P1 Trisaccharide (Galalpha1,4Galbeta1,4GlcNAc) synthesis by enzyme glycosylation reactions using recombinant Escherichia coli

The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galalpha1,4Galbeta1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori beta-l,4-galactosyltransferase and a Neisseria meningitidis alpha-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.     

A vector with the downstream box of the initiation codon can highly enhance protein expression in Escherichia coli

An expression vector, pET-DB, with a perfectly matching downstream box of the initiation codon has been constructed on the basis of the pET system. Any gene of interest can then be inserted into the vector. Four genes were used to test the expression efficiency of the vector. The results show that the vector pET-DB can further increase protein expression level at least up to 35–70% as compared with the initial T7 expression system, indicating that the downstream box can enhance protein expression in Escherichia coli.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Drosophila Free-Running Rhythms Require Intercellular Communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

Application of high-performance liquid chromatography-mass spectrometry to detection of diuretics in human urine

A rapid, sensitive and reliable high-performance liquid chromatographic-mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C(18) column (150 x 2.1 mm, 5 microm). The mobile phase consisted of formic ammonium-formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75-95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25-25 ng/ml for APCI and 0.6-250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC-APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.     

Size and stability of reconstituted sesame oil bodies

Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and oil-body proteins. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil-body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted oil bodies via their central hydrophobic domains. The stability of reconstituted oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross-linked by glutaraldehyde or genipin.     

Effect of IFNgamma on caspase-3, Bcl-2 and Bax expression, and apoptosis in rabbit placenta

The purpose of this study was to determine whether apoptosis in placenta was affected by IFNgamma, which can induce abortion, and whether the effect of IFNgamma on apoptosis resulted from an intrinsic program of apoptosis, which was regulated by Bcl-2 and Bax. DNA fragmentation analysis indicated that cleavage of DNA into 180 bp and its polymers were recognized in placenta in control and IFNgamma treated groups. Quantitative analysis of low molecular weight fragments of DNA revealed a significant increase in cases of 100,000 IU IFNgamma treatment compared with those in normal pregnancy (P<0.05). An analysis in situ revealed that apoptosis occurred predominantly in syncytiotrophoblast. Expression of Bcl-2 and Bax in placenta was evaluated by immunoblot analysis and immunohistochemistry study. Bcl-2 was expressed predominantly in syncytiotrophoblast, and was not expressed in cytotrophoblast of all cases. Whereas Bax was expressed in cytotrophoblast, syncytiotrophoblasts were found to be negative for Bax protein expression in all cases. Both Bcl-2 and Bax expression was decreased 0.44 fold and 0.46 fold by 50,000 IU IFNgamma and 0.41 fold and 0.03 fold by 100,000 IU IFNgamma. This resulted in change of a 0.07 fold increase in the Bax:Bcl-2 ratio in 50,000 IU IFNgamma treated groups and 0.41 fold increase in 100,000 IU IFNgamma treated groups as compared with those in control groups. The difference in Bax to Bcl-2 ratio between control and 100,000 IU IFNgamma treated groups was significant (P<0.05). The localization of caspase-3, the executioner of apoptosis, was detected in some cytotrophoblast and syncytiotrophoblast and increased 0.03 fold and 0.68 fold in 50,000 IU IFNgamma and 100,000 IU IFNgamma treated groups, respectively. There was significant difference between control and 100,000 IU IFNgamma treated groups (P<0.05). The results showed that high dose of IFNgamma administration increased the extent of apoptosis in placenta, the Bax to Bcl-2 ratio, and the activated caspase-3.     

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

Effect of synthetic oligopeptides on osteoporosis

The objective of this study was to establish the solution method of GHRPS, the synthetic oligopeptides Tyr-Gly-Gly-Phe-Met-NH2, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Gly-NH2, and Tyr-Gly-Gly-Phe-Gly-OH, to verify their effect on osteoporosis. Male ICR mice (20 +/- 2 g) were used. The intramuscular injection dose of 6.3 mg/kg prednisone induced a significant decrease of body and femur weight of the animals. The subcutaneous injection dose of 18 microg/kg synthetic peptide was not effective to prevent the decrease of body and femur weight of the animals. The subcutaneous injection dose of 6.3 mg/kg prednisone elicited a decrease in content of femur calcium and in the level of serum calcium of the animals. The subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Leu-NH2, or Tyr-Gly-Gly-Phe-Leu-OH, or Tyr-Gly-Gly-Phe-Gly-NH2, significantly increased the content of femur calcium and decreased the level of serum calcium of the animals. It was also observed that the subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Gly-OH, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Met-NH2 significantly increased the content of femur phosphorous and decreased the activity of ALP of the animals.