Further studies on the resolution of human mercapt- and nonmercaptalbumin and on human serum albumin in the elderly by high-performance liquid chromatography

High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on Asahipak GS-520 showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the secondary one to nonmercaptalbumin (HNA). HPLC analysis of HSA on Asahipak ES-520 N showed three peaks, the principal component corresponding to HMA, the secondary one to HNA having mixed disulfide with cysteine or glutathione and the tertiary one to HNA oxidized higher than mixed disulfide. Two kinds of rapid HPLC for the resolution of HSA into HMA and HNA were developed by the present authors. Using these HPLC, the present authors found a significant decrease in the fraction of HMA in the elderly.     

Resolution of human mercapt- and nonmercaptalbumin by high-performance liquid chromatography

Gel-exclusion high-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on PGP 2000 column (0.10 M sodium phosphate buffer, 0.30 M NaCl, pH 6.86) showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the second one to human nonmercaptalbumin (HNA). Mechanism for the separation of HMA and HNA might be due to weak resin-HSA interaction. HPLC analysis of bovine plasma albumin (BPA) showed a single peak on PGP 2000 column. The elution volume of HSA was larger than that of BPA, resulting in a clear resolution of HSA and BPA.     

Two roads diverged: the structure of hydroxymandelate synthase from Amycolatopsis orientalis in complex with 4-hydroxymandelate

The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation.     

Comparative expression analysis of heavy metal ATPase subfamily genes between Cd-tolerant and Cd-sensitive turnip landraces

The heavy metal ATPase(HMA)subfamily is mainly involved in heavy metal(HM)tolerance and transport in plants,but an understanding of the definite roles and mechanisms of most HMA members are still limited.In the present study,we identified 14 candidate HMA genes named BrrHMAl—BrrHMA8 from the turnip genome and analyzed the phylogeny,gene structure,chromosome distribution,and conserved domains and motifs of HMAs in turnip(Brassica rapa var.rapa).According to our phylogenetic tree,the BrrHMAs are divided into a Zn/Cd/Co/Pb subclass and Cu/Ag subclass.The BrrHMA members show similar structural characteristics within subclasses.To explore the roles of BrrHMAs in turnip,we compared the gene sequences and expression patterns of the BrrHMA genes between a Cd-tolerant landrace and a Cd-sensitive landrace.Most BrrHMA genes showed similar spatial expression patterns in both Cd-tolerant and Cd-sensitive turnip landraces;some BrrHMA genes,however,were differentially expressed in specific tissue in Cd-tolerant and Cd-sensitive turnip.Specifically,BrrHMA genes in the Zn/Cd/Co/Pb subclass shared the same coding sequence but were differentially expressed in Cd-tolerant and Cd-sensitive turnip landraces under Cd stress.Our findings suggest that the stable expression and up-regulated expression of BrrHMA Zn/Cd/Co/Pb subclass genes under Cd stress may contribute to the higher Cd tolerance of turnip landraces.     

Apoptosis induced by Na+/H+ antiport inhibition activates the LEI/L-DNase II pathway

L-DNase II is derived from its precursor leucocyte elastase inhibitor (LEI) by post-translational modification. In vitro, the conversion of LEI into L-DNase II can be induced by incubation of LEI at an acidic pH. In this study, we proposed to analyze the effects of intracellular acidification on this transformation. Amiloride derivatives, like hexamethylene amiloride (HMA), are known to provoke a decrease of cytosolic pH by inhibiting the Na(+)/H(+) antiport. In BHK cells, treatment with HMA-induced apoptosis accompanied by an increase in L-DNase II immunoreactivity and L-DNase II enzymatic activity. Overexpression of L-DNase II precursor led to a significant increase of apoptosis in these cells supporting the involvement of L-DNase II in HMA induced apoptosis. As previously shown in other cells, etoposide-induced apoptosis did not activate L-DNase. On the contrary, LEI overexpression significantly increased cell survival in etoposide-induced apoptosis. Together these results suggest differential roles of LEI and L-DNase II in response to different types of apoptotic inducers.     

应用异源双链泳动分析法快速确定(HIV-1)基因亚型

应用异源双链泳动分析法（Ｈｅｔｅｒｏｕｄｕｐｌｅｘ ｍｏｂｉｌｉｔｙ ａｓｓａｙ,ＨＭＡ）,对来自我国云南、四川、新疆、浙江、广东、福建、天津７省市７３份ＨＩＶ感染者样品,进行了ＨＩＶ－１膜蛋白（ｅｎｖ）基因片段亚型分析。通过ＨＭＡ能确定亚的有７０份,占样品总数的９５．８９％（７０／７２）。其中Ａ亚型２．８６％（２／７０）,Ｆ亚型２．８６％（２／７０）,泰国Ｂ亚型１８．５８％（１３／７０）,欧美Ｂ     

Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery

Human serum albumin is a mixture of mercapt- (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). We studied the mercapt↔nonmercapt conversion of human serum albumin, which reflects the redox state of the extracellular fluids, in cardiac and other common surgical patients using high-performance liquid chromatography. Mean values of [(HMA)/(HMA + HNA)] ± standard deviation [f_HMA ± σ], for patients who received common surgery (group 1) and cardiac surgery (group 2) at the start of anesthesia were0.636±0.50(n=83) and 0.615±0.062(n=14), respectively. f_HMA values were markedly lower than those for healthy male adults of 0.750±0.028(n=28). f_HMA values increased at 24 h after the start of anesthesia and decreased on the 4th postoperative day in most of the patients. These postoperative changes were prominent in surgical cardiac patients. Although f_HMA values after the 7th postoperative day recovered to those at the start of anesthesia in almost all of common surgical patients, those in cardiac surgical patients, never recovered even on the 21st postoperative day.     

