Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases

The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 → 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 → 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.     

Spider toxin (JSTX) on the glutamate synapse

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.     

Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain

By using a cultured neuroblastoma cell line, the present authors recently showed that the N protein of virulent rabies virus fixed strain Nishigahara (Ni), but not that of the attenuated derivative Ni‐CE, mediates evasion of induction of type I interferon (IFN). In this study, to determine whether Ni N protein indeed fulfills this function in vivo, the abilities to suppress IFN responses in the mouse brain of Ni‐CE and the virulent chimeric virus CE(NiN), which has the N gene from Ni in the genetic background of Ni‐CE, were compared. It was demonstrated that CE(NiN) propagates and spreads more efficiently than does Ni‐CE in the brain and that IFN response in brains infected with CE(NiN) is weaker than in those infected with Ni‐CE. It was also shown that amino acids at positions 273 and 394 in the N protein, which are known as pathogenic determinants, affect the ability of the viruses to suppress IFN response in the brain. These findings strongly suggest that, in the brain, rabies virus N protein plays important roles in evasion of innate immune responses and thereby in efficient propagation and spread of virus leading to lethal outcomes of infection.     

Human T-cell leukemia virus type 1 (HTLV-1) infection of mice: proliferation of cell clones with integrated HTLV-1 provirus in lymphoid organs

Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 10(5) spleen cells. Cellular sequences flanking the 3' long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5' LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Abnormal iron delivery to the bone marrow in neonatal hypotransferrinemic mice

Hypotransferrinemic (HP) mice have a splicing defect inthe transferrin gene, resulting in <1% of the normal plasma levels of transferrin. They have severe anemia, suggesting that transferrin is essential for iron uptake by erythroid cells in the bone barrow. To clarify the significance of transferrin on iron delivery to the bone marrow, iron concentration and ⁵⁹Fe distribution were determined in 7-day-old HP mice. Iron concentration in the femur, bone containing the bone marrow, of HP mice was approximately twice higher than in wild type mice. Twenty-four h after injection of ⁵⁹FeCl₃, ⁵⁹Fe concentration in the bone and bone marrow of HP mice was also twice higher than in wild type mice. The present findings indicate that iron is abnormally delivered to the bone marrow of HP mice. However, the iron seems to be unavailable for the production of hemoglobin. These results suggest that transferrin-dependent iron uptake by erythroid cells in the bone marrow is essential for the development of erythrocytes.     

The flavan-3-ol fraction of cocoa powder suppressed changes associated with early-stage metabolic syndrome in high-fat diet-fed rats

Aims

Previous epidemiological studies have suggested that ingestion of chocolate reduces the risk of cardiovascular disease. In the present study, we examined the effects of flavan-3-ols derived from cocoa on blood pressure, lipolysis, and thermogenesis in rats fed a high-fat diet and that showed early signs of metabolic syndrome.

Main methods

The rats were divided into three groups, and fed either normal diet (normal), 60% fat high-fat diet (HFD), or HFD containing 0.2% flavan-3-ols (HFD-flavan) for 4 weeks. At the end of the feeding period, blood pressure was measured and animals were sacrificed under anesthesia. Lipolysis and thermogenesis-related protein levels were measured in several tissues by Western blotting, and mitochondrial DNA copy number was measured by RT-PCR.

Key findings

Mean blood pressure and epididymal adipose tissue weight of HFD-flavan were significantly lower compared with those of HFD. Uncoupling protein (UCP)1 in brown adipose tissue and UCP3 in gastrocnemius of HFD-flavan were significantly increased compared with those of HFD group. Carnitine palmitoyltransferase (CPT) 2 levels in liver and medium-chain acyl-CoA dehydrogenase (MCAD) levels in gastrocnemius and liver were significantly increased by the supplementation of flavan-3-ols.

Significance

In addition to having hypotensive effects, flavan-3-ols enhance thermogenesis and lipolysis and consequently reduce white adipose tissue weight gain in response to high-fat diet feeding.     

A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.