Divergent mechanisms of action of the inflammatory cytokines interleukin 1-beta and tumour necrosis factor-alpha in mouse cremasteric venules.

1. Protein synthesis dependency and the role of endogenously generated platelet activating factor (PAF) and leukotriene B(4) (LTB(4)) in leukocyte migration through interleukin-1beta (IL-1beta)- and tumour necrosis factor-alpha (TNFalpha)-stimulated mouse cremasteric venules was investigated using established pharmacological interventions and the technique of intravital microscopy. 2. Based on previously obtained dose-response data, 30 ng rmIL-1beta and 300 ng rmTNFalpha were injected intrascrotally (4 h test period) to induce comparable levels of leukocyte firm adhesion and transmigration in mouse cremasteric venules. 3. Co-injection of the mRNA synthesis inhibitor, actinomycin D (0.2 mg kg(-1)), with the cytokines significantly inhibited firm adhesion (49+/-13.6%) and transmigration (67.2+/-4.2%) induced by IL-1beta, but not TNFalpha. 4. In vitro, TNFalpha (1-100 ng ml(-1)), but not IL-1beta, stimulated L-selectin shedding and increased beta(2) integrin expression on mouse neutrophils, as quantified by flow cytometry. 5. The PAF receptor antagonist, UK-74,505 (modipafant, 0.5 mg kg(-1), i.v.), had no effect on adhesion induced by either cytokine, but significantly inhibited transmigration induced by IL-1beta (66.5+/-4.5%). 6. The LTB(4) receptor antagonist, CP-105,696 (100 mg kg(-1), p.o.), significantly inhibited both IL-1beta induced adhesion (81.4+/-15.2%) and transmigration (58.7+/-7.2%), but had no effect on responses elicited by TNFalpha. Combined administration of the two antagonists had no enhanced inhibitory effects on responses induced by either cytokine. 7. The data indicate that firm adhesion and transmigration in mouse cremasteric venules stimulated by IL-1beta, but not TNFalpha, is protein synthesis dependent and mediated by endogenous generation of PAF and LTB(4). Additionally, TNFalpha but not IL-1beta, can directly stimulate mouse neutrophils in vitro. The findings provide further evidence to suggest divergent mechanisms of actions of IL-1beta and TNFalpha, two cytokines often considered to act via common molecular/cellular pathways.

A transcript encoding a nucleic acid-binding protein specifically expressed in maize seeds.

A cDNA clone has been obtained for a low-abundance, seed-specific mRNA that encodes a polypeptide which defines a novel family of plant proteins with some similarities to the DnaJ class of molecular chaperones. The MEM1 (Maize Endosperm Motif binding protein) protein is capable of binding to the endosperm motif and activating transcription in the yeast one-hybrid system. Recombinant MEM1 was shown to bind in vitro to nucleic acids, with a preference for RNA over DNA. MEM1 is capable of forming homodimers, a property that is dependent on a domain close to the C-terminus of the protein. The protein is expressed in mid- to late-term endosperm cells. Subcellular fractionation and size fractionation under non-denaturing conditions indicate that the protein is present in the cytosol of endosperm cells. Possible roles of MEM1 in endosperm and protein body development are discussed.

Identification of allantoin, uric acid, and indoxyl sulfate as biochemical indicators of filth in food packaging by LC.