HMA6 and HMA8 are two chloroplast Cu+-ATPases with different enzymatic properties

Copper (Cu) plays a key role in the photosynthetic process as cofactor of the plastocyanin (PC), an essential component of the chloroplast photosynthetic electron transfer chain. Encoded by the nuclear genome, PC is translocated in its apo-form into the chloroplast and the lumen of thylakoids where it is processed to its mature form and acquires Cu. In Arabidopsis, Cu delivery into the thylakoids involves two transporters of the P_IB-1 ATPases family, heavy metal associated protein 6 (HMA6) located at the chloroplast envelope and HMA8 at the thylakoid membrane. To gain further insight into the way Cu is delivered to PC, we analysed the enzymatic properties of HMA8 and compared them with HMA6 ones using in vitro phosphorylation assays and phenotypic tests in yeast. These experiments reveal that HMA6 and HMA8 display different enzymatic properties: HMA8 has a higher apparent affinity for Cu⁺ but a slower dephosphorylation kinetics than HMA6. Modelling experiments suggest that these differences could be explained by the electrostatic properties of the Cu⁺ releasing cavities of the two transporters and/or by the different nature of their cognate Cu⁺ acceptors (metallochaperone/PC).     

Impairing both HMA4 homeologs is required for cadmium reduction in tobacco

In tobacco, the heavy metal P1B‐ATPases HMA4.1 and HMA4.2 function in root‐to‐shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root‐to‐shoot transfer by >90%. Analysis of plants with segregating null and wild‐type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild‐type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.     

Chloroflexi bacteria are more diverse, abundant, and similar in high than in low microbial abundance sponges

Some marine sponges harbor dense and phylogenetically complex microbial communities [high microbial abundance (HMA) sponges] whereas others contain only few and less diverse microorganisms [low microbial abundance (LMA) sponges]. We focused on the phylum Chloroflexi that frequently occurs in sponges to investigate the different associations with three HMA and three LMA sponges from New Zealand. By applying a range of microscopical and molecular techniques a clear dichotomy between HMA and LMA sponges was observed: Chloroflexi bacteria were more abundant and diverse in HMA than in LMA sponges. Moreover, different HMA sponges contain similar Chloroflexi communities whereas LMA sponges harbor different and more variable communities which partly resemble Chloroflexi seawater communities. A comprehensive phylogenetic analysis of our own and publicly available sponge-derived Chloroflexi 16S rRNA gene sequences (>?780 sequences) revealed the enormous diversity of this phylum within sponges including 29 sponge-specific and sponge-coral clusters (SSC/SCC) as well as a 'supercluster' consisting of >?250 sponge-derived and a single nonsponge-derived 16S rRNA gene sequence. Interestingly, the majority of sequences obtained from HMA sponges, but only a few from LMA sponges, fell into SSC/SCC clusters. This indicates a much more specific association of Chloroflexi bacteria with HMA sponges and suggests an ecologically important role for these prominent bacteria.     

HMA3 is a key factor for differences in Cd- and Zn-related phenotype between <Emphasis Type="Italic">Arabidopsis</Emphasis> Ws and Col-0 ecotypes

Columbia-0 (Col-0) appears to be less tolerant to cadmium (Cd) than the Wassilewskija (Ws) ecotype that exhibits the full Heavy Metal ATPase3 (HMA3) coding sequence. However, the physiological and molecular mechanisms of HMA3 encoded by point mutation genes in Col-0 remain unknown. In this study, we investigate whether the different metal-related phenotype observed in Col-0 (with HMA3 mutation) when compared to that of Ws (functional HMA3) is a result only of the HMA3 mutation. This investigation was carried out with a further study using plant materials as follows: Ws and Col-0 ecotypes, two HMA3 (Ws) overexpressing lines in Col-0, hma3 knock-out line in Ws. The results indicate that the Col-0 and hma3 mutant in Ws were less tolerant to Cd and Zn because HMA3 has lost the function of sequestration of Cd and Zn into the root vacuoles, thereby readily translocating Cd and Zn to the aerial parts. In addition, the root-to-shoot metal translocation rates of the Ws- and HMA3-overexpressing lines were lower than those of the Col-0 and hma3 mutants. These results indicate that HMA3 is important for the Cd and Zn detoxification in Arabidopsis.     

