Rapid Determination of Bacteriological Water Quality by Using Limulus Lysate

The Limulus lysate assay was used to measure the endotoxin content in stream water and was found to reflect the degree of bacterial contamination as measured by coliform, enteric, gram-negative, and heterotrophic bacteria. The firm-clot method was found to be a less sensitive and reproducible technique for the detection of endotoxin than was the spectrophotometric modification of the Limulus lysate assay. Bound endotoxin, as determined by the spectrophotometric modification of the Limulus lysate assay, was found to be a better measure of the endotoxin associated with bacterial cells than was total endotoxin.     

鲎试剂在抗淋巴细胞免疫球蛋白热原检测中的应用

报道了鲎试剂在抗淋巴细胞免疫球蛋白内毒素检查中的应用。在用鲎试剂检测细菌内毒素时 ,按中国药典的要求对鲎试剂灵敏度进行标定 ,并作干扰实验 (增强或抑制实验 ) ,然后用鲎试法与家兔法同时测定 15批抗淋巴细胞免疫球蛋白。结果发现三批鲎试剂的灵敏度标示值均正确 ,五批抗淋巴细胞免疫球蛋白半成品在最大有效稀释度 (MVD)时对试验无干扰 ,这两种方法测定结果的符合率达到 91.7%。从而表明鲎试法很有可能代替家兔法     

Preparation, Sensitivity, and Specificity of Limulus Lysate for Endotoxin Assay

Limulus amoebocyte lysate was prepared from a total of 180 crabs during 1971 and 1972 by using a slightly modified lysate preparation procedure. Marked variability of lysate potency was noted both years. In addition, lysate quality appeared diminished in 1972 as compared with 1971. Different lysate batches were evaluated for potency by using a variety of endotoxin preparations. Variations in batch potencies were observed, but little variation in reactivity among different endotoxin preparations was noted. Use of potent lysate batches allowed detection of endotoxin concentrations as low as 100 pg/ml. No endotoxinlike activity was observed from 11 different strains of yeast by use of the Limulus assay.     

The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.     

Evaluation of the Limulus Amebocyte Lysate and Recombinant Factor C Assays for Assessment of Airborne Endotoxin

As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) samplers, Button samplers, closed-face cassettes, and cyclone samplers. Field sampling was performed in 10 livestock production facilities, including those housing swine, chicken, turkey, dairy cows, cattle, and horses. Laboratory sampling was performed in exposure chambers using resuspended airborne dust collected in five livestock facilities. Paired samples were extracted in pyrogen-free water with 0.05% Tween 20 and analyzed using LAL and rFC assays. In 402 field sample pairs there was excellent agreement between endotoxin concentrations determined by LAL and rFC (r = 0.93; P < 0.0001). In 510 laboratory sample pairs there was also excellent agreement between the two assays (r = 0.86; P < 0.0001). Correlations for subgroups of facility or dust type ranged from 0.65 to 0.96. Mixed-model analysis of variance (ANOVA) for the field studies showed significant interactions of facility-sampler and facility-assay. rFC/LAL ratios of the geometric means were 0.9 to 1.14 for the samplers (not significantly different from 1.0). The data from this study demonstrate that the LAL assay and the rFC assay return similar estimates of exposure in livestock facilities. Both methods provided suitable lower limits of detection such that all but 19 of 1,824 samples were quantifiable.Endotoxin, or lipopolysaccharide, is a pathogen-associated molecular pattern of Gram-negative bacteria that associates with MD-2 (lymphocyte antigen 96) to act as a ligand for Toll-like receptor 4 (10). Through this process, inhaled endotoxin induces lung inflammation and can both increase neutrophilic asthma and decrease allergy and atopic asthma (2, 8, 9, 17, 26, 33). Endotoxin is an inflammatory component of most organic dusts (6). Exposure to endotoxins in agricultural dusts, including swine, poultry, and grain, has been associated with asthma, chronic bronchitis, organic dust toxic syndrome (toxic pneumonitis), chronic obstructive pulmonary disease, and declines in pulmonary function (5, 18, 24, 25, 35, 36, 38). High endotoxin exposure also occurs in municipal composting (37), seed and bulb handling (27), and wastewater treatment plants (13, 28), to name a few. In addition to occupational environments, endotoxin exposure in domestic environments is a risk factor for asthma (33), with high occupancy, poverty, pets, pests, and household cleanliness being the major predictors of exposure (32).The kinetic chromogenic Limulus amebocyte lysate (LAL) assay is the most widely used assay for endotoxin measurement for environmental samples (6). This assay uses an endotoxin-triggered enzyme cascade from the Atlantic horseshoe crab (Limulus polyphemus) to cleave a colorimetric substrate. Although the LAL assay is exquisitely sensitive, variability can arise from interlot variations in the Limulus lysate and differences in laboratory methods for sample collection, sample handling and storage, sample extraction, and sample analysis (7, 12, 14, 15, 19, 21, 29, 30, 34). In addition, some implementations of the LAL assay may experience interference from other molecules, such as fungal (1→3)-β-d-glucans (3, 22). A recombinant factor C (rFC) assay that uses rFC reagent produced from the cDNA of the Mangrove horseshoe crab (Cacinoscorpius rotundicauda) was recently developed (4). Since the rFC assay uses a recombinant reagent, the reactivity to endotoxins should vary less between lots than for the Limulus lysate.The goal of this study was to determine the comparability, across a wide range of endotoxin levels, of the kinetic chromogenic LAL and the fluorometric rFC assays for assessing airborne endotoxin. To accomplish this, we performed air sampling in 10 livestock environments and in chamber studies using resuspended dust collected from these livestock environments. Livestock environments are recognized as containing considerable amounts of airborne endotoxin arising from a variety of Gram-negative bacteria. Eight samples were collected simultaneously on a rotating mannequin using pairs of four types of commonly used samplers: two for inhalable dust (Institute of Occupational Medicine [IOM] and Button), one for total dust (closed-face cassette), and one for respirable dust (SKC aluminum cyclone). This resulted in 912 pairs of samples (total n = 1,824).     

