0.95 W high-repetition-rate, picosecond 335 nm laser based on a frequency quadrupled, diode-pumped Nd:YVO(4) MOPA system.

An efficient all-solid-state picosecond (ps) ultraviolet (UV) laser at 335 nm was demonstrated based on frequency quadrupling of a mode-locked 1342 nm MOPA system. An output power of 0.95 W was obtained under a fundamental wave power of 16.38 W, corresponding to a conversion efficiency of 5.8% from infrared to UV. The repetition rate and pulse duration were 77 MHz and 20.2 ps, respectively. The beam quality factor M(2) was measured to be 1.56. This is, to the best of our knowledge, the highest output power at 335 nm.

An electrochemical surface plasmon resonance imaging system targeting cell analysis.

This paper presents an electrochemical-surface plasmon resonance imaging (EC-SPRI) system, enabling the characterization of optical and electrical properties of cells, simultaneously. The developed surface plasmon resonance (SPR) imaging system was capable of imaging micro cavities with a dimension of 10 µm × 10 µm and differentiated glycerol solutions with a group of refractive indices (RIs). Furthermore, the EC-SPRI system was used to image A549 cells, suggesting corresponding RI and morphology changes during the cell death process. In the end, electrochemical and SPR methods were used in combination, recording oxidation peaks of A549 cells in the cyclic voltage curves and SPR response unit increase, simultaneously.

Apparatus for measuring the Seebeck coefficients of highly resistive organic semiconducting materials.

A Seebeck coefficient measurement apparatus for high resistance organic semiconductor materials has been designed and built. It can measure materials with resistance over 7 × 10(12) Ω. This is the highest material resistance value ever reported for Seebeck coefficient measurement. A cyclic temperature gradient generation technique and a corresponding algorithm are proposed to eliminate the negative effects of the long term drift of Seebeck voltage. Sources of errors in these measurements are discussed.

A poly(dimethylsiloxane) based prism for surface plasmon resonance imaging system and its application for gas detection.

This paper presents a surface plasmon resonance (SPR) imaging system based on a low-cost, convenient poly(dimethylsiloxane) (PDMS) prism featured with a close contact with the gold film. Compared to conventional glass prism, both numerical simulations and experimental studies indicated a deeper but wider absorption peak with a higher coupling angle for the PDMS based prism. System repeatability was quantified by the cycled detection of helium and air, with the effect of the flow rate investigated. Furthermore, five types of gases (nitrogen, air, oxygen, hydrogen, and helium) were detected and differentiated by the SPR system, with a calculated sensitivity of 5 × 10(-6) RIU.

A micro gas chromatography column with a micro thermal conductivity detector for volatile organic compound analysis.

In this paper, a micro gas chromatography (µGC) system contained a µGC column and a micro thermal conductivity detector (µTCD) was proposed. In order to reduce the volume of the system, some micro heaters were integrated on the surface and backside of the GC column, which could provide a robust temperature programming capability and rapidly increase the temperature of the µGC column. In addition, a silicon-glass µTCD with four-thermistor thermal conductivity cells that can offer significant advantages over previously reported designs including low dead volume, good thermal isolation, and elimination of the thermal noise was proposed in this paper. Experimental results have indicated that the µGC system with a detection limit of several ppm concentration levels separated and detected the benzene, toluene, and styrene in less than 3 min, and the µGC system also exhibited a good linear response in the test range.

Toll-like receptor 2 type 1 and type 2 polymorphisms in different chicken breeds.