Fluorescence properties of the Na⁺/H⁺exchanger inhibitor HMA (5-(N,N-hexamethylene)amiloride) are modulated by intracellular pH

HMA (5-(N,N-hexamethylene)amiloride), which belongs to a family of novel amiloride derivatives, is one of the most effective inhibitors of Na+/H+ exchangers, while uneffective against Na+ channels and Na+/Ca2+ exchangers. In this study, we provided evidence that HMA can act as a fluorescent probe. In fact, human retinal ARPE19 cells incubated with HMA show an intense bluish fluorescence in the cytoplasm when observed at microscope under conventional UV-excitation conditions. Interestingly, a prolonged observation under continuous exposure to excitation lightdoes not induce great changes in cells incubated with HMA for times up to about 5 min, while an unexpected rapid increase in fluorescence signal is observed in cells incubated for longer times. The latter phenomenon is particularly evident in the perinuclear region and in discrete spots in the cytoplasm. Since HMA modulates intracellular acidity, the dependence of its fluorescence properties on medium pH and response upon irradiation have been investigated in solution, at pH 5.0 and pH 7.2. The changes in both spectral shape and amplitude emission indicate a marked pH influence on HMA fluorescence properties, making HMA exploitable as a self biomarker of pH alterations in cell studies, in the absence of perturbations induced by the administration of other exogenous dyes.     

Inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition

Human intestinal maltase (HMA) is an ??-glucosidase responsible for the hydrolysis of ??-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with IC₅₀ of 20 ± 1.0 and 31.5 ± 1.0 ??M, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants (K _i ) calculated from a Dixon plot were 5.93 ± 0.26 and 7.88 ± 0.57 ??M, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.     

Effect of perfusate ionized calcium on mechanical activity of the rat heart in deep hypothermia

Effects of different concentrations of ionized calcium ([Ca2+]) in perfusion fluid and of the heart temperature on the systolic pressure (Psyst), heart rate (Vc) and their product as general index of the heart mechanical activity (HMA) of the rat isovolumetrically contracting isolated heart preparations were examined in condition of retrograde perfusion at constant volume with a modified Krebs-Heseleit buffer. An increase of [Ca2+] from 0.62 to 2.5 mM (mmol/l) in the normothermia (38 degrees C) augments Psyst and HMA. Heart rate does not change. An increase of [Ca2+] from 0.62 to 4.5 mM at 20 degrees C augments Vc and HMA. An increase [Ca2+] at 15-11 degrees C decreases psyst and HMA. [Ca2+] does not affect the temperature of the heart arrest.     

横断山区草本沼泽湿地水体氮含量空间分布特征

草本沼泽是横断山区重要的湿地类型,为探明横断山区草本沼泽湿地水体中总氮、硝氮和氨氮的空间分布特征及其控制因素,于2014—2017年在横断山区对典型草本沼泽湿地开展水样采集和分析工作,同时收集降水量、地表大气温度、水温、水深、人口数量、GDP、家畜数量等环境数据。结果表明：1）横断山区草本沼泽湿地水体中总氮含量表现出明显的纬向地带性和垂直地带性：总氮含量随纬度和海拔增加而下降,水温是控制这种地带性特征的主要因素。同时,随经度增加,水体中总氮含量也显著增加,社会经济发展因素是导致这种分布特征的主要原因;2）横断山区沼泽湿地水体中硝氮和氨氮含量在海拔梯度上的变化趋势不明显;3）随经度和纬度增加,沼泽湿地水体中硝氮含量逐渐增加,导致这种现象的主要原因是水深和水温;4）随经度增加,沼泽湿地水体中氨氮的含量逐渐增加,但氨氮含量与自然环境因素和社会经济发展因素均无显著相关性。     

Discovery of novel inhibitors for human intestinal maltase: virtual screening in a WISDOM environment and in vitro evaluation

Human intestinal maltase (HMA) is an α-glucosidase that hydrolyses α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA is an important target to discover of new drugs for the treatment of type 2 diabetes. In this study, 308,307 compounds were virtually screened with HMA using Autodock 3.0.5 in a WISDOM production environment to discover novel inhibitors. The 42 top-scoring free binding energy compounds, representing 17 groups containing potential hydrogen bonding with key residues in the active site pocket of HMA, were tested in vitro for their inhibitory activities against recombinant HMA expressed from Pichia pastoris. Compounds 17 and 18 were competitive inhibitors exclusively for HMA without any in vitro inhibition for human pancreatic α-amylase. The K _i values were 20 μM for both compound 17 and 18.     

Rheology and nanostructure of hydrophobically modified alginate (HMA) gels and solutions

The effect of hydrophobic modification on the mechanical and structural characteristics of hydrophobically modified alginate (HMA) solutions and hydrogels were evaluated. The HMA systems consisted of alkyl chains, C₈, grafted onto alginate backbones. With an increase in degree of substitution of hydrophobic tails, the association became stronger in solution, but same was not true for gels. The contribution of ionic crosslinking was found to be the dominant factor in determining the mechanical strength of hydrogels. Rheological measurements of 2 wt% HMA gels reveal formation of a strongly crosslinked network with an elastic modulus close to 100 kPa. Small-angle X-ray scattering (SAXS) experiments indicate that HMA assembles into a disordered structure with regions rich in the hydrophobic domain surrounded by a crosslinked hydrophilic network.