Selective chemical modification of amino acid residues in the flavin adenine dinucleotide binding site of NADPH-ferredoxin reductase.

1. An apo-NADPH-ferredoxin reductase was prepared from holo-NADPH-ferredoxin reductase (EC 1.18.1.2) from bovine adrenocortical mitochondria. 2. Amino acid residues of the apo-reductase were modified selectively, to identify the FAD-binding site of the reductase, with chemical reagents such as diethylpyrocarbonate, 5,5'-dithiobis(2-nitrobenzoate), tetranitromethane, pyridoxal 5'-phosphate, p-nitrophenylglyoxal, diisopropylfluorophosphate and N-bromosuccinimide. The binding of FAD to the apo-reductase was measured as quenching of the fluorescence of FAD caused by the binding between apo-reductase and FAD. The quenching was blocked when the apo-reductase was modified with diethylpyrocarbonate and restored on the addition of hydroxylamine. 3. The blocking of the quenching occurred in a competitive manner as to FAD in the presence of diethylpyrocarbonate. However, when the apo-reductase was modified with 5,5'-dithiobis(2-nitrobenzoate), the blocking of the quenching occurred in a non-competitive manner. 4. These results suggested that a histidyl residue of the apo-reductase is essential for the binding of FAD to the reductase. This was confirmed by amino acid sequencing of the modified apo-reductase.     

Function of the propeptide region in recombinant expression of active procathepsin L in Escherichia coli.

In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.     

Arabidopsis VPS35, a Retromer Component, is Required for Vacuolar Protein Sorting and Involved in Plant Growth and Leaf Senescence

The above article     

Changes in mechanism of PACAP-induced relaxation in longitudinal muscle of the distal colon of Wistar rats with age

Mechanisms of relaxation of longitudinal muscle of the distal colon induced by exogenously added pituitary adenylate cyclase activating peptide (PACAP) were studied in 2- to 30-week-old Wistar rats. Exogenous PACAP induced very significant relaxation of the longitudinal muscle in 2-week-old rats, but this effect decreased significantly with age. The cyclic AMP-cyclic AMP-dependent protein kinase (PKA) pathway and the tyrosine kinase-small conductance Ca2+-activated K+ channel (SK channel) pathway were found to be involved in the mechanism of PACAP-induced relaxation. In 2-week-old rats, PACAP-induced relaxation was significantly inhibited by tetrodotoxin (TTX). Since relaxation was also significantly inhibited by NG-nitro-L-arginine (N5-nitro-amidino-L-2,5-diamino-pentanoic acid: L-NOARG), the neurogenic effect of PACAP seems to be mediated mainly through nitric oxide neurons. In 8-week-old rats, L-NOARG and TTX had little effect on PACAP-induced relaxation, suggesting that the relaxant effect in 8-week-old rats is a direct action on longitudinal smooth muscle cells. Changes in the mechanisms of PACAP-induced relaxation with age were examined in the distal colon in relation to changes in the neurogenic and the direct effects of PACAP. The neurogenic effect in the exogenous PACAP-induced relaxation of the longitudinal muscle of the Wistar rat distal colon is dominant in tissue isolated from 2-week-old and lost in tissue isolated from 8-week-old rats.     

Structure,regulation, and function of <Emphasis Type="Italic">micro1</Emphasis> in the sea urchin <Emphasis Type="Italic">Hemicentrotus pulcherrimus</Emphasis>

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear -catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of -catenin is highly suggestive. We demonstrated that micro1 is a direct target of -catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.     

Fish oil supplementation in F1 generation associated with naproxen, clenbuterol, and insulin administration reduce tumor growth and cachexia in Walker 256 tumor-bearing rats

Weanling female Wistar rats were supplemented with fish oil (1 g/kg body weight) for one generation. The male offspring received the same supplementation until to adult age. Rats supplemented with coconut fat were used as reference. Some rats were inoculated subcutaneously with a suspension (2 x 10(7) cells/mL) of Walker 256 tumor. At day 3, when the tumor was palpable, rats were treated with naproxen (N) (0.1 mg/mL), clenbuterol (Cb) (0.15 mg/kg body weight), and insulin (I) (10 U/kg body weight). At day 14 after tumor inoculation, the animals were killed. Tumor was removed and weighed. Blood, liver, and skeletal muscles were also collected for measurements of metabolites and insulin. In both tumor-bearing untreated rats and tumor-bearing rats supplemented with coconut fat, tumor growth, triacylglycerol, and blood lactate levels were higher, and glycogen content of the liver, blood glucose, cholesterol and HDL-cholesterol levels were lower as compared with the non-tumor-bearing and fish oil supplemented groups. Fish oil supplementation of tumor-bearing rats led to a partial recovery of the glycogen content in the liver and a full reversion of blood glucose, lactate, cholesterol, and HDL-cholesterol levels. The treatment with N plus Cb plus I attenuated cancer cachexia and decreased tumor growth in both coconut fat and fish oil supplemented rats. In conclusion, chronic fish oil supplementation decreased tumor growth and partially recovered cachexia. This beneficial effect of fish oil supplementation was potentiated by treatment with naproxen plus clenbuterol plus insulin.     

A molecularly imprinted catalyst designed by a computational approach in catalysing a transesterification process

A computational approach was developed to optimize the monomer formulation of molecularly imprinted catalysts. A virtual library of the intermediates of a lipase-catalysed transesterification process was constructed using Chem3D software with p-nitrophenyl acetate as substrate. The energies of the intermediates were minimized using the semi-empirical MOPAC method with the most stable intermediate expected to lead to a higher turn over rate. According to the optimization results, a MIC was prepared by co-polymerising 4(5)-vinylimidazole and itaconic acid with trimethylpropanol trimethacrylate micro spheres in the presence of p-nitrophenyl acetate. The MIC achieved of the transesterification process between p-nitrophenyl acetate and hexanol with a turn over rate of 26.2 min(-1), and showed substrate specificity towards its template with a 6.5-fold preference for p-nitrophenyl acetate over p-nitrophenyl salicylate.