Toll-like receptors mediate immune responses via recognition of pathogen-associated molecular patterns. Polymorphisms of toll-like receptors may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known about the polymorphisms of chicken toll-like receptors (ChTLR) among breeds. In this study, we cloned ChTLR2 type 1 and type 2 genes from 7 chicken breeds and analyzed their sequences. It was found that there were 10 amino acid polymorphism sites in ChTLR2 type 1 and type 2, among which 6 sites were in type 1 (5 sites in the extracellular domain and 1 site in the cytoplasmic domain) and 4 sites were in type 2 (all 4 sites in the extracellular domain). These results demonstrate that ChTLR2 type 1 and type 2 genes are polymorphic among chicken breeds, suggesting a varied resistance among breeds of chicken. We found that Laiwu Black chicken breeds have distinctive polymorphic sites at I699T in the type 1 and H561R in the type 2 ChTLR2 gene. Beijing Fatty chickens have distinctive sites at Q45R in type 1 and V66L in type 2. Nongda No.3 Chickens have distinctive sites at L115P, H232Y, and T494A in type 1. Hy-Line variety brown chickens have distinctive sites at I311V in type 2. Beijing White 939 chickens have distinctive sites at E284G in type 1 and A22V in type 2. These findings may provide some clues toward understanding the resistance of lighter chickens to salmonellosis.

A novel method for polypeptide design to prepare specific antibody of the peptide and applied to immunoassay.

Peptide was polymerized by a novel method: compound containing double bond between two carbons such as acdryloyl chloride was introduced into peptide during peptide synthesis, then it was transformed to a polymer which has a poly propionyl core matrix with peptide branches by radical polymerization either after being cleaved from resin or before being cleaved from resin according to the peptide physical-chemical character. According to this design, the macromolecules with average MW about 40 kD for poly-Osteogenic Growth Peptide (poly-OGP), 25 KD for poly-penetratin could be produced. Poly-OGP was further applied for antibody preparation and immunoassays. Immunizing New Zealand rabbits, we obtained the antiserum with titer of 2.5x10(4), examined by enzyme-linked immunosorbent assay (ELISA), without cross-reaction with bovine serum albumin (BSA), while the antiserum produced by using BSA as peptide carrier strongly reacted with BSA (with titer of more than 50x10(4) for BSA). Based on competitive ELISA, the anti-poly-OGP antibody showed much better immunoreactive sensitivity than anti-BSA-OGP antibody by comparing their IC(50) toward OGP. The antigen determinant of OGP and the OGP content in the serum of mice, determined by anti-poly-OGP antibody, showed that the anti-polypeptide antibody can be used as tools for immunoassay. Thus, the polypeptide system is not only a new approach for preparing synthetic peptide antibody for immunoassays but also provided the prospect for preparing synthetic peptide-based vaccine.

Rapid microscale enzymic semisynthesis of epidermal growth factor (EGF) analogues.

Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFalpha, VGF, heparin-binding EGF, and betacellulin), suggests that Leu47, which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu47 results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF1-45 core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF1-45 core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF1-45 in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF1-47 from EGF1-45 with equivalent mitogenic and receptor binding activity to EGF1-47 generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or nonphysiological amino acids (e.g. allo-Ile) for Asp46, Leu47 or Arg48. Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.

Monomeric destetrapeptide human insulin from a precursor expressed in Saccharomyces cerevisiae.

Destetrapeptide insulin (DTI, human insulin with B27-30 removed) was obtained from a monomeric precursor (MIP) expressed in Saccharomyces cerevisiae through tryptic transpeptidation in the presence of synthetic tetrapeptide Gly-Phe-Phe-Tyr. The in vivo biological activity of DTI, determined by mouse convulsion assay, is 22 IU/mg. Its binding activity with insulin receptor on human placental membrane is 80% and its in vitro biological activity, determined by free fat cell assay, is 77%. Compared with native insulin, DTI molecules do not associate in solution but exist in the monomeric form, thus leading to its rapid utilization in vivo.

Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.

Effects of orphanin FQ on endomorphin-1 induced analgesia.

