Distinct cellular expression pattern of annexins in Hydra vulgaris. The annexins are a structurally related family of Ca2+ and phospholipid binding proteins whose function has not been clearly defined. Further investigations of annexin function may be enhanced by studying simpler organisms that express fewer annexin gene products. We previously characterized annexin XII from the freshwater cnidarian Hydra vulgaris (Schlaepfer, D. D., D. A. Fisher, M. E. Brandt, H. R. Bode, J. Jones, and H. T. Haigler. 1992. J. Biol. Chem. 267:9529-9539). In this report, we detected one other hydra annexin (40 kD) by screening hydra cell extracts with antibodies raised against peptides from highly conserved regions of known annexins. The 40-kD protein was expressed at less than 1% of annexin XII levels. These biochemical studies indicate that hydra contain a very limited number of annexin gene products. The cellular hydra annexin distribution was analyzed by indirect immunofluorescence. Using affinity-purified antibodies to annexin XII, the epithelial battery cells were stained throughout the tentacle. A lower level of annexin XII staining was detected in peduncle region epithelial cells. No other cell types showed detectable annexin XII staining. The anti-peptide antibody that specifically detected the 40-kD hydra annexin, maximally stained the cytoplasm of nematocytes. The immunofluorescent results showed that annexin XII and the 40-kD annexin were not co-expressed in the same cells. Since the hydra annexins localized to specific subsets of the total hydra cell types, it is likely that these proteins perform specialized biological roles, and not general "housekeeping" functions which are part of the essential molecular machinery of all cells.


Enteric defensins: antibiotic peptide components of intestinal host defense. Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.


Proteolytic processing of endogenous and recombinant beta 4 integrin subunit. The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.


Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells. We have synthesized an antisense oligonucleotide primer that matches a supposedly conserved sequence in messages for heparan sulfate proteoglycans with transmembrane orientations. With the aid of this primer we have amplified partial and selected full-length copies of a message from human lung fibroblasts that codes for a novel integral membrane heparan sulfate proteoglycan. The encoded protein is 198 amino-acids long, with discrete cytoplasmic, transmembrane, and amino-terminal extracellular domains. Except for the sequences that represent putative heparan sulfate chain attachment sites, the extracellular domain of this protein has a unique structure. The transmembrane and cytoplasmic domains, in contrast, are highly similar to the corresponding domains of fibroglycan and syndecan, the two cell surface proteoglycans that figured as models for the design of the antisense primer. This similarity includes the conservation of four tyrosine residues, one immediately in front of the stop transfer sequence and three in the cytoplasmic segment, and of the most proximal and most distal cytoplasmic sequences. The cDNA detects a single 2.6-kb message in cultured human lung fibroblasts and in a variety of human epithelial and fibroblastic cell lines. Polyclonal and monoclonal antibodies raised against the encoded peptide after expression as a beta-galactosidase fusion protein react with the 35-kD coreprotein of a cell surface heparan sulfate proteoglycan of human lung fibroblasts and decorate the surface of many cell types. We propose to name this proteoglycan "amphiglycan" (from the Greek words amphi, "around, on both sides of" and amphoo, "both") referring to its domain structure which extends on both sides of the plasmamembrane, and to its localization around cells of both epithelial and fibroblastic origin.


Behaviour of ejaculated spermatozoa from bull, boar and ram during thin-layer countercurrent partition in aqueous two-phase systems. Ejaculated spermatozoa from bulls, boars and rams were subjected to thin-layer countercurrent partition in aqueous two-phase systems composed of dextran T500 and polyethylene glycol 6000 (PEG) in sucrose-based Hepes-buffered media. In the basal system, the great majority of spermatozoa tended to partition with the dextran-rich bottom phase; however, by including very low levels of phosphate, they could be made to partition increasingly with the PEG-rich top phase (complete at 10 mM phosphate). A procedure was developed for carrying out four separations simultaneously under identical conditions, whereby it could be shown that distribution varied with the number of spermatozoa in the sample. In the case of bull, the effect of cell number could be reduced considerably by inclusion of small quantities of seminal plasma in the phase system, but no such effect was found for ram or boar. Considerable variation in distribution pattern was seen between samples, which did not appear to be due to technical inconsistency. Livability in the phase systems was also variable, and we believe that PEG may exert a detergent-like effect on the sperm surface that is exacerbated in highly defined media free of protective proteins.