A liquid chromatographic (LC) method was developed for the determination of allantoin, uric acid, and indoxyl sulfate in mammalian urine contaminated packaging material including paper bagging, corrugated cardboard, grayboard, and burlap bagging. The procedure involves solvent extraction and isolation of the 3 analytes by reversed-phase LC with ultraviolet detection at 225 nm for allantoin and 286 nm for uric acid and indoxyl sulfate. The composition of authentic mammalian urine such as mouse, rat, cat, dog, and human were also determined with regard to the 3 compounds of interest. A linear concentration range of 0.11-20.4, 0.02-10.0, and 0.04-30.0 microg/mL was obtained for allantoin, uric acid, and indoxyl sulfate, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.0104 and 0.0345 microg/mL for allantoin; 0.0018 and 0.0060 microg/mL for uric acid; and 0.0049 and 0.0165 microg/mL for indoxyl sulfate, respectively. Interday relative standard deviation values for a mixture of standard allantoin, uric acid, and indoxyl sulfate (n = 5) were 0.97, 0.80, and 0.94%, respectively. Analyte composition for 5 types of authentic mammalian urine varied from 0.19-6.88 mg/mL allantoin; 0.08-0.57 mg/mL uric acid; and 0.03-0.78 mg/mL indoxyl sulfate. Analyte content for 8 samples including 2 samples each for paper, cardboard, grayboard, and burlap bagging each contaminated with mouse or rat urine ranged from

Determination of phenolic disinfectant agents in commercial formulations by liquid chromatography.

Using liquid chromatography (LC), a convenient and versatile method was developed for the assay of disinfectant products containing the commonly used phenolic agents, alone or in combination, including phenol, 2-phenylphenol, 4-t-amylphenol, 4-chloro-3,5-dimethylphenol (PCMX), 2-benzyl-4-chlorophenol, and triclosan. The procedure involves a simple dilution or dissolution of the product aliquot or portion followed by membrane filtration and LC. The method was applied to 19 different products representing 17 manufacturers and containing from one to 4 phenolic agents. Chromatography was performed using a Zorbax SB-C18 column, acetonitrile-0.05M KH2PO4 (55 + 45, v/v) pH 3.00, as mobile phase and UV detection at 280 nm. The intralaboratory precision of the product assays ranged form 0.34-5.35% (n = 5) and recoveries via fortification varied from 96.1-103.8%. The limits of quantitation (LOQ) and detection (LOD) ranged from 5.58 x 10(-5) to 2.50 x 10(-4) mg/mL and from 1.76 x 10(-5) to 0.67 x 10(-4) mg/mL, respectively, for the 6 analytes. The response for all analytes was observed to be linear for at least a 50-fold range in concentration (0.001-0.05 mg/mL). The proposed method provides an efficient means for the isolation and quantitation of these phenolic compounds.

Liquid chromatographic determination of methyl anthranilate in artificially flavored nonalcoholic beverages.

A liquid chromatographic method was developed that provides a simple and rapid means of determining methyl anthranilate (MA) in carbonated and noncarbonated, artificial grape-flavored, nonalcoholic beverages. The proposed procedure, which was applied to 12 different products, uses a Nova-Pak C18 column, a mobile phase containing acetonitrile-0.025M KH2PO4 (40 + 60), pH 3.00, and UV detection at 220 nm. Assay values ranged from 0.35 to 16.6 microg MA/mL. The intralaboratory precision (relative standard deviation) for the products ranged from 0.51 to 2.23% (n = 5), and recoveries via fortification ranged from 83.6 to 102.4%. The limits of quantitation and detection were 0.00417 and 0.00125 microg/mL, respectively, and the analyte response was linear over a 100-fold concentration range (0.0001-0.01 mg/mL).

Maize endosperm secretes a novel antifungal protein into adjacent maternal tissue.

A series of endosperm transfer layer-specific transcripts has been identified in maize by differential screening of a cDNA library of transcripts at 10 days after pollination. Sequence comparisons revealed among this class of cDNAs a novel, small gene family of highly diverged sequences encoding basal layer antifungal proteins (BAPs). The bap genes mapped to two loci on chromosomes 4 and 10. So far, bap-homologous sequences have been detected only in maize, teosinte and sorghum, and are not present in grasses outside the Andropogoneae tribe. BAP2 is synthesized as a pre-proprotein, and is processed by successive removal of a signal peptide and a 29-residue prodomain. The proprotein can be detected exclusively in microsomal membrane-containing fractions of kernel extracts. Immunolocalization reveals BAP2 to be predominantly located in the placentochalazal cells of the pedicel, adjacent to the basal endosperm transfer layer (BETL) cells, although the BAP2 transcript is found only in the BETL cells. The biological roles of BAP2 propeptide and mature peptide have been investigated by heterologous expression of the proprotein in Escherichia coli, and by tests of its fungistatic activity and that of the fully processed form in vitro. The mature BAP2 peptide exhibits potent broad-range activity against a range of filamentous fungi, including several plant pathogens.