Orphanin FQ (also known as nociceptin) is a 17-amino-acid peptide which acts as a potent endogenous agonist of the orphan opioid receptor-like (ORL1) receptor. Endomorphin-1, a 4-amino-acid peptide discovered recently, is a potent and selective endogenous agonist for the mu-opiate receptor. In the present study, the effect of OFQ or/and endomorphin-1 on the response to noxious thermal stimuli was observed using the tail-flick test in rats. Intracerebroventricular (i.c.v.) administration of OFQ (1, 5 microg) could shorten tail-flick latency; In contrast, intrathecal (i.t.) administration of OFQ (1, 2 or 10 microg) could increase the latency; i.c.v. (1, 2, 5 microg) or i.t. (0.2, 2, 5 microg) administration of endomorphin-1 dose-dependently increased the latency, indicating an analgesic effect. Furthermore, OFQ (0.1-5 microg) when intraventricularly injected together with endomorphin-1 (5 microg), could dose-dependently reverse the analgesia induced by the latter. On the contrary, OFQ (1 microg) intrathecally injected together with endomorphin-1 (0.2 microg) could further increase the tail-flick latency. The results showed that OFQ at the supraspinal level produces hyperalgesia and is antagonistic to endomorphin-1, while at the spinal level it produces analgesia and is synergic with endomorphin-1. Different interaction mechanism between OFQ and endomorphin-1 in the brain and the spinal cord is thus suggested.

Chaperone activity of DsbC.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.

Antagonistic effect of orphanin FQ on opioid analgesia in rat.

AIM: To study the effect of orphanin FQ (OFQ), a newly discovered heptadecapeptide, on nociception and opioid analgesia. METHODS: The intracerebroventricular (i.c.v.) and intrathecal (i.t.h.) injections were used to give the drugs. The tail-flick model of rats were used to test the pain threshold. RESULTS: OFQ (i.c.v. or i.t.h.) 0.1 microgram had no effect on nociception but 0.5-10 micrograms induces hyper-reaction of rat to noxious electric stimulus; the decapeptide (OFQ1-10 i.c.v.), a fragment of the OFQ, did not affect the pain reaction of rats. Fentanyl (1 microgram, i.c.v. or i.t.h.), a selective mu-receptor agonist, DSLET (5 micrograms, i.c.v. or i.t.h.), a selective delta-receptor agonist, or U50488H (1 microgram, i.t.h.), a kappa-receptor agonist, induced an increase in pain threshold, when OFQ (0.1 or 1 microgram) was added together with one of them (except for the ith injection of DSLET), the increase of pain threshold was reduced obviously. CONCLUSION: OFQ induces hyperalgesia and antagonizes opioid analgesia mediated by mu- and delta-receptors in the brain and by mu- and kappa- but not delta-receptors in the spinal cord of rats.

[Effect of intrathecal or intracerebroventricular administrition of OFQ on pain threshold and acpuncture analgesia in rats].

Orphanin FQ (OFQ) is a newly discovered 17-amino-acid peptide capable of inducing hyperalgesia. In the present study, the effects of OFQ on basal pain threshold and acupuncture anlgesia (AA) in rats were observed using the tail-flick test. It was found that intrathecal (i.t.) or intracerebroventricular (i.c.v.) administrition of 0.1 microgram OFQ had no effect on basal pain threshold of rats, while 1 microgram OFQ could lower the threshold. However, OFQ at both the doses (0.1 or 1.0 microgram) administered by either i.t. or i.c.v. injection could antagonize AA with that occuring in the brain being more prominent then in the spinal cord. When the rats were repeatedly treated with antisense oligonucleotide to block synthesis of OFQ receptor, pain threshold increased significantly. At such instance, when the OFQ was combined with acupuncture, the effect of AA showed no obvious change. The above results show that the OFQ at small dose has no effect on pain threshold but can lower it at larger dose; while in both cases OFQ can antagonize AA.

Antagonistic action of orphanin FQ on acupuncture analgesia in rat brain.

The influence of orphanin FQ (OFQ) (a newly discovered 17-amino acid peptide) on acupuncture analgesia (AA) was assessed in rat tail-flick model. Intracerebroventricular (i.c.v.) injection of OFQ (1 microgram) elicited a significant decrement of pain threshold which was abolished by the repeated pretreatment with antisense oligonucleotide (ASO) to OFQ receptor. Electroacupuncture (EA) induced an obvious analgesic effect; when OFQ was used combined with EA, it showed a dose-dependent effect on antagonizing the EA analgesia. When rat was repeatedly i.c.v. injected with ASO to block the synthesis of OFQ receptor, the EA analgesia was enhanced markedly. In this instance, the OFQ did not show antagonistic effect on EA analgesia any more. The results suggest that the OFQ play its antagonistic role on EA analgesia via activating OFQ receptor.

