A MEMS SOI-based piezoresistive fluid flow sensor.

In this paper, a SOI (silicon-on-insulator)-based piezoresistive fluid flow sensor is presented; the presented flow sensor mainly consists of a nylon sensing head, stainless steel cantilever beam, SOI sensor chip, printed circuit board, half-cylinder gasket, and stainless steel shell. The working principle of the sensor and some detailed contrastive analysis about the sensor structure were introduced since the nylon sensing head and stainless steel cantilever beam have distinct influence on the sensor performance; the structure of nylon sensing head and stainless steel cantilever beam is also discussed. The SOI sensor chip was fabricated using micro-electromechanical systems technologies, such as reactive ion etching and low pressure chemical vapor deposition. The designed fluid sensor was packaged and tested; a calibration installation system was purposely designed for the sensor experiment. The testing results indicated that the output voltage of the sensor is proportional to the square of the fluid flow velocity, which is coincident with the theoretical derivation. The tested sensitivity of the sensor is 3.91 × 10-4 V ms2/kg.

Induction of Cytochrome P450 Activity by the Interaction of Chlorantraniliprole and Sinigrin in the Spodoptera exigua (Lepidoptera: Noctuidae).

The joint toxicity of chlorantraniliprole, a novel insecticide that acts on ryanodine receptors, and sinigrin, a natural plant defense compound from brassicaceous vegetables, to the larvae of Spodoptera exigua (Hübner) was determined in this paper. Additionally, the joint effects of the two compounds on cytochrome P450 enzyme activity and on the expression levels of mRNA of three P450 genes (including CYP9A9, CYP6B, and CYP4G37) and an NADPH cytochrome P450 reductase gene (HQ852049) were investigated. The toxicity of the mixture of chlorantraniliprole and sinigrin to fourth-instar S. exigua larvae was 1.60-fold higher than the toxicity of the chlorantraniliprole-only treatment after 24 h. Induced by chlorantraniliprole and sinigrin, the specific activity of the P450 O-deethylase was affected in a time-, dose-, and organ-specific manner in fifth-instar S. exigua larvae. The effects were more pronounced in the midgut than in the fat body. The specific activity of the P450 O-deethylase in almost all treatments increased at 12, 24, and 36 h posttreatment compared with that in the control. Based on real-time PCR analyses, the expression levels of the P450 genes CYP9A9, CYP6B, and CYP4G37 and the NADPH cytochrome P450 reductase gene HQ852049 in fifth-instar S. exigua larvae were induced by chlorantraniliprole and sinigrin, and the trends were similar to the specific activity of the P450 O-deethylase. Therefore, the CYP9A9, CYP6B, and HQ852049 in the tested genes were the most inducible genes that were expressed when the S. exigua larvae were exposed to chlorantraniliprole and sinigrin.

The relationship between IL-6 in CSF and occurrence of vasospasm after subarachnoid hemorrhage.

BACKGROUND: It is hypothesized that inflammatory response after subarachnoid hemorrhage (SAH) may play a relevant role in the development and maintenance of vasospasm. This research investigated the correlation between IL-6 in cerebrospinal fluid (CSF) after SAH and the occurrence of vasospasm. METHODS: We analyzed both daily clinical manifestation and laboratory data of CSF in 46 patients who suffered from intracranial aneurismal subarachnoid hemorrhage during a period of 14 days, studied the relationship between the development of vasospasm and the quantities of the inflammatory factor, revealing potential power of IL-6 for predicting vasospasm detected by transcranial doppler (TCD). RESULTS: The incidence of vasospasm developed in 43.5% of the patients, with a mean onset of 6.1±4.6 days after intracranial aneurysm treatment. Patients with vasospasm demonstrated statistically significant higher median values of IL-6CSF on Day 1, 2, 3, 5 and 7 (P<0.05). The cut-off value is settled in 400 pg/ml on Day 3 after treatment. On the other hand, gender, Hunt & Hess scale (H&H) and Fisher scale of CT after SAH were proved to be the correlation factor with vasospasm. CONCLUSION: IL-6CSF seems to be a reliable early marker for predicting vasospasm after subarachnoid hemorrhage on Days 3 after treatment before clinical onset.

