Age-dependent aluminum accumulation in the human aorta and cerebral artery

The purpose of this study was to determine the extent of aluminum (Al) accumulation in the human aorta and cerebral arteries. The Al contents in the aortae and in the cerebral arteries from 23 human subjects was determined by inductively coupled plasma atomic emission spectrophotometry (ICP-AES). The subjects' age range was 45–99-yr-old; 15 of the subjects were males and 8 were females. Al was detected in twelve aortae and in six cerebral arteries, when the entire specimen was analyzed. Two specimens where Al was found in the cerebral arteries contained no Al in the aorta. No relationship to the subject's sex was found. When related to age, two groups were established. Group L (45–75 yr old) and group H (>75 yr old), which exhibited aortal Al concentrations of 33.3 and 72.7%, respectively. When the aortic wall was dissected into the tunica intima, media, and adventitia, Al was found mainly in the tunica media. In the aorta, significant relationships were found between Al and phosphorus (P) levels (r=0.801,p<0.01) and between Al and calcium (Ca) (r=0.661,p<0.05). We have concluded that Al accumulation is age-dependent and that it occurs both in the aorta and in the cerebral artery. In the aorta, accumulation occurs mainly in the tunica media. Both P and Ca appear to enhance aortal Al accumulation.     

Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity

NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC₅₀ of 0.95 μM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.     

Improvement of Intracellular Traffic System by Overexpression of KDEL Receptor 1 in Antibody‐Producing CHO Cells

The localization of soluble endoplasmic reticulum (ER) chaperones in the cell organelle is mediated by the C‐terminal KDEL (lysine, aspartic acid, glutamic acid and leucine) motif. This motif is recognized by the KDEL receptor, a seven‐transmembrane protein that cycles between the ER and cis‐Golgi to capture missorted KDEL chaperones from post‐ER compartments in a pH‐dependent manner. The KDEL receptor's target chaperones have a substantial role in protein folding and assembly. In this study, the gene expression level of KDEL receptor 1 shows a moderate upregulation during either ER stress or growth of Chinese hamster ovary (CHO) cells in batch culture, while the ER chaperones show higher upregulation. This might indicate the possibility of saturation of the ER retention machinery or at least hindered retention during late stage batch culture in recombinant CHO cells. KDELR1 is overexpressed in a monoclonal antibody‐producing CHO cell line to improve the intracellular chaperone retention rate in the ER. An increase in the specific productivity of IgG1 by 13.2% during the exponential phase, and 23.8% in the deceleration phase of batch culture is observed. This is the first study to focus on the ER retention system as a cell engineering target for enhancing recombinant protein production.     

Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions

Enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(n)Galbeta1,4GlcNAcbeta-pNP (n=1-4) was demonstrated using a transglycosylation reaction of Escherichia freundii endo-beta-galactosidase. The enzyme catalyzed a transglycosylation reaction on GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1), which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl beta-glycosides GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(1)Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(2)Galbeta1,4GlcNAcbeta-pNP (3), GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(3)Galbeta1,4GlcNAcbeta-pNP (4) and GlcNAcbeta1,3(Galbeta1,4GlcNAcbeta1,3)(4)Galbeta1,4GlcNAcbeta-pNP (5). When 2 was used as an initial substrate, it led to the preferential synthesis of nonasaccharide beta-glycoside 4 to heptasaccharide beta-glycoside 3. This suggests that 4 is directly synthesized by transferring the tetrasaccharide unit GlcNAcbeta1,3Galbeta1,4GlcNAcbeta1,3Gal to nonreducing end GlcNAc residue of 2 itself. The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-beta-galactosidase was significantly enhanced by the addition of BSA and by a low-temperature condition. Resulting 2 and 3 were shown to be useful for studying endo-beta-galactosidase-catalyzed hydrolytic and transglycosylation reactions.     

