The effect of 4-isothiocyanatobenzoic acid on the oxygen-linked chloride binding sites in human hemoglobin: Influence on the alkaline Bohr effect

Human oxyhemoglobin reacted with 4-isothiocyanatobenzoic acid shows a decreased oxygen affinity that does not change with increasing chloride concentration indicating that all of the oxygen-linked chloride binding sites are blocked in the modified protein. By contrast, reaction of oxyhemoglobin with 4-isothiocyanatobenzenesulfonamide produces a modified protein with increased oxygen affinity below pH 7.3 that shows the expected decrease in oxygen affinity with increasing chloride concentration. The latter result demonstrates the importance of the negatively charged moiety in producing both the decrease in oxygen affinity and the effect on the oxygen-linked chloride binding sites produced by 4-isothiocyanatobenzoic acid. Reduction in the alkaline Bohr effect by 50% in the protein modified by 4-isothiocyanatobenzoic acid indicates that contribution to the alkaline Bohr effect is evenly divided between chloride dependent and chloride independent groups.     

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.     

Acquirement of fetal properties in hepatoma on glutathione metabolism

Glutathione peroxidase/glutathionè reductase activity ratio was determined in the high-speed supernatant fraction of the rat livers. The ratio was dependent on age and the ratio increase gradually with the increase in age. The fetal liver showed a ratio of 1.5–2.0, which was almost the same value to those of the 4-dimethylaminoazobenzene-induced primary hepatoma and some transplantable hepatomas originating from the azodye-induced hepatoma. Four cell lines of transplantable ascites hepatoma examined in this study showed the value of 1.2–1.8 for the activity ratio, however, the values of two strains were found to be 2.8–3.0, even though these cell lines were also originated from the azodye-induced hepatoma.Glutathione contents of azodye-induced hepatoma and ascites hepatomas were also similar with those of fetal rat livers.The acquirement of the fetal properties in hepatoma was discussed in relation to glutathione metabolism.     

Identification of 4-acetoxyaminoquinoline from the hydrolysis of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline,in vitro and in vivo properties

4-Acetoxyaminoquinoline (Ac-4-HAQ) (1) was identified as a hydrolysis product of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline (diAc-4-HAQO). The reaction allowing the obtention of (1) obeys to a reduction mechanism implying the N₁-O cleavage. The carcinogenic properties of (1) observed by Sato et al. (Japan J. Exp. Med., 40 (1970) 475) in mice were studied in rats with the in vivo system we used previously with 4-nitroquinoline-1-oxide (4-NQO) and 4-hydroxyaminoquinoline-1-oxide (4-HAQO). In rats (1) does not covalently bind DNA. It was, therefore, possible to propose an interpretation of the results obtained by Enomoto et al. (Proc. Soc. Exp. Biol. Med., 136 (1971) 1206) who injected diAc-4-HAQO s.c. to mice and rats. Compound 1 could be responsible for the carcinogenic effects observed through the following pathway: (1) should be formed by hydrolysis of diAc-4-HAQO and reactivated by an enzymatic system to N-oxide derivative, the 4-acetoxyaminoquinoline-1-oxide (Ac-4-HAQO), which constitutes an ultimate carcinogen model of 4-NQO.     

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, β-sitosterol, Δ5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1515 mg/100 g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100 g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100 g oil) was detected in H52 at 14 DAF. During linseed development, β sitosterol (830 mg/100 g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100 g oil) and stigmasterol (265 mg/100 g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Synthesis of metabolism-resistant substrates for the transport system for cationic amino acids; their stimulation of the release of insulin and glucagon, and of the urinary loss of amino acids related to cystinuria

Several amino acids have been synthesized as model transport substrates building on the piperidine and cyclohexane rings. Only when the distal N atom is part of an unambiguously cationic structure are these compounds transported predominantly by the cationic amino acid system. These amino acids in labeled form are excreted rather slowly in unmodified state, very little ¹⁴CO₂ being released. Those which are unambiguously cationic (including also homoarginine) led to a greatly increased excretion of arginine, lysine, ornithine and citrulline. Those which might be expected to act as lysine analogs had little effect on the excretion of the basic amino acids, although the excretion of citrulline and the sum of glutamine plus asparagine was accelerated. Certain of the analogs intensified the excretion of citrulline in dissociation from effects on resorption of the basic amino acids, also in dissociation from effects on cystine resorption. These results indicate citrulline resorption does not occur principally by the same agency serving for the basic amino acids, nor by the agency serving for cystine, despite the observed interactions for resorption. The injection of either of three transport analogs for arginine into the rat leads to early increases in the circulating levels of immunologically reactive insulin and glucagon.     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Ontogenic expression pattern and genetic polymorphisms of the retinol-binding protein 4 (RBP4) gene in Erlang mountainous chickens