Development and functions of seed transfer cells.

In secretion or absorption processes, solutes are transported across the plasmalemma between the symplastic and apoplastic compartments. For this purpose, certain plant cells have developed a specialised transfer cell morphology characterised by wall ingrowths, which amplify the associated plasmalemma surface area up to 20-fold. Detailed studies on the function and development of transfer cells in the context of seed filling have been carried out mainly in cereal endosperm, and for the cotyledon and seed coat cells of legumes. The major solutes transferred are amino acids, sucrose and monosaccharides. The contributions of recently identified symporter proteins to solute transfer are reviewed here, as is the role of apoplastic invertases in promoting solute assimilation. Expression of invertase and monosaccharide transporters early in both cereal and legume seed development orchestrates the distribution of free sugars which play an important role in regulating transfer cell function and determining final endosperm or embryo cell number. Transfer cell differentiation is subject to developmental control, and may also be modulated by sugar levels. The most abundant genes specifically expressed in the transfer layer of maize endosperm encode small antipathogenic proteins, pointing to a role for these cells in protecting the developing endosperm against pathogen ingress. The functional characterisation of the corresponding transfer layer-specific promoters has provided a tool for dissecting transfer cell functions. Transfer cells are highly polar in their organisation, the characteristic cell wall ingrowths developing on one face only. The presence of cytoskeletal components bordering wall ingrowths is documented, but their role in establishing transfer cell morphology remains to be established.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1)-deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane.

Studies with neutralizing antibodies have indicated roles for platelet-endothelial cell adhesion molecule-1 (PECAM-1) in leukocyte migration through the endothelium and the perivascular basement membrane. Because some of these findings have been contentious, this study aimed to explore the role of PECAM-1 in leukocyte migration by analyzing leukocyte responses in interleukin 1beta (IL-1beta)- and tumor necrosis factor-alpha (TNFalpha)-activated cremasteric venules of PECAM-1-deficient mice using intravital and electron microscopy. Although no differences in levels of leukocyte rolling flux or firm adhesion were observed, a delay in leukocyte transmigration in response to IL-1beta, but not TNFalpha, was detected in PECAM-1-deficient mice. Electron microscopy indicated that this delay occurred at the level of perivascular basement membrane. To address the cytokine specificity of PECAM-1 dependence, in vitro experiments demonstrated that TNFalpha, but not IL-1beta, could induce rapid adhesion of murine neutrophils to protein-coated surfaces, suggesting that TNFalpha elicited leukocyte transmigration in wild-type mice via direct stimulation of leukocytes. In summary, the results suggest a regulatory role for PECAM-1 in leukocyte migration through the perivascular basement membrane, a role that appears to be cytokine-specific and associated with the ability of the cytokine to stimulate rapid neutrophil adhesion.

Liquid chromatographic determination of dehydroepiandrosterone (DHEA) in dietary supplement products.

A liquid chromatographic (LC) method was developed to assess the potency of products that contain dehydroepiandrosterone (DHEA), a precursor hormone synthesized from cholesterol by the human adrenal cortex and converted to potent androgens and/or estrogens in peripheral tissues. Forty-five commercial products (both single and multi-ingredient) were subjected to analysis by the proposed method. A Zorbax Rx C18 column with a mobile phase containing acetonitrile-0.025 M phosphate buffer (60 + 40), pH 3.50, and UV detection at 292 nm was used for 87% of the products. An alternative mobile phase containing methanol-0.025 M phosphate (75 + 25), pH 3.50, was used to isolate DHEA from more complex product mixtures. Assay values varied from 0 to 109.5% of the declared amount with an overall mean value of 91.1%. The recoveries based on fortified products ranged from 96.4 to 101.2%, and the intraday precision (RSD, n = 5) varied from 0.50 to 1.66%.

rgf1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development.

