Localization of receptors and early events of phagocytosis in the macrophage

We studied the attachment of MRBC, 1.1 μm latex (foreign surface receptor), hemocyanin-DNP antiDNP Ab and sensitized SRBC (Fc receptor) to mouse peritoneal macrophages in culture, and the phagocytosis of 1.1 μm latex, AbAg complex, and sensitized SRBC by macrophages. Both types of receptors appeared over the whole cell surface. The particles attached mostly in the central area of the cell, but peripheral attachment was also observed. Internalization of 1.1 μm latex beads appeared to be mostly perinuclear, by sinking in of the cytoplasm with the attached particles, rather than a flow of membrane process over the beads. Also in the case of the AbAg complex attached to the Fc receptor internalization seemed mostly perinuclear and similar in mode to the latex ingestion. When attachment occurred in the periphery, active ruffling was observed. Sensitized SRBC internalization was seen mostly in the extreme periphery of the cells after the attached SRBC were moved from the perinuclear area where many of them first attached.     

Catalase-positive particles (“microperoxisomes”) from rat preputial gland and bovine adrenal cortex

Catalase activity was detected histochemically within membrane-bound cell organelles in epithelial cells of rat preputial gland and bovine adrenal cortex. These particles are oval to worm-like in rat preputial gland, 0.08 – 0.15 μm thick and up to 1.0 μm long. In bovine adrenal cortex the shape of catalase-positive particles is rather spherical (diameter 0.1 to 0.3 μm). Particles of both organs lack crystalline or dense cores.Biochemical examination of cell fractions prepared from tissue homogenates by differential centrifugation revealed the presence of two typical peroxisomal oxidases, viz. α-hydroxy acid and -amino acid oxidase, with maximal relative specific activities in the ‘microsomal’ fraction (preputial gland) and in the ‘lysosomal’ fraction (adrenal cortex), respectively. Urate oxidase is absent in both tissues.The concomitant occurrence of catalase and hydrogen peroxide producing oxidases in the particles described characterizes them as true peroxisomal systems (‘microperoxisomes’).     

THE EFFECTS OF HALOTHANE ON CULTURED MOUSE NEUROBLASTOMA CELLS : I. Inhibition of Morphological Differentiation

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3–2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40–80 Å in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any cells and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40–80 Å diameter microfilaments.     

Association between fibronectin receptor and the substratum: Spare receptors for cell adhesion

We have investigated the association of the recently described 140-kDa cell membrane receptor for fibronectin with the cytoskeleton or with substratum-bound fibronectin. Using a monospecific polyclonal antibody to the 140-kDa receptor, we have demonstrated that most of the receptor molecules are soluble in nonionic detergent either in suspension culture CHO cells or in CHO cells attached to and spread on a fibronectin-coated substratum. This may suggest that putative linkages of the receptor either to fibronectin or to detergent-insoluble cytoskeletal components are labile to nonionic detergent and thus are rather weak. Alternatively, it may mean that only a small fraction of the cell's receptors are needed to mediate adhesion. In order to explore this latter concept, we have coated substrata with various concentrations of PB1, a monoclonal antibody with a high affinity for fibronectin receptor. We demonstrate that coating the substratum with increasing concentrations of PB1 results in increasing amounts of 140-kDa receptor becoming bound to the substratum in detergent-insoluble form. However, the amount of receptor bound does not necessarily correlate with the degree of cell adhesion and spreading. Thus, coating the substratum with 5 μg/ml of PB1 results in essentially complete attachment and spreading of CHO cells, but only a small fraction of the 140-kDa receptor becomes substratum bound. Coating with 50 μg/ml of PB1 produces no further increase in cell adhesion and spreading, but results in the detergent-stable association of a large fraction of the total receptor pool with the substratum. These observations suggest the possibility of there being “spare” receptors for cell adhesion processes.     