Retinol-binding protein 4 (RBP4) is the only circulatory transport protein for vitamin A. Based on the essential role of vitamin A in chicken reproduction, we measured RBP4 mRNA abundance in Erlang mountainous chickens. We also identified and analyzed the gene polymorphism and its effect on reproduction traits among 349 chickens. The expression of RBP4 mRNA showed specific developmental changes and striking differences among tissues. The mRNA abundance was greatest (P < 0.05) in the liver, intermediate in the ovary, kidney, small intestine, oviduct and heart, and lowest in the hypothalamus and pituitary, as compared to all other tissues (P < 0.05). We detected one single nucleotide polymorphism (g.19942455C>G) in intron 2 of the RBP4 gene. Three genotypes (CC, CG and GG) were identified, with a significant effect of genotype on the age at first egg (AFE), first egg weight (FEW), total eggs at 300 days (TE300), highest continuous laying days (HCLD) and average laying interval (ALI). The GG genotype, where chickens display earlier AFE, more TE300, longer HCLD and shorter ALI, would be genetically advantageous and its selection may improve reproduction traits. These results suggested that the RBP4 gene might play an important role in reproduction traits in chickens.     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。     

4,4,4-三氟乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

对实验室保藏菌种进行筛选,得到一株葡萄汁酵母Saccharomyces uvarum SW-58,对其产酶条件进行优化,其发酵培养基组成:醋酸钠60 g/L,玉米浆30 g/L,KH2PO46 g/L,MgSO4.7H2O 1 g/L;培养条件为:发酵温度30℃,初始pH 6.0,发酵周期36 h。4,4,4-三氟乙酰乙酸乙酯羰基还原酶酶活最高可达388.1 U/L,产物的浓度由优化前的3.5 g/L提高到4.6 g/L,所得产物的光学纯度由优化前的60.8%e.e.提高到85.0%e.e。     

COL4A3 mutations cause focal segmenta glomerulosclerosis

Focal segmental glomerulosclerosis （FSGS） is a histologically identifiable gtomerular injury often leading to proteinuria and renal failure. To identify its causal genes, whole-exome sequencing and Sanger sequencing were performed on a large Chinese cohort that comprised 40 FSGS families, 50 sporadic FSGS patients, 9 independent autosomal recessive Atport＇s syndrome （ARAS） patients, and 190 ethnically matched healthy controls. Patients with extrarenal manifestations, indicating systemic diseases or other known hereditary renal diseases, were excluded. Heterozygous COL4A3 mutations were identified in five （12.5%） FSGS families and one （2%） sporadic FSGS patient. All identified mutations disrupted highly conserved protein sequences and none of them was found in either public databases or the 190 healthy controls. Of the FSGS patients with heterozygous COL4A3 mutations, segmental thinning of the glomerular base membrane （GBM） was only detected in the patient with electronic microscopy examination results available. Five ARAS patients （55.6%） had homozygous or compound-heterozygous mutations in COL4.43 or COL4A4. Serious changes in the G BM, hearing loss, and ocular abnormalities were found in 100%, 80%, and 40% of the ARAS patients, respectively. Overall, a new sub- group of FSGS patients resulting from heterozygous C01.4A3 mutations was identified. The mutations are relatively frequent in famiUes diagnosed with inherited forms of FSGS. Thus, we suggest screening for C01.4A3 mutations in familial FSGS patients.     

Studies on the mechanism of action of the aromatase inhibitor, 4-hydroxyandrostenedione

[1 beta-3H], [1 alpha,2 alpha-3H] and [1 beta,2 beta-3H] 4-Hydroxyandrostenedione (4-OH-A) were synthesized to study the mechanism of inhibition of aromatase by 4-OH-A. Incubations of [1 beta-3H] and [1 beta,2 beta-3H] 4-OH-A with placental microsomes in the presence of NADPH showed very little loss of tritium, with aromatization of 4-OH-A ranging from 0.3 to 0.6 percent. No loss of tritium was observed in the absence of NADPH. The extent of covalent binding of 4-OH-A to microsomal proteins was higher with incubations in the absence of NADPH than with those in the presence of NADPH. These results are discussed in light of what has been proposed for the mechanism of androgen aromatization.     

Anti-tubercular activity of novel 4-anilinoquinolines and 4-anilinoquinazolines

We screened a series of 4-anilinoquinolines and 4-anilinoquinazolines and identified novel inhibitors of Mycobacterium tuberculosis (Mtb). The focused 4-anilinoquinoline/quinazoline scaffold arrays yielded compounds with high potency and the identification of 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (34) with an MIC₉₀ value of 0.63–1.25 µM. We also defined a series of key structural features, including the benzyloxy aniline and the 6,7-dimethoxy quinoline ring, that are important for Mtb inhibition. Importantly the compounds showed very limited toxicity and scope for further improvement by iterative medicinal chemistry.     

The effect of 4-isothiocyanatobenzene sulfonic acid on the oxygen affinity of human hemoglobin

Human hemoglobin reacts with 4-Isothiocyanatobenzene sulfonic acid at the four amino groups of the N-terminal valines. The modified protein shows a decreased oxygen affinity over a wide pH range, a reduced alkaline Bohr effect, decreased co-operativity, and a reduced effect of inositol hexasulfate on the oxygen affinity.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.