The maize cob presents an excellent opportunity to screen visually for mutations affecting assimilate partitioning in the developing kernel. We have identified a defective kernel mutant termed rgf1, reduced grain filling, with a final grain weight 30% of the wild type. In contrast with most defective endosperm mutants, rgf1 shows gene dosage-dependent expression in the endosperm. rgf1 kernels possess a small endosperm incompletely filling the papery pericarp, but embryo development is unaffected and the seeds are viable. The mutation conditions defective pedicel development and greatly reduces expression of endosperm transfer layer-specific markers. rgf1 exhibits striking morphological similarities to the mn1 mutant, but maps to a locus approximately 4 cM away from mn1 on chromosome 2 of maize. Despite reduced starch accumulation in the mutant, no obvious lesion in starch biosynthesis has been detected. Free sugar levels are unaltered in rgf1 endosperm. Rates of sugar uptake, measured over short (8 h) periods in cultured kernels, are increased in rgf1 compared to the wild type. rgf1 and wild-type kernels, excised at 5 DAP and cultured in vitro also develop differently in response to variations in sugar regime: glucose concentrations above 1% arrest placentochalazal development of rgf1 kernels, but have no effect on cultured wild-type kernels. These findings suggest that either uptake or perception of sugar(s) in endosperm cells at 5-10 DAP determines the rgf1 kernel phenotype.

Divergent effects of platelet-endothelial cell adhesion molecule-1 and beta 3 integrin blockade on leukocyte transmigration in vivo.

The final stage in the migration of leukocytes to sites of inflammation involves movement of leukocytes through the endothelial cell layer and the perivascular basement membrane. Both platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and the integrin alphavbeta3 have been implicated in this process, and in vitro studies have identified alphavbeta3 as a heterotypic ligand for PECAM-1. In the present study we have addressed the roles of these molecules by investigating and comparing the effects of PECAM-1 and alphavbeta3 blockade on leukocyte migration in vivo. For this purpose we have examined the effects of neutralizing Abs directed against PECAM-1 (domain 1-specific, mAb 37) and beta3 integrins (mAbs 7E3 and F11) on leukocyte responses in the mesenteric microcirculation of anesthetized rats using intravital microscopy. The anti-PECAM-1 mAb suppressed leukocyte extravasation, but not leukocyte rolling or firm adhesion, elicited by IL-1beta in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg 37 Fab), but had no effect on FMLP-induced leukocyte responses. Analysis by electron microscopy suggested that this suppression was due to an inhibition of neutrophil migration through the endothelial cell barrier. By contrast, both anti-beta3 integrin mAbs, 7E3 F(ab')2 (5 mg/kg) and F11 F(ab')2 (5 mg/kg), selectively reduced leukocyte extravasation induced by FMLP (38 and 46%, respectively), but neither mAb had an effect on IL-1beta-induced leukocyte responses. These findings indicate roles for both PECAM-1 and beta3 integrins in leukocyte extravasation, but do not support the concept that these molecules act as counter-receptors in mediating leukocyte transmigration.

Analyte loss due to membrane filter adsorption as determined by high-performance liquid chromatography.

