Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L.

NADP-malic enzyme (EC 1.1.1.40 [EC] ), which is involved in Crassulaceanacid metabolism (CAM), was purified to electrophoretic homogeneityfrom the leaves of the inducible CAM plant Mesembryanthemumcrystallinum. The NADP-malic enzyme, which was purified 1,146-fold,has a specific activity of 68.8 µmol (mg protein)^–1min^–1. The molecular weight of the subunits of the enzymewas 64 kDa. The native molecular weight of the enzyme was determinedby gel-filtration to be 390 kDa, indicating that the purifiedNADP-malic enzyme is a hexamer of identical subunits. The optimalpH for activity of the enzyme was around 7.2. Double-reciprocalplots of the enzymatic activity as a function of the concentrationof L-malate yielded straight lines both at pH 7.2 and at pH7.8 and did not reveal any evidence for cooperativity of bindingof L-malate. The K_m value for L-malate was 0.35 mM. Hill plotsof the activity as a function of the concentration of NADP⁺indicated positive cooperativity in the binding of NADP⁺ tothe enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value(the concentration giving half-maximal activity) of 9.9 µMfor NADP⁺ was obtained. Oxaloacetate inhibited the activityof the NADP-malic enzyme. Effects of succinate and NaHCO₃ onthe activity of NADP-malic enzyme were small. (Received October 30, 1991; Accepted May 1, 1992)     

Impacts of 3 years of elevated atmospheric CO2 on rhizosphere carbon flow and microbial community dynamics

Carbon (C) uptake by terrestrial ecosystems represents an important option for partially mitigating anthropogenic CO₂ emissions. Short‐term atmospheric elevated CO₂ exposure has been shown to create major shifts in C flow routes and diversity of the active soil‐borne microbial community. Long‐term increases in CO₂ have been hypothesized to have subtle effects due to the potential adaptation of soil microorganism to the increased flow of organic C. Here, we studied the effects of prolonged elevated atmospheric CO₂ exposure on microbial C flow and microbial communities in the rhizosphere. Carex arenaria (a nonmycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown at defined atmospheric conditions differing in CO₂ concentration (350 and 700 ppm) for 3 years. During this period, C flow was assessed repeatedly (after 6 months, 1, 2, and 3 years) by ¹³C pulse‐chase experiments, and label was tracked through the rhizosphere bacterial, general fungal, and arbuscular mycorrhizal fungal (AMF) communities. Fatty acid biomarker analyses and RNA‐stable isotope probing (RNA‐SIP), in combination with real‐time PCR and PCR‐DGGE, were used to examine microbial community dynamics and abundance. Throughout the experiment the influence of elevated CO₂ was highly plant dependent, with the mycorrhizal plant exerting a greater influence on both bacterial and fungal communities. Biomarker data confirmed that rhizodeposited C was first processed by AMF and subsequently transferred to bacterial and fungal communities in the rhizosphere soil. Over the course of 3 years, elevated CO₂ caused a continuous increase in the ¹³C enrichment retained in AMF and an increasing delay in the transfer of C to the bacterial community. These results show that, not only do elevated atmospheric CO₂ conditions induce changes in rhizosphere C flow and dynamics but also continue to develop over multiple seasons, thereby affecting terrestrial ecosystems C utilization processes.     

p21, an important mediator of quiescence during pituitary tumor formation, is dispensable for normal pituitary development during embryogenesis

A delicate balance between proliferation and differentiation must be maintained in the developing pituitary to ensure the formation of the appropriate number of hormone producing cells. In the adult, proliferation is actively restrained to prevent tumor formation. The cyclin dependent kinase inhibitors (CDKIs) of the CIP/KIP family, p21, p27 and p57, mediate cell cycle inhibition. Although p21 is induced in the pituitary upon loss of Notch signaling or initiation of tumor formation to halt cell cycle progression, its role in normal pituitary organogenesis has not been explored. In wildtype pituitaries, expression of p21 is limited to a subset of cells embryonically as well as during the postnatal proliferative phase. Mice lacking p21 do not have altered cell proliferation during early embryogenesis, but do show a slight delay in separation of proliferating progenitors from the oral ectoderm. By embryonic day 16.5, p21 mutants have an alteration in the spatial distribution of proliferating pituitary progenitors, however there is no overall change in proliferation. At postnatal day 21, there appears to be no change in proliferation, as assessed by cells expressing Ki67 protein. However, p21 mutant pituitaries have significantly less mRNA of Myc and the cyclins Ccnb1, Ccnd1, Ccnd2 and Ccne1 than wildtype pituitaries. Interestingly, unlike the redundant role in cell cycle inhibition uncovered in p27/p57 double mutants, the pituitary of p21/p27 double mutants has a similar proliferation profile to p27 single mutants at the time points examined. Taken together, these studies demonstrate that unlike p27 or p57, p21 does not play a major role in control of progenitor proliferation in the developing pituitary. However, p21 may be required to maintain normal levels of cell cycle components.     