Behavioral responses to the alarm pheromone of the ant Camponotus obscuripes (Hymenoptera: Formicidae)

The alarm pheromone of the ant Camponotus obscuripes (Formicinae) was identified and quantified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Comparisons between alarm pheromone components and extracts from the major exocrine gland of this ant species revealed that the sources of its alarm pheromone are Dufour's gland and the poison gland. Most components of Dufour's gland were saturated hydrocarbons. n-Undecane comprised more than 90% of all components and in a single Dufour's gland amounted to 19 microg. n-Decane and n-pentadecane were also included in the Dufour's gland secretion. Only formic acid was detected in the poison gland, in amounts ranging from 0.049 to 0.91 microl. This ant species releases a mixture of these substances, each of which has a different volatility and function. When the ants sensed formic acid, they eluded the source of the odor; however, they aggressively approached odors of n-undecane and n-decane, which are highly volatile. In contrast, n-pentadecane, which has the lowest volatility among the identified compounds, was shown to calm the ants. The volatilities of the alarm pheromone components were closely related to their roles in alarm communication. Highly volatile components vaporized rapidly and spread widely, and induced drastic reactions among the ants. As these components became diluted, the less volatile components calmed the excited ants. How the worker ants utilize this alarm communication system for efficient deployment of their nestmates in colony defense is also discussed herein.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.     

Absorption, migration and kinetics in peripheral blood of orally administered ovalbumin in a mouse model

Intestinal absorption of food proteins is well known, whereas its physiological significance remains to be investigated. Various amounts (1, 10 and 50 mg) of ovalbumin were orally administered to mice and the blood kinetics were subsequently analyzed by two-site ELISA. The blood ovalbumin concentration consistently reached its maximum (7-90 ng/ml) about 20 min after the oral administration and then gradually decreased in a dose-dependent manner. Only intact (45 kDa) and truncated (40 kDa) ovalbumins were always detected in the blood independently of the administration site, intra-stomach or intra-intestine, while various fragments of the protein were observed in the gastrointestinal lumen after the oral administration. Recognition by a specific monoclonal antibody and an acidic shift of its pI value suggested that the 40-kDa truncated ovalbumin was produced by intracellular limited proteolysis at its C-terminus. Such stable absorption and blood kinetics of undigested ovalbumin in normal mice suggest some sort of physiological significance for the intestinal uptake of intact food proteins.     

Increases of Calcium,Phosphorus, and Magnesium in Both the Right and Left Fibrous Trigones of Human Heart with Aging

We previously reported that reduced platelet endogenous antioxidant enzymes activities are related to the low plasma zinc level in patients with end-stage renal failure (ESRF). In this study, we attempt to evaluate whether dietary zinc deprivation reduces the activities of endogenous antioxidant and then enhances oxidative stress in the unstimulated platelet of normal and 5/6 nephrectomized (Nx) rats because increased platelet oxidative stress is suggested to involve in the incidence of thrombotic and atherosclerotic diseases. Male Sprague–Dawley rats (n = 48) were fed a zinc-deficient diet and deionized distilled water for 1 week to induce reduction of plasma zinc level. Half of the rats continued on this diet for 4 weeks as zinc-deplete group, and the other half were maintained on the same diet but with zinc-supplemented water (120 mg/L zinc sulfate solution) to correct the reduction of plasma zinc level as zinc-replete group. Half of each group underwent 5/6 Nx, while the other half underwent sham operation. Another 12 normal rats were fed standard rat chow (containing 23.4% protein and 50 ppm zinc) and drank deionized distilled water as normal control rats. In zinc-deplete rats including sham-operated and 5/6 Nx rats exhibited lower endogenous antioxidant enzymes activities such as reduced glutathione (GSH), superoxide dismutase (SOD), and glutathione peroxidase (GPX) and higher malondialdehyde (MDA) levels than normal control rats in the unstimulated platelets. However, in zinc-replete rats including sham-operated and 5/6 Nx rats have a normal endogenous antioxidant enzymes activity and normal MDA levels in the unstimulated platelets. We suggest that in uremia, the low plasma zinc level may be a risk factor for thrombotic and atherosclerotic diseases because it reduces the activities of endogenous antioxidant enzymes and increases oxidative stress in the unstimulated platelet. Supported by grant 92-117 from Taipei Veterans General Hospital