The phenomenon of membrane filter adsorption in high-performance liquid chromatography (HPLC) is investigated utilizing 16 brands of filters representing 3 polymeric materials: cellulose acetate (CA), nylon, and polyvinylidene difluoride in a variety of diameters (3, 4, 7, 13, and 25 mm). Sixteen compounds commonly encountered in drug preparations are selected as sample analytes and classified as acidic, basic, and neutral in chemical behavior. Six mobile phase/sample solvent mixtures are included: 3 with methanol-water and 3 with acetonitrile-water as major constituents. When using methanol as the mobile phase organic component, CA, nylon, and polyvinylidene difluoride (PVDF) filters exhibit negligible to moderate adsorption levels with regard to the neutral and basic drug compounds. The acidic drug test compounds are adsorbed by 50% of all 3 filter materials tested in methanol-water. In acetonitrile, neutral compounds are affected by 31.4%, basic compounds are affected by 47.0%, and acidic compounds are affected by 53.6% of the nylon and PVDF filters. CA is incompatible with acetonitrile and is excluded from the study with this solvent.

Recovery and sporicidal resistance of various B. subtilis spore preparations on porcelain penicylinders compared with results from AOAC test methods.

Sporicidal test results obtained from carriers inoculated with 4 types of defined Bacillus subtilis spore preparations were compared with the standard AOAC sporicidal test using soil extract nutrient broth (SENB) B. subtilis 19659 spores. Recoveries of spores inoculated on penicylinders from B. subtilis clean spores (washed and suspended in water) and B. subtilis 19659 spores inoculated from culture filtrates according to the AOAC method were compared. Spores were exposed to 6 concentrations (0.5-3.0% w/v) of glutaraldehyde in phosphate buffer (pH 7.5) for 10 h. Concentrations were established by titrimetry and liquid chromatography. Recoveries of surviving spores were determined for 3 types of clean B. subtilis var. niger preparations, one clean B. subtilis 19659 preparation, and the SENB B. subtilis 19659 filtrates. Spore carriers, inoculated by the standard AOAC protocol, resulted in as much as a 2-log number difference in runs 1-12, but not more than 0.5 log number for each clean spore preparation. The SENB spores varied most in resistance to glutaraldehyde, with no growth in recovery media from 3 different batches of 1, 1.5, and 2% glutaraldehyde. Separate batches of SENB preparations of B. subtilis 19659 were resistant and destroyed by 1.0% glutaraldehyde, with 3.98 and 6.0 log numbers of spores on penicylinders, respectively. Clean spore preparations of B. subtilis 19659 on porcelain penicylinders were more resistant to glutaraldehyde than were SENB spores. Nutrient agar/Mg/Ca and nutrient agar/Mg spore preparations of B. subtilis var. niger showed the most uniform resistance to glutaraldehyde. Spores with calcium added showed increased resistance to glutaraldehyde. B. subtilis 19659 spores from the Columbia broth spore preparation were the most resistant and were recovered after exposure to 3.0% glutaraldehyde.

Evidence for factors regulating transfer cell-specific expression in maize endosperm.

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (< 100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.

Sporicidal testing of commercial germicides using a chemical standard and a calibrated bioindicator.

The AOAC sporicidal method uses as a standard the resistance of spores on carriers to 2.5N HCl. This resistance is variable at exposure times ranging from 2 to 20 min. The method described in this paper uses a glutaraldehyde standard and distinguishes various levels of sporicidal activity in the presence of 1-5% glutaraldehyde by using appropriate spore strains, spore preparations, and spore levels. The resistances of 2 Bacillus subtilis 19659 spore preparations cultured in 10% Columbia broth plus manganese and nutrient agar plus minerals, as well as that of B. subtilis var. niger cultured on Lab-Lemco agar, were tested. T-soy broth was a better recovery medium than fluid thioglycollate or modified fluid thioglycollate for B. subtilis 19659 spores exposed to HCl. Sporicidal tests were done on B. subtilis 19659 spores with 2 types of spore preparations. A commercial glutaraldehyde germicide was used for comparison of the sporicidal activity of the glutaraldehyde standard. Two strains of B. subtilis spores and 4 levels of spores (20,000-80,000, 100,000-400,000, 500,000-800,000, and 1,000,000 and up) were removed from check penicylinders from the same batches used for sporicidal tests. B. subtilis var. niger spores were the most resistant to HCl, while B. subtilis 19659 spores were more resistant to glutaraldehyde. Sporicidal activities of a commercial germicide containing 2.5% glutaraldehyde with additives and another containing 5% glutaraldehyde in phosphate buffer were similar. Both totally destroyed high levels of B. subtilis 19659 spores cultured in 10% Columbia broth plus manganese. Results indicate that use of a glutaraldehyde standard, calibrated numbers of spores on penicylinders (bioindicators), and appropriate spore strains and preparations can reduce the variability of sporicidal testing of commercial germicides.