Proteolytic activation of membrane-bound guanylate cyclase.

Membrane-bound guanylate cyclase-A (GC-A), the receptor for atrial natriuretic factor (ANF), has been shown to be regulated by its kinase-like domain. To resolve the nature of this regulation, we measured the effects of various proteases on the activity of guanylate cyclase in rat lung membranes, and on the activity of the bacterial-expressed catalytic domain (GC-c) and on a recombinant peptide composed of both the kinase-like and catalytic domain (GC-kc) of guanylate cyclase. Pronase increased rat guanylate cyclase activity in a biphasic manner with a maximal effect at about 10-20 microg per assay tube. Thermolysin had effects similar to those of pronase on the activity of guanylate cyclase in rat lung membranes. In the case of bacterial-expressed proteins, pronase increased the activity of GC-kc, but not GC-c. These results indicate that GC-A contains an autoinhibitory site on its kinase-like domain, and that removal of the autoinhibitory site by limited proteolysis leads to enzyme activation. GC-A was poorly activated by ANF and ATP after the rat lung membrane was pretreated with pronase, suggesting that ANF/ATP and pronase activate guanylate cyclase through the same mechanism. It is suggested that the binding of ANF and ATP to GC-A may induce a conformational change of the receptor that releases the inhibitory constraint on enzyme activity leading to enzyme activation.

Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene.

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

Mutational inactivation of the catalytic domain of guanylate cyclase-A receptor.

Guanylate cyclase-A, the receptor for atrial natriuretic factor, contains a protein kinase-like domain and a catalytic domain in the intracellular region. To investigate the active site (the catalytic cavity) of guanylate cyclase-A, we amplified the catalytic domain plus three amino acids from the kinase-like domain of guanylate cyclase-A (GC-c) with polymerase chain reaction (PCR) and expressed it in Escherichia coli. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Results of cDNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutated protein. The mutated GC-c bound to GTP-agarose as well as the wild-type protein, indicating that the binding capability of mutated GC-c to GTP is not significantly affected by the Arg substitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weight complex. Since mutation of Leu 817 to Arg abolishes the catalytic activity, Leu 817 is likely located near the active site of guanylate cyclase-A. These results demonstrate that the carboxyl fragment of guanylate cyclase-A is an ideal system for studying the active site of guanylate cyclase-A.

Structural requirements of mastoparan for activation of membrane-bound guanylate cyclase.

Mastoparan activated membrane-bound guanylate cyclase and potentiated the effect of atrial natriuretic factor (ANF) and ATP on guanylate cyclase activity in rat lung membranes. Mastoparan is a cationic, amphiphilic tetradecapeptide with an amidated carboxyl terminus. It takes the alpha-helical conformation upon interacting with the membrane. Several analogs were synthesized to study the role of the positive charges, the carboxyl amino group and the alpha-helical conformation of mastoparan in the activation of guanylate cyclase. The results showed that substitution of the C-terminal amide group of mastoparan with a carboxyl group significantly reduced its potency on the activation of guanylate cyclase. Replacement of three lysine residues of mastoparan with aspartic acid or serine residues completely abolished the stimulatory effect of mastoparan. When the alanine at position 10 of mastoparan was substituted by a proline, the resulting analog had no effect on guanylate cyclase activity. These results demonstrate that the positive charges and the helical structure of mastoparan are critical determinants for the activation of guanylate cyclase.

Melittin potentiates guanylate cyclase activation stimulated by atrial natriuretic factor and ATP.

