Brønsted analysis reveals Lys218 as the carboxylase active site base that deprotonates vitamin K hydroquinone to initiate vitamin K-dependent protein carboxylation

The vitamin K-dependent (VKD) carboxylase converts Glu's to carboxylated Glu's in VKD proteins to render them functional in a broad range of physiologies. The carboxylase uses vitamin K hydroquinone (KH(2)) epoxidation to drive Glu carboxylation, and one of its critical roles is to provide a catalytic base that deprotonates KH(2) to allow epoxidation. A long-standing model invoked Cys as the catalytic base but was ruled out by activity retention in a mutant where every Cys is substituted by Ala. Inhibitor analysis of the cysteine-less mutant suggested that the base is an activated amine [Rishavy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13732-13737], and in the present study, we used an evolutionary approach to identify candidate amines, which revealed His160, His287, His381, and Lys218. When mutational analysis was performed using an expression system lacking endogenous carboxylase, the His to Ala mutants all showed full epoxidase activity but K218A activity was not detectable. The addition of exogenous amines restored K218A activity while having little effect on wild type carboxylase, and pH studies indicated that rescue was dependent upon the basic form of the amine. Importantly, Br?nsted analysis that measured the effect of amines with different pK(a) values showed that K218A activity rescue depended upon the basicity of the amine. The combined results provide strong evidence that Lys218 is the essential base that deprotonates KH(2) to initiate the reaction. The identification of this base is an important advance in defining the carboxylase active site and has implications regarding carboxylase membrane topology and the feedback mechanism by which the Glu substrate regulates KH(2) oxygenation.     

塔克拉玛干沙漠南缘柽柳和胡杨水势季节变化研究

对塔克拉玛干沙漠南缘的柽柳和胡杨生长周期内的清晨水势和水势日变化的连续野外测定表明,两种植物在整个生长期内均未出现明显的水分亏缺.清晨水势的季节变化幅度不大,正午水势有不同程度的降低;一次性人工灌溉对植物水分状况没有明显影响.采伐利用方式不影响植物的水分状况.地下水是柽柳和胡杨生存与生长的先决条件.维持该区域地下水位的基本稳定是保证该区域柽柳和胡杨恢复重建的重要前提.     

Non‐destructive dry matter estimation of Alhagi sparsifolia vegetation in a desert oasis of Northwest China

Question: Can above‐ground biomass of naturally growing Alhagi sparsifolia shrubs be estimated non‐destructively? Location: Qira oasis (37° 01′N, 80° 48′E, 1365 ma.s.l.) at the southern fringe of the Taklamakan desert, Xinjiang, NW China. Methods: Two methods were compared to estimate above‐ground biomass (AGB) of Alhagi. At first shrub AGB was estimated by manual ground measurements (called ‘allometric approach’) of length, width and height of 50 individuals. Subsequently regression equations were established between calculated shrub canopy volume and shrub AGB (r²= 0.96). These equations were used to calculate AGB from manual ground measurements in 20 sample plots within the Alhagi field. Secondly, kite‐based colour aerial photography coupled with the use of a Geographic Information System (called ‘GIS approach’) was tested. First and second order polynomial regressions between AGB data of the 50 individual shrubs and their respective canopy area allowed to automatically calculate the AGB of all remaining shrubs covered by the photograph (r²= 0.92 to 0.96). The use of non‐linear AGB regression equations required an automatised separation of shrubs growing solitary or in clumps. Separation criteria were the size and shape of shrub canopies. Results: The allometric approach was more reliable but also more time‐consuming than the GIS‐based approach. The latter led to an overestimation of Alhagi dry matter in densely vegetated areas. However, this systematic error decreased with increasing size of the surveyed area. Future research in this field should focus on improvements of AGB estimates in areas of high shrub density.     

The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent K_m = 4 μM and saturation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is approx. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

^99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, ^99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a ^99mTc-labeled HYNIC-DAPI compound with that of ^99mTc pertechnetate (^99mTcO₄ ⁻). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by ^99mTc-HYNIC-DAPI (1.03) was twice that caused by ^99mTcO₄ ⁻ (0.51), and the number of DSBs increased fivefold in the ^99mTc-HYNIC-DAPI-treated sample compared with the ^99mTcO₄ ⁻ treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the ^99mTcO₄ ^– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the ^99mTc-HYNIC-DAPI-treated samples. These results indicated that ^99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the ^99mTc-labeled compound with DNA. In contrast to these results, ^99mTcO₄ ⁻ induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of ^99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.     

Water use by perennial plants in the transition zone between river oasis and desert in NW China

The study aimed at establishing the role of two possible water sources (inundation, ground water) for the water supply to the perennial plant species Alhagi sparsifolia, Calligonum caput-medusae, Populus euphratica and Tamarix ramosissima growing in the transition zone between a river oasis and the open desert at the southern fringe of the Taklamakan desert (Xinjiang province, NW China). The basic hypothesis was that inundations, which normally occur in summer when rivers from a nearby mountain range carry high water, contribute significantly to the plants’ water supply. When, in the first summer, inundations did not occur, four sites, each of which covered by a relatively dense stand of one species, were artificially flooded. Soil and plant water relations as well as meteorological variables were measured during two growing seasons. Water use efficiency of production (WUE_P) was calculated by relating biomass production, which was determined using allometric regressions, to water use.The effects of artificial flooding on the plant water relations were negligible. Water use was relatively high, especially in the A. sparsifolia and the P. euphratica stands and in a dense stand of T. ramosissima (up to approx. 500 kgH₂O m⁻² year⁻¹). Using the total above-ground biomass in the calculation, WUE_P was highest in C. caput-medusae and P. euphratica, and lowest, in A. sparsifolia. From soil and plant water relations, and against the background of the climate and the productivity of the vegetation, it is concluded that all perennial plants in the transition zone between oases and desert in that region must have sufficient access to ground water to ensure long-term survival. Management of ground water such that it remains continuously accessible to the perennial plants is a prerequisite for the conservation and sustainable use of the vegetation in the transition zone.     

Histochemische Untersuchungen zur Frage der endokrinen Aktivität der Hodenzwischenzellen während der Fetalzeit beim Menschen

Zusammenfassung An Hoden von menschlichen Feten (Scheitel-Fersenlänge 20–42 cm) wurden die Fettfärbung mit Sudanschwarz B, der Cholesterinnachweis und die Reaktion auf Ketongruppen (NAHD) ausgeführt. Alle Reaktionen fielen in den Zwischenzellen am stärksten, in den Sertolizellen schwächer aus. Alle übrigen Gewebsbestandteile des Hodens verhielten sich negativ.Die vorgelegten Ergebnisse machen wahrscheinlich, daß in den Zwischenzellen des fetalen menschlichen Hodens Wirkstoffe vom Charakter der Ketosteroide gebildet und gespeichert werden. Die zum Teil feste Bindung der Ketosteroide an zytoplasmatische Strukturen spricht dafür, daß die Wirkstoffe nur in sehr geringem Maße an die Blutbahn abgegeben werden. Ein gewisses Quantum ist jedoch für die normale Entwicklung der männlichen Genitalorgane während der Fetalzeit notwendig.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

A contribution to the mathematical biophysics of cell growth and shapes: I

Under the assumption that the elongated shape assumed by a growing nerve cell is caused by surface tension forces between the cell, its fluid medium, and a fibrous substrate track along which the cell grows, equations of elongation and conditions insuring elongation are derived. One specific type of cell-substrate contact is considered. Equations of elongation and conditions for elongation are treated in both the nonfrictional and frictional types of motion of the cell-process.