Sensitivity and specificity of radioisotope right-left shunt measurements and pulse oximetry for the early detection of pulmonary arteriovenous malformations.

STUDY OBJECTIVES: To assess the effectiveness of pulse oximetry and radioisotope measurement of right-to-left (R-L) shunt for the early detection of pulmonary arteriovenous malformations (PAVMs) in patients with hereditary hemorrhagic telangiectasia (HHT). DESIGN: Patients with HHT had serial measurements of the following: (1) arterial oxygen saturation (SaO2) by pulse oximetry in erect and supine positions, and on maximal exercise using cycle ergometry; (2) quantitative radioisotope measurements of R-L shunt using IV 99mTc-labeled macroaggregates of albumin; and (3) routine pulmonary function. After percutaneous transcatheter embolization of all PAVMs with feeding vessel diameters > 3 mm, residual PAVMs were assessed with selective digital subtraction pulmonary angiography. Using postembolization angiography as the "gold standard," SaO2 and radioisotope shunt measurements after embolization were analyzed retrospectively using logistic regression to assess the ability of each test to predict for the presence of residual PAVMs. RESULTS: Of the 66 patients included, 40 had small PAVMs remaining postembolization. Using univariate logistic regression, radioisotope shunt and erect saturation showed a significant relationship with the presence of residual PAVMs (p=0.001, 0.005, respectively). Erect SaO2 < or = 96% had 73% sensitivity and 35% specificity for detecting PAVMs. Radioisotope shunt >3.5% of cardiac output had 87% sensitivity and 61% specificity for detecting PAVMs. CONCLUSIONS: These results confirm that noninvasive measurements are useful in the screening of patients with HHT for the presence of PAVMs without need for angiography and its associated risks, and that radionuclide scanning is better than pulse oximetry.

Determination of phosphine residues in whole grains and soybeans by ion chromatography via conversion to phosphate.

An ion chromatographic (IC) method was developed for determining phosphine (PH3) in whole grains (barley, corn, oats, rice, rye, and wheat) and soybeans. The method converts phosphine to phosphate (i.e., orthophosphate) and isolates the phosphate by IC with eluent-suppressed conductivity detection. Recoveries of unbound phosphine by the method were similar to those obtained by an established colorimetric method for 7 different products fortified at 3 levels. Mean recoveries were low (i.e., 30-60%) and varied with product type and level of fortification. Recoveries of PH3 from previously fumigated products fortified with aluminum phosphide ranged from 19.0% for barley fortified at 0.734 ppm to 88.3% for corn fortified at 1.691 ppm. Precision data from 3 products based on replicate analyses (n = 4 or 5) gave relative standard deviations of 1.78-4.66% for mean laboratory-fumigated PH3 levels of 0.679-1.309 ppm. Estimated limits of detection (LOD) and quantitation (LOQ) for PH3 were 0.010 microgram/g (10 ppb) and 0.0275 microgram/g (27.5 ppb) at signal-to-noise ratios (S/N) of 4:1 and 10:1, respectively. These values were also determined for a nonchemically suppressed IC system with LOD of 0.02 microgram/g (20 ppb) and LOQ of 0.055 microgram/g (55 ppb) at S/N of 4:1 and 10:1, respectively. Phosphate response was linear over the concentration range equivalent to 0.30-10.0 micrograms P/mL, with a mean correlation coefficient of 0.9988 based on replicate standard curves. The relationship of product composition to recovery from various products was also examined.