The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface

A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

A critical assessment of standard processing methods for the preparation of palynological samples

Standard processing techniques for the isolation of organic walled dinoflagellate cysts from geological samples are examined, with particular attention to the size and type of sieve mesh used. Variations within the ‘standard’ processing techniques used by different laboratories are identified, and an assessment of the retention capacities of meshes of different sizes and different materials is carried out. Some dinoflagellate cysts and large numbers of Lycopodium spores, used for the calculations of absolute abundance data, were found to pass through 20 μm meshes. This is due to a combination of factors including: the diagonal aperture diameter of a 20 μm mesh measuring over 28 μm; the three-dimensional properties of different mesh weaves (nylon and polyester); and the non-spherical shape of the particles. Experiments demonstrate that the maximum mesh size that should be used in palynological processing is 15 μm. Nylon mesh is more practical to use than polyester as processing time is reduced, but nylon is degraded by contact with acid solutions. Meshes with apertures < 15 μm may be used, though this may be impractical for large samples containing significant quantities of fine siliciclastic or organic material.     

Sustained induction of ERK,protein kinase B,and p70 S6 kinase regulates cell spreading and formation of F-actin microspikes upon ligation of integrins by galectin-8, a mammalian lectin

Galectin-8, a member of the galectin family of mammalian lectins, is a secreted protein that promotes cell adhesion and migration upon binding to a subset of integrins through sugar-protein interactions. Ligation of integrins by galectin-8 triggers a distinct pattern of cytoskeletal organization, including formation of F-actin-containing microspikes. This is associated with activation of integrin-mediated signaling cascades (ERK and phosphatidylinositol 3 kinase (PI3K)) that are much more robust and are of longer duration than those induced upon cell adhesion to fibronectin. Indeed, formation of microspikes is enhanced 40% in cells that overexpress protein kinase B, the downstream effector of PI3K. Inhibition of PI3K activity induced by wortmannin partially inhibits cell adhesion and spreading while largely inhibiting microspike formation in cells adherent to galectin-8. Furthermore, the inhibitory effects of wortmannin are markedly accentuated in cells overexpressing PKB or p70S6K (CHO(PKB) and CHO(p70S6K) cells), whose adhesion and spreading on galectin-8 (but not on fibronectin) is inhibited approximately 25-35% in the presence of wortmannin. The above results suggest that galectin-8 is an extracellular matrix protein that triggers a unique repertoire of integrin-mediated signals, which leads to a distinctive cytoskeletal organization and microspike formation. They further suggest that downstream effectors of PI3K, including PKB and p70 S6 kinase, in part mediate cell adhesion, spreading, and microspike formation induced by galectin-8.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown “petite-negative” yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40°C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles.

2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerolgrown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae.

3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the “petite-positive” yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126.

4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 μM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 μM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor.

5. We conclude that “petite-positive” and “petite-negative” yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Zooplankton feeding ecology: bacterivory by metazoan microzooplankton

Bacterivory by the rotifer Brachionus plicatilis Müller, nauplii and copepodites of the copepods Centropages Krøyer sp. and Acartia tonsa Dana, and the tintinnid Favella panamensis Kofoid & Campbell was examined using fluorescently labelled bacteria (FLB) and epifluorescence microscopy. FLB were < 1 μm in diameter, and were offered at environmental concentrations (1.47−9.08 × 10⁶ cells·ml⁻¹). FLB were visible within rotifers, nauplii, copepodites, and tintinnids, confirming ingestion. Rotifer clearance rates (32–418 μl·animal⁻¹·h⁻¹) exhibited no relation with FLB concentration. In some cases rates of clearance of FLB by rotifers were different with alternative phytoplankton food (Nanochloris Naumann sp.) than in replicates with FLB alone, whereas in other cases presence of alternative food exhibited no clear effects on rates of ingestion of FLB. Clearance rates for all six naupliar stages of A. tonsa nauplii (0–320 μl·animal⁻¹·h⁻¹) were stage-related, with higher rates by NIII-VI nauplii than NI-II nauplii. Nauplii had higher rates of clearance of FLB in the absence of alternative phytoplankton food (Isochrysis Parke sp.). Clearance rates of FLB by a single stage of Centropages sp. nauplii, A. tonsa CI copepodites and F. panamensis (each obtained at only a single food concentration of either 1.5 or 5.0 × 10⁶ cells·ml⁻¹) were within the range of 85–142 μl·animal⁻¹·h⁻¹. These ranges were similar to those of rotifers and A. tonsa nauplii. This is the first report of FLB ingestion by metazoan marine microzooplankton. Although rotifers and ciliates might be expected to ingest small particles such as FLB using ciliary induced feeding currents, the means by which nauplii and copepodites eat FLB is less clear. We propose that they may “eat” bacteria as they “drink” to osmoregulate.     

