Quantitative analyses of ultrastructure and vascularization of the slow muscle fibres of the anchovy

A quantitative study has been made of the ultrastructure and vascularization of slow fibres in the lateral muscles of the European anchovy (Engraulis encrasicolus). Mitochondria and myofibrils occupy 45.5 and 44.3% of total fibre volume respectively. More than 95% of all myofibrils are adjacent to mitchondria. A total of 51 % of the sarcolemma is in direct contact with capillaries with a mean of 12.9 capillaries per fibre. In transverse sections anchovy slow fibr es are considerably flattened (long to short axis 12:1) such that the surface to volume ratio is more than twice that of a cylindrical fibre of the same area (1115 μm²). The capillary surface required to supply l μm³ of mitochondria is 0.18 μm² and the maximum distance between any capillary and mitochondrion 8 μm. T-system and sarcoplasmic reticulum occupy 0.43 and 2.7% of fibre volume respectively. Adaptations for increasing the capacity of skeletal muscle for aerobic work are discussed.     

Multiplex Amplicon Genotyping by High-Resolution Melting

High-resolution amplicon melting is a simple method for genotyping that uses only generic PCR primers and a saturating DNA dye. Multiplex amplicon genotyping has previously been reported in a single color, but two instruments were required: a carousel-based rapid cycler and a high-resolution melting instrument for capillaries. Manual transfer of capillaries between instruments and sequential melting of each capillary at 0.1°C/s seriously limited the throughput. In this report, a single instrument that combines rapid-cycle real-time PCR with high-resolution melting [LightScanner-32 (LS-32), Idaho Technology, Salt Lake City, UT] was used for multiplex amplicon genotyping. The four most common mutations associated with thrombophilia, F5 (factor V Leiden 1691G>A), F2 (prothrombin 20210G>A), and methylenetetrahydrofolate reductase (MTHFR; 1298A>C and 677C>T) were genotyped in a single homogeneous assay with internal controls to adjust for minor chemistry and instrument variation. Forty temperature cycles required 9.2 min, and each capillary required 2.2 min by melting at 0.3°C/s, 3× the prior rate. Sample volume was reduced from 20 μl to 10 μl. In a blinded study of 109 samples (436 genotypes), complete concordance with standard assays was obtained. In addition, the rare variant MTHFR 1317T>C was genotyped correctly when present. The LS-32 simplifies more complex high-resolution melting assays by reducing hands-on manipulation, total time of analysis, and reagent cost while maintaining the resolution necessary for multiplex amplicon genotyping.     

In vitro phagocytosis by Limulus blood cells.

Limulus blood cells maintained in culture are able to phagocytose particles under conditions where bacterial endotoxin is absent. In the presence of endotoxin, phagocytosis is inhibited because the cells are immobile under these conditions and because the extracellular gel found in the presence of endotoxin prevents cell-particle contact. It is suggested that Limulus blood cells respond to Gram-negative organisms by the formation of an extracellular gel matrix that entraps the bacteria and handles other types of foreign particles by phagocytosis.     

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent K_m for bilirubin of 61.7 μM. Release of the bilirubin from the brain capillaries to the medium was observed at 37°C but not at 4°C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent K_i for the pigment of 137 μM.These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.     

Effect of Escherichia coli endotoxin on adenine nucleotide translocation in canine heart mitochondria

The in vitro effect of Escherichia coli endotoxin on the translocation of adenine nucleotides in dog heart mitochondria was studied. Mitochondrial adenine nucleotides were labeled with ¹⁴C by incubating mitochondrial preparations in the presence of [¹⁴C]ADP. The exchange reaction was initiated by addition of unlabeled ADP, proceeded for 5 to 60 s at 4 °C, and was terminated by addition of atractyloside. The results showed that preincubation of mitochondria with endotoxin (50 μg/mg protein) for 10 min at 23 °C decreased the exchange reaction by 21.2% (P < 0.05). The inhibitory effect of endotoxin was increased with increasing concentrations of endotoxin with an I₅₀ value of 45 μg/mg protein. The initial rate and the total extent of exchange were both affected. Double reciprocal plots showed that only the V but not the K_m for ADP was affected by endotoxin, indicating that the inhibition was noncompetitive in nature. The exchange of adenine nucleotide remained depressed by endotoxin in the presence of either oligomycin or antimycin A, indicating that the inhibitory effect of endotoxin was independent of the action of endotoxin on oxidative phosphorylation. The leakage of labeled adenine nucleotides from mitochondria at 23 °C was increased by 100% by endotoxin (100 μg/mg protein) in the absence of added unlabeled ADP, and this increase in the leakage could not be blocked by atractyloside. The endotoxin-induced changes in adenine nucleotide exchange and leakage were either partially or completely prevented by hydrocortisone, heparin, dibucaine, or EDTA. Since most of these agents have in common an effect on lipid metabolism, it is suggested that endotoxin-induced alterations in the exchange and leakage of adenine nucleotides in heart mitochondria are protected through a mechanism involving membrane lipid reorganization.     