Detection of Microbial Translocation in HIV and SIV Infection Using the Limulus Amebocyte Lysate Assay is Masked by Serum and Plasma

Objective

Microbial translocation (MT) is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS) from Gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results.

Methods

Specimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175) and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and –uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection.

Results

When exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100%) samples at 20% plasma concentration compared to control; in contrast 5/10 (50%) samples at 2% plasma concentration (p = 0.07) and 0/10 (0%) at 0.1% plasma concentration (p = 0.004) showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42%) of samples at 2% serum concentration compared to control, while 0/12 (0%) of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07). Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17%) human samples in 2% serum, ranging from 3,436–4,736 pg/mL, compared to 9/12 (75%) samples in 0.1% serum, ranging from 123 pg/mL –60,131 pg/mL (p = 0.016). In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p<0.05).

Conclusions

Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.     

Synthetic Studies on Cyclopentane Derivatives

A new synthetic route to prostaglandin-F₁ skeleton from readily accessible 2-carboxyhexyl-cyclopentane-1,3,4-trione was achieved. The route included 2-alkyl-3-cyano-4-hydroxy-2-cyclopenten-1-one as an intermediate.     

Synthetic Studies on Cyclopentane Derivatives

Reactions of enol ethers of cyclopentane-1,3-dione derivatives (I) with cyanide ion were investigated in order to develope new synthetic routes to 3-functionalized-2-cyclopenten-1-one derivatives from I.

I could be converted to the 3-cyano-2-cyclopenten-1-one skeleton by several procedures for hydrocyanation, among which Nagata’s reagents (HCN-triethylaluminium, diethylaluminium cyanide) were proved to be potent ones.

Reactions of enol ethers of 4-hydroxy-cyclopentane-1,3-dione derivatives were also investigated. From 4-hydroxy-3-methoxy-2-cyclopenten-1-one derivatives (V) 1,4-addition type products with the 4-hydroxy-3-cyano-2-cyclopenten-1-one skeleton (VIII) were obtained as sole isolatable products. NMR studies of some hydroxy-cyclopentenone derivatives were also described.     

Synthetic Studies on Cyclopentane Derivatives

An alternative route to dl-prostaglandin-B₁ using the Grignard reaction of 2-(6′-tert-butyloxycarbonylhexyl)-3-methoxy-2-cyclopenten-1-one(XII) with 3-tetrahydropyranyloxy-1-octyne was developed.

An easy synthesis of dihydrojasmone was also described.     

New Synthetic Tools for Peptide-Tetraphenylporphyrin Derivatives

Summary New metal-tetraphenylporphyrins and Fmoc-lysine-metalloporphyrin derivatives have been used to prepare peptide-porphyrin and peptide-metalloporphyrin compounds via solid-phase peptide synthesis. A water-soluble peptide, covalently bound to a manganese(III)-porphyrin, has been used as a catalyst to promote the oxidation of ABTS by hydrogen peroxide ort-butylhydroperoxide.     

New Synthetic Tools for Peptide-Tetraphenylporphyrin Derivatives

New metal-tetraphenylporphyrins and Fmoc-lysine-metalloporphyrin derivatives have been used to prepare peptide-porphyrin and peptide-metalloporphyrin compounds via solid-phase peptide synthesis. A water-soluble peptide, covalently bound to a manganese(III)-porphyrin, has been used as a catalyst to promote the oxidation of ABTS by hydrogen peroxide or t-butylhydroperoxide.     