Assignment of the binding site for tissue plasminogen activator on human fibronectin.

The tissue-type plasminogen activator (t-PA) has been found to bind reversibly to human fibronectin (Fn). To locate the binding site on Fn for t-PA, the Fn was degraded with N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin, and the resulting fragments were monitored by the enzyme-linked immunosorbent assay method for t-PA binding activities. A 20-kDa fragment with t-PA binding activity was identified, separated, and purified. It was subjected to further degradation with Staphylococcus aureus proteinase V8. An active 10-kDa fragment was finally purified by reverse-phase high pressure liquid chromatography on a C3 column. The dissociation constants of the binding of Fn and the 10-kDa fragment to t-PA were estimated by Scatchard plot to be 1.13 x 10(-8) and 2.08 x 10(-8) M, respectively. The 10-kDa fragment was sequenced and proved to be located at the 8-9th domains of type I homology of Fn. Based on the structural analysis of the 8-9th domains, a heptadecapeptide corresponding to the sequence Thr535-Glyl551 of Fn, which resided at the large disulfide loop of domain (I-9), was designed and synthesized. Both the 10-kDa fragment and the synthetic peptide could competitively inhibit the binding of Fn to t-PA. The synthetic peptide showed about one-tenth of the binding activity of Fn to t-PA with a dissociation constant of 1.35 x 10(-7) M and was proved to be the binding region of Fn for t-PA. In addition, like the intact Fn, both the 10-kDa fragment and the synthetic peptide could remarkably enhance the amidolytic activity of t-PA in a dose-dependent manner, as shown by using S-2288 as a chromogenic substrate.

The structure basis of the poor fibrin specificity of urokinase (II)--The inhibition of urokinase A chain 149-157 on the fibrin stimulated activation of plasminogen by tissue type plasminogen activator.

In view of the similarity of the charge distribution between fibrin A alpha 148-161 and A chain 149-157 of urokinase, the latter might compete with fibrin A alpha 148-161 when single chain pro-urokinase is converted to double chain urokinase. To test this, the stretch of urokinase A chain 135-157 was separated from the low molecular weight urokinase, a competitive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-binding assay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen was demonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149-157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the presence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154 and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fibrin stimulated activation of plasminogen by tPA. These results suggest that the positively charged residues in the stretch 149-157 of urokinase are crucial for the inhibition of fibrin binding with the kringle domain of urokinase.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF): identification of a binding site for a neutralizing antibody.

One approach to the localization of functionally active regions of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal antibodies. We have defined the epitope recognized by one neutralizing antibody (LMM102) using proteolytic fragments obtained by enzymic digestion of bacterially synthesized hGM-CSF. RP-HPLC fractionation of a tryptic digest resulted in the identification of an immunoreactive "tryptic core" peptide containing 66 amino acids (52% of the protein). Further digestion of this "tryptic core" with S. aureus V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. The individual peptides, generated by reduction with dithiothreitol, were not recognized by the antibody. An analog of this peptide has been synthesized chemically and shown to have similar immunoreactivity to the epitope obtained by enzymic digestion. A series of modified peptides has also been synthesized to identify further the region required for antibody recognition.

Structure and activity of the B-chain of insulin.

Despentapeptide (B26-30)-insulinamide (B25) prepared by a semisynthetic procedure was found to have about 65% of the hypoglycaemic activity of natural insulin. In contrast, the binding of the modified insulin analogue to insulin specific receptors was markedly increased. The discrepancy between the loss of biological potency and the apparent increase in binding affinity for membrane receptors reveals that not all of the biological activity of insulin is regulated by the receptor-binding system. The tetrapeptidamide of the B-chain of insulin (Arg-Gly-Phe-Phe-NH2) was clearly shown to have both insulin-like and insulin-potentiating actions in vivo although it had no effect on insulin receptor function in vitro. Evidence suggests that the small peptide fragment of insulin may be internalized and acts at the post-binding site(s) of the glucose metabolic pathway in target tissues. The present data support the general concept that insulin may exert its complex molecular actions through internalized hormonal fragment as well as the transmembrane mediators generated from receptor binding.