CR3 (CD11b/CD18) activation of nasal neutrophils: a measure of upper airway endotoxin exposure

Inhaled endotoxin (lipopolysaccharide, LPS) initiates an inflammatory response and leads to the expression of CR3 (CD11b/CD18) receptors on polymorphonuclear leukocytes (PMNs). We determined if PMN activation in nasal lavage fluid (NLF) is a possible biomarker of occupational endotoxin exposure. Seven subjects exposed to endotoxin provided NLF samples that were split into three aliquots (negative control – 1?M nicotinamide; sham; positive control – 11 ηg of exogenous LPS) and PMN activation was measured using a chemiluminometer. Differences in mean PMN activation were apparent, negative control: 548?±?15.65 RLU 100 μl^?1; sham: 11469?±?2582 RLU 100 μl^?1; positive control: 42026?±?16659 RLU 100 μl (n?=?7; p <0.05). This technique shows promise as a diagnostic method for measuring upper airway LPS exposure.     

Sensitive detection of C-reactive protein in serum by immunoprecipitation–microchip capillary gel electrophoresis

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP–MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R² = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP–MCGE bears potential for point-of-care diagnosis.     

Delta endotoxin inhibits a phosphatase in midgut epithelial membranes of Heliothis virescens

A vanadate- and delta endotoxin-sensitive phosphatase copurified with plasma membranes and brush border membranes from the midgut epithelium of Heliothis virescens. Phosphatase activity was stimulated under alkaline conditions. Total phosphatase activity was inhibited 60% in the midgut membranes by 360 nM delta endotoxin with a K_i = 76 nM. Brush border membranes were inhibited 75% by 1.47 μM delta endotoxin with a K_i = 64.7 nM. Vanadate (200 μM) completely inhibited the toxinsensitive alkaline phosphatase. A 72 kDa protein was phosphorylated when membranes were incubated with Mg²⁺ and [³²P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.     

Microcapillary reactors using solid-phase DNA sequencing for direct sample introduction into slab gels

Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d. = 100 microns; o.d. = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule. With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary. Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp). The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer. The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel. It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles. The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP). It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format. However, ethanol precipitation was required before gel loading to remove excess terminator.     

Anti-ethylene Effects of cis-2-butene and cyclic olefins

The ability of butenes and cyclic olefins to induce an ethylene response or to counteract ethylene's action in etiolated pea seedlings was investigated. 1-Butene gave an ethylene-like response at 25 OOOμl/l. cis-Butene exhibited no ethylene-like response but appeared to overcome the action of 0.3 μl/l. ethylene at the same concentration. trans-Butene was without effect. 2,5-Norbornadiene competitively inhibited ethylene's action with a K_i of 170 μl/l. (computed as a gas). The K_i for norbornene was 360 μl/l. This value represents about one-half of the activity given by 2,5-norbornadiene. Other K_i values obtained were 488 μl/l. for 1,3-cyclohexadiene, 870 μl/l. for 1,3-cycloheptadiene, 1100 μl/l. for cyclopentene, 4700 μl/l. for 1,4-cyclohexadiene, and 6 ml/l. for cyclohexene. Cyclohexane and benzene were without efrect.     

Development of an electrochemical Limulus amebocyte lysate assay technique for portable and highly sensitive endotoxin sensor

Here, we report the development of an electrochemical detection method for endotoxin based on the Limulus amebocyte lysate (LAL) assay. A mixture of LAL reagent and endotoxin sample solution was incubated for 1 h. The endotoxin activated a cascade reaction of zymogens contained in the LAL to generate p-nitroaniline (pNA) which was then electrochemically detected by differential pulse voltammetry (DPV). The generated pNA gave a clear peak at -0.75 V vs. silver/silver chloride (Ag/AgCl), which increased with the concentration of endotoxin in the LAL assay solution. This DPV detection was performed using an electrode chip device fabricated from a diamond-like carbon-coated glass substrate. This chip device could detect as low as 10 endotoxin units l(-1) at room temperature within 1 h. This novel electrochemical method for the detection of endotoxin appears promising for the development of compact, low-cost and easy-to-use sensors for on-site monitoring of potentially contaminated medical supplies, including dialysis fluid, transplanted tissue and culture medium for assisted reproduction.     

Determination of maprotiline in plasma by high-performance liquid chromatography with chemiluminescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of maprotiline, an antidepressant, in plasma. After a single-step extraction from plasma (100 μl) with n-hexaneisoamylalcohol (19:1, v/v), the drug and desipramine (internal standard) are converted into their chemiluminescent derivatives by reaction with 6-isothiocyanatobenzo[g]phthalazine-1,4(2H,3H)-dione, a new chemiluminescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSKgel ODS-80, using isocratic elution with acetonitrile-100 mM acetate buffer (pH 3.2), and produced chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline medium. The detection limit for maprotiline added to plasma is 0.36 pmol (0.1 ng)/ml plasma (1.5 fmol on column), at a signal-to-noise ratio of 3.     