The biologically relevant receptor for atrial natriuretic factor (ANF) has been shown to be membrane-bound guanylate cyclase. While guanylate cyclase is known to be activated by ANF and ATP, the molecular mechanism of the enzyme activation remains unclear. We now show that melittin, the main peptide toxin of bee venom, activates membrane-bound guanylate cyclase and potentiates ANF- and ATP-stimulated guanylate cyclase activity in rat lung membranes. Melittin stimulated basal guanylate cyclase activity by increasing the Vmax without significantly affecting the Km of the substrate, GTP. However, melittin enhances ANF- and ATP-stimulated enzyme activity by altering both the Vmax and the EC50 of ANF and ATP. Although melittin activates guanylate cyclase in crude membranes, it has little effect on the activity of the purified enzyme. The effect of melittin on guanylate cyclase activation in rat lung membranes is attenuated by the Ca2+ chelator, EGTA. These results suggest that the effects of melittin on guanylate cyclase activation may require the participation of accessory proteins or nonprotein factors. Therefore, melittin would be a valuable tool for exploring the molecular mechanisms of ANF-mediated guanylate cyclase activation.

Suppression of experimental autoimmune myasthenia gravis with new immunosuppressants: 15-deoxyspergualin and actinobolin.

In the search for a new drug to treat myasthenia gravis, we studied the efficacy of new immunosuppressants on experimental autoimmune myasthenia gravis (EAMG). 15-Deoxyspergualin (15-DSP), bactobolin and actinobolin were administered to some groups at the time of immunization and to other groups 10 days after. The most effective results were achieved with doses of 2.5 mg/kg daily of 15-DSP and 30 mg/kg daily of actinobolin administered from day 1. In both groups, the body weights of the rats increased as normally as those of controls and signs of myasthenia were mild. Immunoelectron microscopic examination of the neuromuscular junctions in rats treated with 2.5 mg/kg of 15-DSP appeared normal, even in the chronic phase (induced by a booster at week 4). Levels of anti-acetylcholine receptor antibodies were almost completely suppressed. Although the effects of these drugs were more remarkable when administered from day 1 than from day 10, the results suggest that they may prove useful in treating myasthenic patients.

Formycin triphosphate as a probe for the ATP binding site involved in the activation of guanylate cyclase.

Formycin A triphosphate (FTP), a fluorescent analog of ATP, slightly increased basal guanylate cyclase activity, but significantly potentiated guanylate cyclase activity stimulated by atrial natriuretic factor (ANF) in rat lung membranes. FTP potentiated ANF-stimulated guanylate cyclase activity with an EC50 at about 90 microM and inhibited ATP-stimulated guanylate cyclase activity with an IC50 at about 100 microM. These results indicate that FTP binds more tightly than ATP for the same binding site. Therefore, FTP would be an excellent tool for studying the ATP binding site.

Pharmacokinetic studies in the rat on a o-raffinose polymerized human hemoglobin.

We have studied the plasma half-life (T 1/2), oxygen-binding affinity (P50), organ distribution, and excretion of the individual molecular weight (MW) components of human hemoglobin polymerized with periodate-oxidized, ring-opened raffinose (oR poly-Hb), following transfusion in the rat. The model was an isovolemic 50% exchange transfusion in the conscious, chronically catheterized rat. Total plasma Hb levels yielded a (T 1/2) of 10 to 11 hr for oR poly-Hb. The T 1/2 values of individual MW components of the poly-Hb as determined by size-exclusion HPLC were approximately: 4 hr for the monomeric fraction (Hb)1, 9 hr for the dimer (Hb)2, and 15 hr for the fraction representing trimers to nanomers (Hb)3-9. The P50 values of plasma samples containing oR poly-Hb (collected from 0-24 hr after exchange) remained unchanged at 28 +/- 3 mmHg. oR stabilized and polymerized Hb were not excreted via the kidneys. Hepatic and renal distribution as well as plasma and renal clearance were determined by liquid scintillation counting using individual tritium [3H] labelled MW components purified from [3H]-oR poly-Hb: (Hb)1/2, (Hb)1, (Hb)2, (Hb)3&4, and (Hb) greater than 9. In kidney, uptake (determined by the relative concentration of radioactivity) decreased with increasing MW of the labelled component. Conversely, in liver, uptake increased with increasing MW. Plasma and renal clearance results were consistent with those obtained by HPLC analysis. Hematocrit levels returned from a 20% post-transfusion level to normal pre-transfusion levels (44%) within 10 days after the exchange.