Correction to: Lentinula edodes as a Source of Bioelements Released Into Artificial Digestive Juices and Potential Anti-inflammatory Material

Biological Trace Element Research - The original version of this article unfortunately contained a mistake.     

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).     

Comparative studies on the mechanism of cytotoxic action of novel platinum II complexes with pyrazole ligands

In search for new platinum-based anticancer drugs, four cisplatin analogues, which contain pyrazole rings as non-leaving ligands, have been synthesized: cis-PtCl(2)(3,5-DM HMPz)(2), cis-PtCl(2)(Pz)(2), cis-PtCl(2)(ClMPz)(2), and cis-PtCl(2)(HMPz)(2), where Pz=pyrazole, H=hydroxyl, M=methyl. We tested their cytotoxicity, apoptosis induction ability, DNA damaging and modification properties comparing them in respect to the parent compound. The cytotoxic activity of these platinum pyrazole complexes toward the murine leukemia cell line was 2.9-3.8 times lower than actvity of cisplatin. The tested compounds varied in their mechanism of action by producing different DNA lesions. The most interesting compound seems to be the complex with chloromethyl groups at N1 of pyrazole rings, which exhibited the highest ability to form bifunctional adducts with DNA in vitro.     

Spectral analysis of Delayed Luminescence from human skin as a possible non-invasive diagnostic tool

In vivo measurements of Delayed Luminescence (DL), the low-level photo-induced emission which lasts for a longer time after switching off the excitation light, have been performed on human skin, with the aim to develop a technique for optical biopsy. Preliminary tests have been performed on healthy volunteers, measuring the time decays of the spectral components (λ_emiss = 400–800 nm) starting 10 μs after switching off the excitation (λ_exc = 337 nm). Significant differences in the decay trends of DL from different subjects were revealed and quite a good reproducibility for the same subject was observed. The modeling of experimental data has been examined in detail in order to get parameters, characterizing the theoretical fit, whose changes may be correlated with age differences and seasonal variations. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Paleoenvironmental conditions preceding the Messinian Salinity Crisis in the Central Mediterranean: Integrated data from the Upper Miocene Trave section (Italy)

Integrated data of calcareous plankton and benthic foraminifers from the pre-evaporitic interval of Trave section (Central Italy) allowed the reconstruction of surface and bottom-water conditions in the Central Mediterranean during the interval from 7.61 to 6.33 Ma, preceding the Messinian Salinity Crisis.Our data point out a three-step paleoenvironmental evolution. During the first stage (7.61-7.02 Ma) benthic foraminiferal assemblages depict stable, well-oxygenated and ventilated bottom-water conditions, while the surface water records variable temperature and high nutrient conditions, probably associated with strong seasonality. The second stage (7.02-6.70 Ma) points to unfavourable bottom-water condition, triggered by deep-sea stagnation. This is witnessed by a significant decrease in oxygen concentration and biotic diversity, and by the presence of stress-tolerant taxa. A general warming of the surface water and a strongly stratified water column, characterized by an expanded mixed layer, are also recorded.From 6.70 Ma onwards (third stage), a prominent change to more restricted, low-oxygenated, hypersaline conditions at the sea floor is testified by the total disappearance of deep-dwelling planktonic foraminifers and the increasing abundance of stress-tolerant species. Calcareous plankton reflects high instability of the surface water in terms of nutrients, temperature and salinity. During this stage the environmental deterioration reaches intermediate depths in the water column.The initial change toward a step-wise isolation of the Central Mediterranean bottom-waters is probably related to a general warming, responsible for a first slowing-down of the vertical circulation, favouring stratification of surface and intermediate waters and stagnation of bottom-waters. This warming is related to the restricted connection between the Mediterranean Sea and the Atlantic Ocean, which occurred since 7.146 Ma.In the Trave section, the isolation of bottom-waters most likely occurred at the same time as in other Mediterranean sections. However, due to the presence of a hiatus it cannot be excluded that it occurred with a delay of ~ 100 kyr, probably related to the shallower paleodepth of the basin.