Influence of iron-saturation of plasma transferrin in iron distribution in the brain

Based on the evidence that iron distribution in the peripheral tissues is changed by iron-saturation of plasma transferrin, the influence of iron-saturation of plasma transferrin in iron delivery to the brain was examined. Mouse plasma was pre-incubated with ferric chloride in citrate buffer to saturate transferrin and then incubated with (59)FeCl(3). Peak retention time of (59)Fe was transferred from the retention time of transferrin to that of mercaptalbumin, suggesting that iron may bind to albumin in the plasma in the case of iron-saturation of transferrin. When mice were intravenously injected with ferric chloride in citrate buffer 10 min before intravenous injection of (59)FeCl(3), 59Fe concentration in the plasma was remarkably low. (59)Fe concentration in the liver of iron-loaded mice was four times higher than in control, while 59Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of (59)FeCl(3), brain autoradiograms also showed that (59)Fe concentrations in the brain of iron-loaded mice were approximately 40-50% of those of control mice in all brain regions tested except the choroid plexus, in which (59)Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain.     

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.     

Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.     

Glycosidase-catalyzed deoxy oligosaccharide synthesis. Practical synthesis of monodeoxy analogs of ethyl beta-thioisomaltoside using Aspergillus niger alpha-glucosidase

Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl alpha-D-glucopyranoside (Glc alpha-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glc alpha-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl beta-D-thioglucopyranoside (Glc beta-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger alpha-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH3CN (1:1 v/v) at 37 degrees C. High activity of the enzyme was observed in the reaction between 2D-Glc alpha-O-pNP and Glc beta-S-Et to afford the monodeoxy analogs of ethyl beta-thiomaltoside and ethyl beta-thioisomaltoside that contain a 2-deoxy alpha-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-alpha-D-arabino-hexopyranosyl-(1,4)-beta-D-thioglucopyranoside and ethyl 2-deoxy-alpha-D-arabino-hexopyranosyl-(1,6)-beta-D-thioglucopyranoside, in 6.72% and 46.6% isolated yields (based on 2D-Glc alpha-O-pNP), respectively. Moreover, from 3D-Glc alpha-O-pNP and Glc beta-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl beta-thioisomaltoside that was modified at the glycon alpha-D-glucopyranose moiety, namely ethyl 3-deoxy-alpha-D-ribo-hexopyranosyl-(1,6)-beta-D-thioglucopyranoside, in 23.0% isolated yield (based on 3D-Glc alpha-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glc alpha-O-pNP and Glc beta-S-Et.     

pH-induced conformational change in an alpha-helical coiled-coil is controlled by His residues in the hydrophobic core

An alpha-helical coiled-coil structure is one of the basic structural units in proteins. Hydrophilic residues at the hydrophobic positions in the coiled-coil structure play important roles in structures and functions of natural proteins. We reported here a peptide that formed a triple stranded alpha-helical coiled-coil showing the pH-dependent structural change. The peptide was designed to have two His residues at the hydrophobic positions of the center of the coiled-coil structure. The peptide folded into a triple stranded coiled-coil at neutral pH, while it unfolded at acidic pH. This construct is useful to create a protein that the structure or function is controlled by pH.     

Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle

To determine the molecular mechanism underlying hyperglycemia-induced insulin resistance in skeletal muscles, postreceptor insulin-signaling events were assessed in skeletal muscles of neonatally streptozotocin-treated diabetic rats. In isolated soleus muscle of the diabetic rats, insulin-stimulated 2-deoxyglucose uptake, glucose oxidation, and lactate release were all significantly decreased compared with normal rats. Similarly, insulin-induced phosphorylation and activation of Akt/protein kinase B (PKB) and GLUT-4 translocation were severely impaired. However, the upstream signal, including phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and -2 and activity of phosphatidylinositol (PI) 3-kinase associated with IRS-1/2, was enhanced. The amelioration of hyperglycemia by T-1095, a Na(+)-glucose transporter inhibitor, normalized the reduced insulin sensitivity in the soleus muscle and the impaired insulin-stimulated Akt/PKB phosphorylation and activity. In addition, the enhanced PI 3-kinase activation and phosphorylation of IR and IRS-1 and -2 were reduced to normal levels. These results suggest that sustained hyperglycemia impairs the insulin-signaling steps between PI 3-kinase and Akt/PKB, and that impaired Akt/PKB activity underlies hyperglycemia-induced insulin resistance in skeletal muscle.     

Characterization and amino acid sequences of cytochromes c6 from two strains of the green alga Chlorella vulgaris

Cytochromes c6 from the green algae Chlorella vulgaris CK-5 (CK5cyc6) and C. vulgaris CK-22 (CK22cyc6) were characterized and their amino acid sequences were analyzed. CK5cyc6 had a molecular mass of 9.3 kDa, isoelectric points of 3.0 (reduced) and 3.6 (oxidized), and a redox potential of +362 mV at pH 7.0. CK22cyc6 had a molecular mass of 9.5 kDa, isoelectric points of 2.9 (reduced) and 3.5 (oxidized), and a redox potential of +355 mV at pH 7.0. The absorption spectra of both cytochromes c6 showed 4 maxima in reduced form, and 2 maxima and a weak peak at 695 nm in oxidized form. The pyridine ferrohemochrome spectra indicated that their prosthetic group was heme c. These physicochemical properties were similar to those of other algal cytochromes c6. The amino acids (88 residues) of CK5cyc6 and CK22cyc6 were sequenced and the sequence motif -CXXCH-, which is typical of the heme-binding site of c-type cytochrome, was clearly confirmed in both cytochromes. Twenty-six amino acid residues were substituted, and the similarity score of each of them was 70.45%.     

Hydrolytic activity of alpha-galactosidases against deoxy derivatives of p-nitrophenyl alpha-D-galactopyranoside

The four possible monodeoxy derivatives of p-nitrophenyl (PNP) alpha-D-galactopyranoside were synthesized, and hydrolytic activities of the alpha-galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger alpha-galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters (K(m) and Vmax) of the enzymes for the hydrolysis of PNP alpha-D-galactopyranoside and its deoxy derivatives were obtained from kinetic studies.     

Hepatic zinc response via metallothionein induction after tumor transplantation

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.