Oxidatively induced Cu for Mn exchange in protein phosphatase 1gamma: a new method for active site analysis.

Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.

Protein phosphatase inhibitory activity of tautomycin photoaffinity probes evaluated at femto-molar level.

Herein we describe the further improvement of our in-house developed firefly bioluminescence assay system for the determination of inhibition of protein phosphatase (PP). The advantage with the new system is higher sensitivity as well as being time and sample efficient. The inhibition activity of tautomycin with PP1gamma was determined using the upgraded test system and Ki was found to be 4.5 nM, which compare favorably with the activity reported previously by others using different methods. The test system was then used in order to determine the activity of nine tautomycin (TTM) photoaffinity probes. One of the TTM photoaffinity probes (anti-10) was found to possess higher activity than the natural product itself with a Ki of 3.4 nM, while the remaining photoaffinity probes were found to possess Ki in the range of 8.0-213 nM.

High protein buckwheat flour suppresses hypercholesterolemia in rats and gallstone formation in mice by hypercholesterolemic diet and body fat in rats because of its low protein digestibility.

OBJECTIVE: This study evaluated the physiologic properties of high protein buckwheat flour (PBF) by examining its effects on serum cholesterol and body fat in rats and on cholesterol gallstone formation in mice. METHODS: Animals were fed experimental diets that contained casein, buckwheat protein extract (BWP), or PBF as a protein source (net protein content 200 g/kg). RESULTS: In experiment 1, consumption of PBF and BWP for 10 d caused 33% and 31% decreases, respectively, in serum cholesterol of rats fed cholesterol-enriched diets when compared with consumption of casein (P < 0.05). Dietary PBF caused a significant decrease in liver cholesterol, whereas dietary BWP caused only a slight decrease (P > 0.05). Fecal excretion of neutral and acidic steroids in the PBF group was significantly higher than those in the BWP and casein groups. In experiment 2, consumption of PBF for 10 d significantly suppressed adipose tissue weight and hepatic activity of fatty acid synthase in rats fed cholesterol-free diets compared with consumption of casein (P < 0.05), whereas that of BWP for this period caused only a slight decrease in adipose tissue weight (P > 0.05). In experiment 3, dietary PBF and BWP significantly decreased the incidence of cholesterol gallstones and lithogenic index in mice fed cholesterol-enriched diets for 27 d, which was associated with increased fecal excretion of acidic steroids. CONCLUSION: This study demonstrated that PBF has strong activities against hypercholesterolemia, obesity, and gallstone formation, suggesting a potential usefulness of PBF as functional ingredient.

Two types of non-selective cation channel opened by muscarinic stimulation with carbachol in bovine ciliary muscle cells.

In the ciliary muscle, the tonic contraction requires a sustained influx of Ca2+ through the cell membrane. However, little has hitherto been known about the route(s) of Ca2+ influx in this tissue that lacks voltage-gated Ca2+ channels. To identify ion channels as the Ca2+ entry pathway we studied the effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. Experiments were carried out using pipettes filled with K+ -free solution containing 100 mm caesium aspartate, 5 mm BAPTA and 180 microm GTP (pH 7.0; the intracellular free Ca2+ concentration, [Ca2+]i = 70 nm). CCh evoked an inward current showing polarity reversal at a holding potential near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCCL and NSCCS) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li+, Na+, Cs+, Mg2+, Ca2+, Sr2+ and Ba2+, estimated by cation replacement procedures, were 0.9: 1.0: 1.5: 0.2: 0.3: 0.4: 0.5 for NSCCL, and 1.0: 1.0: 1.8: 2.5: 2.6: 3.2: 5.0 for NSCCS. NSCCS, but not NSCCL, was strongly inhibited by elevation of [Ca2+]i. Both NSCCL and NSCCS were dose-dependently inhibited by 1-100 microm SKF96365, La3+ and Gd3+, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the initial phasic component. NSCCL and/or NSCCS may serve as a major Ca2+ entry pathway required for sustained contraction of the bovine ciliary muscle. RT-PCR experiments in the bovine ciliary muscle (whole tissue) detected mRNAs of several transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated cation channels.

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats.

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold exposure.

It has been shown that norepinephrine (NE) can mediate vasodilatation by stimulating the production of nitric oxide (NO) in brown adipose tissue (BAT), resulting in an increase in BAT blood flow. We speculated that constitutive NO synthase (NOS) is involved in this NO production. However, it is not known whether constitutive NOS is expressed in BAT. To answer this question, we assessed the expression of two types of constitutive NOS, endothelial (eNOS) and neuronal NOS (nNOS), in BAT of rats. eNOS was abundantly expressed in both BAT and isolated brown adipocytes, whereas nNOS was not. Cold exposure, which is known to stimulate NE release from sympathetic nerve terminals in BAT, led to a significant increase in eNOS mRNA in this tissue. In contrast, very low levels of inducible NOS (iNOS) mRNA were expressed, and cold stimulation failed to increase iNOS mRNA levels in BAT. These results suggest that eNOS is the primary isoform that is responsible for NO production in BAT and that its expression may be under sympathetic control.

Contribution of Ventral Blood Island (VBI)-Derived Cells to Postembryonic Liver Erythropoiesis in Xenopus laevis: (erythropoiesis/larval hemoglobin/liver/anemia/Xenopus).

The ventral blood islands (VBI) of Xenopus laevis embryos are known as the hemopoietic site where the initial erythropoiesis takes place at st. 28. To determine the site of postembryonic erythropoiesis, larvae were induced anemic by phenylhydrazine (PHZ) at st. 31 and 40, and the tissue distribution of regenerating erythrocytes was determined with an anti-larval hemoglobin (LHb) monoclonal antibody. Three days after total anemia induction, the LHb+ cells were detected first in the liver and the digestive tract, followed by the appearance of a few LHb+ cells in the blood vessels. The lavae which had been hepatectomized and cardiectomized before the PHZ treatment showed a remarkable reduction in recovery of the LHb+ cells. Induction of anemia in the chimeric individuals containing cytogenetically labelled VBI tissues demonstrated that the VBI-derived cells contribute to the regenerating LHb+ cells in all experimental individuals. These results suggest that the larval liver is the major site where the VBI-derived hemopoietic cells reside and differentiate into erythrocytes.

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins: (sea urchin/micromere/protein synthesis/differentiation).

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [35 S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140-kDa, 105-kDa, 43-kDa, 32-kDa, and 28-kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32-kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo, several hours earlier than the onset of spicule formation. The synthesis of 32-kDa protein was paralleled to active spicule formation and the uptake of Ca2+ . Cell-free translation products directed by poly (A)+ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.