A study of spin-probe solubility in mitochondrial membranes correlated with ATP-dependent conformational changes

Addition of ATP to submitochondrial particles causes considerable changes in the ESR spectra of hydrophobic spin-probe bound to particles which indicate an improvement in probe solubilization in the submitochondrial particle membranes due to energization. This effect is abolished by 2,4-dinitrophenol (10^-4 M) and oligomycin (I μg/mg of protein). At lower concentrations (0.1–0.2 μg/mg), oligomycin, on the contrary, promotes the action of ATP on the submitochondrial particle-bound spinprobe ESR spectra as well as activates the ATP-dependent transhydrogenase reaction in submitochondrial particles.     

Properties of particle movement in the plasma membrane of 3T3 mouse fibroblasts

The effects of cytochalasin B, vinblastine and temperature on particle movement in the plasma membrane of several 3T3 mouse fibroblast lines were investigated. Preincubation of normal 3T3 cells for 24 h in 5–10 μg/ml cytochalasin B had no effect on the mean square relative displacement of marker particles in the membrane (motion constant), but preincubation for 4 h in 40 μg/ml vinblastine reduced the value of this constant by 70%. A 10 °C decrease in temperature decreased the motion constant in normal cells (Q₁₀ = 4) more than in virus-transformed 3T3 cells (Q₁₀ = 1.8). Interpreting the motion constant of the particles as an expression of the viscosity of the membrane material, values of 3 poise for normal 3T3 cells and 6 poise for the transformed cells are obtained for 37 °C and pH = 7.4.A method is suggested to quantitate aggregation of particles on the surface of cells. When this method is applied to gold particles on 3T3 cells, disaggregation of particles is seen to behave as an unordered process, whereas aggregation appears to express cellular control. This consideration and the effect of vinblastine indicate that the interpretation of particle movement as Brownian movement in a viscous membrane material does not cover all phenomena observed.The membrane movement of the flat revertant SVF1 101 [1] was investigated. This cell line occupies an intermediary position between normal 3T3 mouse fibroblasts and the polyoma and SV40 transformed 3T3 cell lines as judged by growth properties. Its membrane movement was found to occupy an intermediary position between the membrane movements of these cell lines, too.     

An optical study of isolated rat adrenal chromaffin cells

Isolated rat adrenal chromaffin cells were studied with laser light scattering and Nomarski differential interference contrast microscopy. Evidence of organelle movement and of the fusion of secretory granules with the plasma membrane was obtained. A specially modified microscope was used to collect, coherently, laser light scattered from groups of 1–3 cells. Autocorrelation analysis of intensity fluctuations of the scattered light indicated that relative movements of cytoplasmic particles of about 0.1 μm or larger took seconds or longer, compared with the millisecond time periods that would be expected for free diffusion of the cytoplasmic organelles of the cells in a medium with a viscosity equal to that of water. Cytochalasin B (CB) and reduced temperature were found to reduce the relatively fast component of the decay of the autocorrelation, indicating decreased motion in the cells. Fixation with formaldehyde reduced the autocorrelation amplitude to zero, indicating the absence of motion. It is suggested that the intensity fluctuations were a consequence of organelle motility within the cells. Time-lapse photomicrography with Nomarski differential interference contrast optics indicated that the movements of the cytoplasmic particles which could be distinguished were highly restricted, except for occasional observations of distinct particle saltations at about 0.2 μm/sec. No change was observed in particle motion during stimulation with secretagogues, but microspikes formed on the cell surfaces, presumably due to the addition of secretory granule membranes to the plasma membrane as a consequence of exocytosis.     