Biological Activity of Novel Synthetic Derivatives of Carnosine

Two novel derivatives of carnosine—(S)-trolox-l-carnosine (STC) and (R)-trolox-l-carnosine (RTC) are characterized in terms of their antioxidant and membrane-stabilizing activities as well as their resistance to serum carnosinase. STC and RTC were synthesized by N-acylation of l-carnosine with (S)- and (R)-trolox, respectively. STC and RTC were found to react more efficiently with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and protect serum lipoproteins from Fe²⁺-induced oxidation more successfully than carnosine and trolox. At the same time, STC, RTC and trolox suppressed oxidative hemolysis of red blood cells (RBC) less efficiently than carnosine taken in the same concentration. When oxidative stress was induced in suspension of cerebellum granule cells by their incubation with N-methyl-d-aspartate (NMDA), or hydrogen peroxide (H₂O₂), both STC and RTC more efficiently decreased accumulation of reactive oxygen species (ROS) than carnosine and trolox. Both STC and RTC were resistant toward hydrolytic degradation by human serum carnosinase. STC and RTC were concluded to demonstrate higher antioxidant capacity and better ability to prevent cerebellar neurons from ROS accumulation than their precursors, carnosine and trolox.     

Amylase Formation by Lysozyme Lysate of Bac. subtilis

Bacterial amylase was formed by a subcellular system that was obtained by treating cells of an amylase-producing bacteria with lysozyme. However, the response of enzyme-forming ability of the lysate to various carbon and nitrogen sources was found to be significantly different from that of vegetative cells. The present paper describes some properties of the lysate and the effects of carbon and nitrogen sources on enzyme formation by the lysate, with a comparison of those by the vegetative cells.     

Delayed Human Neutrophil Apoptosis by Trichomonas vaginalis Lysate

Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis.     

Synthetic Studies on the Isomeric N -Methyl Derivatives of C-Ribavirin

Abstract

The syntheses of all three of the mono-N-methy1 derivatives of C-ribavirin (3-β-D-ribofuranosyl-1, 2, 4-triazole-5-carboxamide, 2) have been accomplished. Reaction of 1-(β-D-ribofuranosyliminomethyl)-2-methyl-hydrazine ( 7 ) with ethyl oxamate (8) in boiling ethanol gave the N′-methyl-C-ribavirin ( 3 ). A similar treatment of β-D-ribofuranosyl-1-carboximidic acid methyl ester ( 6 ) with N′-methyloxamic hydrazide ( 10 ) furnished the N²-methyl-C-ribavirin ( 4 ). Direct methylation of unprotected 2 with methyl iodide in the presence of potassium carbonate in dimethyl sulfoxide gave N ⁴-methyl isomer ( 5 ) as the major product. Structural assignments of 3 , 4 , and 5 were based on the unequivocal synthetic sequences, ¹H and ¹³C NMR data and confirmed by single crystal X-ray diffraction analysis.     

Dextran synthesis by immobilized dextran sucrase

Dextran sucrase has been produced by fermentation of Leuconostoc mesenteroides NRRL B-512, with and without continuous sucrose addition to improve enzyme production. The enzyme preparation has been concentrated from the fermentation broth by ultrafiltration and purified by gel permeation chromatography on Ultrogel. The specific activity of the dextran sucrase was greatly enhanced by calcium chloride addition to the purified enzyme. This enzyme preparation has been immobilized by covalent coupling onto an amino porous silica support (Spherosil) activated with glutaraldehyde. Immobilized dextran sucrase derivatives with an activity up to 830 dextran sucrase units per g. support could thus be obtained. The effect of the support specific area on coupling efficiency and reaction kinetics has been investigated, and the effect of intraparticular diffusion underlined. The molecular weight distribution of the dextran has been determined when varying several parameters.     

Degradation of Dextran by Free Radical Systems

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1) from Thea sinensis seeds (variety: Zairai) was isolated and purified about 1,500-fold using preparative disc electrophoresis. The specific activity was about 3.4 units/mg protein against ethyl alcohol.

Its value was 6.96 S and its molecular weight was approximately 150,000 using gel filtration on Sephadex G–200. The physical, chemical and catalytic properties of the enzyme are described. The oxidoreduction products formed by the enzyme were identified by gas chromatography, and for the unsaturated compounds the conversion of double bond and geometrical isomerization was observed.

The substrate specificity of tea enzyme is discussed in comparison with the enzymes from Leuconostoc mesenteroides, and horse and human livers. Paticularly a tendency for reactivity in the oxidoreduction of unsaturated alcohols and aldehydes were discribed by comparing the effects of geometry, the position of the double bond and the length of chain in substrates.