[Ultrastructural immunocytochemical localization of electron-transport enzymes in mitochondrial myopathy].

Ultrastructural localization of mitochondrial electron transport enzymes in biopsied muscle from a patient with mitochondrial myopathy was studied. We applied the immunoelectron microscopic technique using colloidal-gold labeled antibodies. The results demonstrated a very distinct and dense labeling by gold particles of complexes I, III, and IV in the inner mitochondrial membrane. The density of gold particles reacted to complex IV was cleanly decreased. This result was correlated with the decreased biochemical activity of complex IV. Labeling on the paracrystalline inclusions was substantially decreased in abnormal giant mitochondrial, but gold particles were extensively confined on the membrane of cristae surrounding the inclusions.

Development of more potent atrial natriuretic factor (ANF) analogs.

Four modified atrial natriuretic factor (ANF) analogs were designed and synthesized by the solid-phase method. Using human ANF-(99-126) as the reference compound the receptor binding affinity and biological activity of these analogs were examined by radioreceptor assay and in vivo experiments. PLO68, a 21 amino peptide with structural modifications des-Ser103,104-[Mpr105,D-Ala107,114]APII-amide exhibited 2.5 and 2.2 fold more activity than human ANF-(99-126) in lowering blood pressure and causing natriuresis in urethane anesthetized rats. Receptor binding assays using rat lung membranes showed that PLO68 had a Kd of 200 +/- 12 pM compared to a Kd of 620 +/- 12 pM for human ANF-(99-126). Similar chemical modifications, except for substitution of glycine and alanine at the positions 115 and 118, and 120 by D-alanine resulted in three analogs PLO63 and PLO64, PLO67 respectively. PLO63, PLO64 and PLO69 had similar affinities and in vivo potency as human ANF-(99-126). These data suggest that the structural modifications made in PLO68 can cause an increase in the receptor binding ability and an enhancement of biological activities.

[Study on biosynthesis of indigo involving transferring naphthalene plasmid DNA from Pseudomonas to E. coli].

Indigo is one of the brilliant blue dyes, which was used to be extracted from plants, but now synthesized chemically. Indigo is also produced by bacteria. In recent years, Ensley, B. D. (1983) and Mermod, N. (1986) reported a pathway producing indigo of bacteria. We are currently studying indigo formation by bacteria. Using Pseudomonas sp. S13 harboring naphthalene degradation plasmid as donor and E. coli as recipient, conjugates and transformants with the plasmid were obtained. The conjugates and transformants were not only able to grow in medium with naphthalene as the sole carbon and energy source, but also to synthesize indigo in Lennox medium. The conjugates and transformants were grown in Lennox medium at 30 degrees C for 48 hr then resulted in synthesis of indigo. The production of indigo is increased in the presence of tryptophan or indole. Indigo formation was enhanced if the bacteria was grown in a medium supplemented with either 0.1% of naphthalene or 1% of salicylic acid. The present work opens a field for the synthesis of dyes by microbes and it might shed light on the potential use in controlling environmental pollution by engineering bacteria.

Brain natriuretic factor. Augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding.

The newly discovered peptide, brain natriuretic factor (BNF), caused a concentration-dependent increase (up to 400-fold) in intracellular cyclic GMP levels in cultured endothelial, smooth muscle and fibroblast cells. The extent of cGMP augmentation was comparable to that produced by atrial natriuretic factor (ANF). The activity of the membrane-bound guanylate cyclase of different rat tissues and cultured cells was markedly stimulated by the peptide and the addition of ATP potentiated the stimulation. As opposed to tissue particulate guanylate cyclase, the enzyme in cell membranes was slightly more sensitive to activation by BNF than to stimulation by ANF. On bovine aortic smooth muscle (BASM) cells, specific high-affinity binding sites (Bmax = 398 fmol/10(6) cells, Kd = 0.52 nM) for BNF were observed for which ANF could compete with apparently equal affinity. These results suggest that activation of the cGMP pathway constitutes a common mechanism of action for both BNF and ANF.