Ultrastructural description of the life cycle of Nosema monorchis n. sp. (Microspora, Nosematidae), hyperparasite of Monorchis parvus (Digenea, Monorchiidae), intestinal parasite of Diplodus annularis (Pisces, Teleostei)

Life cycle stages of a new species of the genus Nosema Naegeli, 1857 (Microspora, Nosematidae), were examined by light and electron microscopy. It parasitizes the gut and the uterus of the digenean Monorchis parvus (Monorchiidae), in Diplodus annularis (Pisces, Teleostei). All stages were in close contact with the cytoplasm of the host cell and were probably all diplokaryotic. The divisions of meronts and sporonts were recognizable by the formation of spindle plaques at the surface of the nucleus. Spores were oval, measured 3.2±0.3×2.5±0.2 μm on ultrathin sections, and had a polar filament with 16–17 coils. The polaroplast presented two parts: an anterior region with closely packed lamellae and a posterior part with wider lamellae. This Nosema species is compared with the other microsporidian parasites of digeneans. This new species is named Nosema monorchis n. sp., after the generic name of its host.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

Nuclear maturation of ovine oocytes in cultured preantral follicles

Small (150–250 μm in diameter) and large (251–400 μm in diameter) preantral follicles (PFs) in sheep were cultured for 6 days in four different concentrations of transforming growth factor-alpha (TGF-), epidermal growth factor (EGF), FSH and LH. Proportions of follicles exhibiting growth, antrum formation and increase in follicular and oocyte diameter were the initial indicators of development. The ability of the oocytes isolated from these cultured follicles to mature to metaphase II (MII), after 24 h culture in a known in vitro maturation medium was the final criterion of success. TGF- 2.5 ng ml⁻¹, EGF 50 ng ml⁻¹ and FSH 1 and 2 μg ml⁻¹ supported good initial growth of the PFs. Thirty and seventeen percent of the oocytes from the large PFs cultured in TGF- 2.5 ng ml⁻¹ and FSH 2 μg ml⁻¹ respectively, matured to the MII stage. These proportions for oocytes from small PFs were 11 and 6%, respectively. Oocytes from follicles cultured in EGF did not mature to the MII stage. LH at all concentrations tested and TGF-, EGF and FSH above 5, 50 ng ml⁻¹ and 2 μg ml⁻¹, respectively, induced degeneration of the PFs. It was concluded that (i) TGF- 2.5 ng ml⁻¹ supports development of large PFs in sheep to obtain meiotically competent oocytes, (ii) PFs > 250 μm in initial diameter develop better in vitro, and (iii) in vitro development of sheep PFs could be obtained independent of gonadotropin stimulation.     

A Modified Mallory-Cason Staining Procedure for Large Cryosections

The classic Mallory-Cason staining procedure has been modified for application to sections “on tape” obtained from large deep frozen tissue specimens. These 20 μm cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

Determination of p-chloronitrobenzene in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of p-chloronitrobenzene (p-CNB) in rat plasma. A plasma sample was deproteinized with methanol containing the internal standard (p-bromonitrobenzene). The resulting methanol eluate obtained after centrifugation was filtered and injected into a high-performance liquid chromatograph (50 μl each). A column packed with 5 μm octadecylsilane (ODS) spherical particles was used with isocratic elution of methanol—water (45:55, v/v) at a flow-rate of 1.0 ml/min. The compounds were detected by ultraviolet absorbance at 280 nm. The retention times of p-CNB and the internal standard were 12.5 and 15.5 min, respectively, at a column oven temperature of 30°C. The results were linear from 0.05 to 100 μg/ml (r = 0.999), and the detection limit was 0.01 μg/ml. The relative error and the coefficient of variation on replicate assays were less than 7 and 10%, respectively, for all concentrations studied. The overall recoveries of p-CNB were between 97 and 105%. Plasma samples could be stored for up to one month at −20°C.