliate leaves were exposed to photoinhibitory treatments at PFDs of 800 and 1400 mol · m–2 · s–1 and at temperatures of 12 and 20°C. Chlorophyll fluorescence and photon yields were measured at regular intervals throughout each treatment. Plants grown in 13 h had significantly greater leaf areas than those grown in 8 h. There were no differences in maximum rates of photosynthesis, photon yields and only minor but significant differences in F_v/F_m for plants in the two treatments, showing photosynthetic characteristics were dependent on PFD but not DPR. A significant decline in photosynthesis and F_v/F_m occurred over the 13-h but little change in photosynthesis for plants in the 8 h, indicating some feedback inhibition of photosynthesis was occurring. Plants grown in 8 h were consistently more susceptible to photoinhibition of photosynthesis at all treatments than 13-h plants. Nevertheless, photoinhibition was exacerbated by increases in PFD, and by decreases in temperature for leaves from both treatments. However, for plants from the 8-h day, exposing leaves to 12°C and 1400 mol · m–2 · s–1 caused photo-oxidation and severe bleaching but no visible damage on leaves from 13-h-grown plants. Closure of the photosystem II reaction-centre pool was partially correlated with increasing extents of photoinhibition but the relationship was similar for plants from both treatments. There remains no clear explanation for their wide differences in susceptibility to photoinhibition.Abbreviations and Symbols DPR daily photon receipt - F₀ and F_m initial and maximal fluorescence - F_v/F_m fluorescence ratio in dark-treated leaves - F/F_m intrinsic efficiency of PSII during illumination - PFD photon flux density - _i photon yield (incident basis) - _psi quantum yield of PSII electron transport - P_max maximum rate of photosynthesis - q_N non-photochemical quenching coefficient - q_P photochemical quenching coefficient Many thanks to my colleague William Laing who spent a considerable effort in developing the programme to run the photosynthesis apparatus. I am also indebted to one reviewer with whom I corresponded to resolve some issues in the paper. This project was funded by the New Zealand Foundation for Research, Science and Technology.     

In situ estimation of net CO2 assimilation, photosynthetic electron flow and photorespiration in Turkey oak (Q. cerris L.) leaves: diurnal cycles under different levels of water supply

Diurnal time courses of net CO₂ assimilation rates, stomatal conductance and light-driven electron fluxes were measured in situ on attached leaves of 30-year-old Turkey oak trees (Quercus cerris L.) under natural summer conditions in central Italy. Combined measurements of gas exchange and chlorophyll a fluorescence under low O₂ concentrations allowed the demonstration of a linear relationship between the photochemical efficiency of PSII (fluorescence measurements) and the apparent quantum yield of gross photosynthesis (gas exchange). This relationship was used under normal O₂ to compute total light-driven electron fluxes, and to partition them into fractions used for RuBP carboxylation or RuBP oxygenation. This procedure also yielded an indirect estimate of the rate of photorespiration in vivo. The time courses of light-driven electron flow, net CO₂ assimilation and photorespiration paralleled that of photosynthetic photon flux density, with important afternoon deviations as soon as a severe drought stress occurred, whereas photochemical efficiency and maximal fluorescence underwent large but reversible diurnal decreases. The latter observation indicated the occurrence of a large non-photochemical energy dissipation at PSII. We estimated that less than 60% of the total photosynthetic electron flow was used for carbon assimilation at midday, while about 40% was devoted to photorespiration. The rate of carbon loss by photorespiration (R₁) reached mean levels of 56% of net assimilation rates. The potential application of this technique to analysis of the relative contributions of thermal de-excitation at PSII and photorespiratory carbon recycling in the protection of photosynthesis against stress effects is discussed.     

Differential effects of elevated ultraviolet-B radiation on the photochemical and reproductive performances of dicotyledonous and monocotyledonous arid-environment ephemerals

Four dicotyledonous Astcraceac and four monocotyledonous Iridaceac species were grown from seed under three different effective ultraviolet-B (UV-B, 280–320 nm) fluenccs, which approximated those received daily over the natural growing periods of these species at their southerly and northerly distribution limits under ambient stratospheric ozone, and at the northerly limit assuming a 20% ozone depletion. Diminished photochemical efficiencies, delayed flowering, decreased flower production, pollen germination and tube growth, and reduced numbers of seeds set, accompanied by corresponding increases in mass per seed, were observed mainly in dicotyledons under elevated UV-B radiation, despite their higher foliar and pollen polyphenolic contents, their possession of trinucleatc pollen and their natural occurrence at lower latitudes than monocotyledons with binucleate pollen. The results demonstrate the difficulties in determining plant sensitivity to UV-B radiation from polyphenolic content, pollen type and natural geographic distribution, and suggest that monocotyledons possess other attributes that confer greater resilience to UV-B radiation.