Bac-to-Bac/BmNPV杆状病毒在家蚕幼虫中表达重组鲎C因子

鲎C因子是鲎血细胞中一种对内毒素具有高亲和力的丝氨酸蛋白酶,被内毒素激活后可水解人工合成的三肽底物,因而能替代传统的鲎试剂用于内毒素检测。通过RT-PCR从东方鲎血细胞中扩增出鲎C因子基因(factor C)序列,利用Bac-to-Bac/BmNPV杆状病毒表达系统在家蚕幼虫中表达该蛋白,并对其进行活性测定。结果显示:被感染的家蚕血清稀释后能检测到Factor C活性,稀释500倍的血清可以用于内毒素检测,且其灵敏度能达到0.2 EU/mL,有望开发新型的、低成本的内毒素检测试剂。     

S-(4-Bromo-2,3-dioxobutyl)CoA: an affinity label for certain enzymes than bind acetyl-CoA.

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site(s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μm) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K_i's (and K_m) were in the range, 30 to 120 μm. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with α1-antagonist prazosin

Chronic vasodilatation represents a stimulus for capillary growth associated with increased luminal shear stress. We have examined the ultrastructure of more than 2000 capillaries to establish whether the sequence of angiogenesis in response to this stimulus is similar to that described during development and under pathological circumstances. Administration of the α₁-blocker prazosin to rats for 2 weeks led to a greater capillary length density in extensor hallucis proprius muscles without any change in capillary tortuosity: J _v(c,f)=262±54 compared with 350±17 mm^–2, control compared with prazosin (P<0.002). There were obvious signs of endothelial cell (EC) activation after prazosin treatment, including an increased proportion of capillaries with rough endoplasmic reticulum, large cytoplasmic vacuoles, thickened endothelium and an irregular luminal surface. Capillaries from control muscles had a maximum of three ECs in cross section, whereas four ECs were noted in 0.8+0.5% of capillaries after 1 week (n.s.) and 2.5±0.9% after 2 weeks (P<0.01) of treatment. This could be due to elongation and/or migration of ECs, as cell proliferation has not been described at these time points. There was also an increase in the proportion of capillaries having a narrow, slit-like lumen (1.7±0.8% of controls; 7.1±1.9% at 1 week; 8.8±2.5% at 2 weeks; P<0.02), some of which were smaller in size (less than 2 μm diameter) than in controls (3–5 μm) and/or “seamless”, i.e. lacking EC junctions. These may represent newly formed vessels. Focal discontinuity of the basement membrane and abluminal EC processes were rarely seen, and capillary growth by abluminal sprouting appeared to be very infrequent (less than 0.001% of profiles). Of more importance was growth starting from the luminal side. Significantly more thin cytoplasmic processes were observed protruding into the lumen of capillaries after 1 week (47.5±6.2%, P<0.001) and 2 weeks of prazosin (34.2±5.5%, P<0.05) than in control vessels (16.7±3.9%). Some of these traversed the entire lumen and connected with endothelium of the opposite side, probably involving membrane fusion, resulting in the appearance of a double lumen. Individual capillaries with a complete double lumen were observed after 2 weeks’ prazosin but comparatively rarely, in only four out of six muscles. These findings indicate a pattern of luminal growth which is completely different from intussusceptive growth previously described during development, and from the abluminal capillary sprouting seen under pathological circumstances.     

Inactivation of endotoxin by Limulus amoebocyte lysate.

The inactivation of bacterial endotoxin by aqueous extracts (Limulus amoebocyte lysate) of the circulating blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus, is described. Active extracts were obtained by heating Limulus amoebocyte lystate (LAL) to 60°C for 20 min to denature the clotting enzyme, rendering the LAL incapable of gel formation in the presence of endotoxin. Endotoxin inactivation was assayed using the Limulus amoebocyte lysate test and by rabbit bioassay. Inactivation of endotoxin with heated extracts of LAL was suggestive of enzymatic mediation, as indicated by dependence on time, temperature, pH, and the kinetics of inactivation. Endotoxin inactivation occurred over a broad pH range, 4.5–8.5, with the optimum at a pH of 6.1. Temperature optima were between 37° and 50°C, with observed activity between 0° and 65°C. Ionized calcium was inhibitory to endotoxin inactivation with heated extracts of LAL, with partial inhibition at 0.001 m calcium and complete inhibition at 0.02 m calcium. Other divalent cations (Mg, Ba, Mn, and Cu) were also found to inhibit the inactivation of endotoxin. Similarities between the endotoxin-inactivating system of L. polyphemus and those described to be present in mammalian and lower vertebrate sera are discussed.     

Endotoxin fever in para-chlorophenylalanine (PCPA) treated newborn guinea pigs and kittens.

Guinea pigs aged 0–3 days responded to 0.2 μg icv. E. coli endotoxin with phasic changes in body temperature; two rises separated by a marked fall. This fall was diminished in PCPA treated animals. In 5–10 day-old kittens the otherwise monophasic endotoxin fever was modified by PCPA treatment; the rise was attenuated in the period corresponding to the transient temperature fall of guinea pigs.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension

The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 μM) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 μM safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(τ→0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 μg).