Morphometric studies of pancreatic acinar granule formation in NCTR-Balb/c mice. NCTR-Balb/c mice are afflicted with a cholesterol lysosomal storage disorder stemming from a defect in intracellular cholesterol processing. The clinical and biochemical abnormalities expressed in the mice resemble Niemann-Pick type C and D disorders in humans. One of the proposed mechanisms to explain the pathophysiology of the disorder implies a defect in the process of membrane transport that normally takes place in the vesicular movement of cholesterol to specific target sites in the cell. Secretory granule formation in pancreatic acinar cells is one of the biological processes known to involve massive membrane flow. Thus, we have undertaken a morphometric study of the regranulation mechanism in the pancreatic acinar cells of the mutant mice, as a way of studying cellular membrane movement. Electron micrographs of pancreatic acinar cells from mutant and normal mice were taken at several time points after extensive degranulation induced by pilocarpine injection. Two hours after stimulation the pancreatic cells demonstrated a complete loss of granules, and at later time points newly formed granules appeared. Identical unit granule volumes were observed in both groups, indicating that the progranules were of normal size. However, the rate of granule formation and maturation was reduced in the mutant mice, which might be the result of a defect in membrane function.


Dynamics of transmembrane proteins during Sindbis virus budding. Label-fracture and immunogold fracture-flip techniques are used to address at the ultrastructural level the dynamics of viral and cellular transmembrane proteins during the budding of Sindbis virus on the plasma membrane of infected cells. Immunolabeling with anti-Sindbis spike antibodies shows that the viral proteins are mostly in clusters, all associated with budding viruses. Ultrastructural observation of the unlabeled freeze-fractured plasma membranes shows that membrane particles aggregate over the budding viruses. These results indicate that the concentration of viral transmembrane proteins gives rise to a parallel concentration of membrane particles. Immunolabeling with anti-CD8 antibodies of cells expressing by transfection the CD8 transmembrane protein and infected with Sindbis virus shows absence of labeling on the particle aggregates over the forming virions. These findings indicate the exclusion of CD8 proteins from the portions of the membrane where budding occurs.


Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi. Epimastigote forms of the pathogenic parasite Trypanosoma cruzi were used to study the endocytic process in a protozoon. These elongated unicellular organisms are highly polarized cells: endocytosis occurs only at the anterior region through the cytostome and the flagellar pocket membrane, areas of the plasma membrane where the cell cytoskeleton, formed by sub-pellicular microtubules, is absent. When the cells were incubated at 4 degrees C or 28 degrees C with gold-labeled transferrin, fixed and processed for routine transmission electron microscopy our observations show that this ligand initially binds to the cytosome and the membrane lining the flagellar pocket and is subsequently ingested through a clathrin-independent receptor-mediated endocytotic process, with formation of uncoated pits and vesicles. Ingested complexes are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the posterior end of the cell. Immunocytochemical data from Lowicryl-embedded cells demonstrated that the reservosomes are acidic compartments (pH 6.0, as shown using DAMP as a pH probe) with no acid phosphatase or typical lysosome-associated membrane proteins (LAMP 1, LAMP 2 and lgp 120), but rich in cysteine proteinase. These data suggest that the reservosome is a pre-lysosomal compartment. Since cysteine proteinase of T. cruzi contains no phosphorylated mannose residues and the cation-independent mannose 6-phosphate receptor could not be immunocytochemically detected in the reservosomes, it is possible that lysosomal enzymes in the epimastigote forms of T. cruzi are targeted to compartments related to the endocytic pathway through a mechanism different from that which occurs in other eukaryotic cells.


Identification of the intermediate filament-associated protein gyronemin as filamin. Implications for a novel mechanism of cytoskeletal interaction. In a previous paper, a monoclonal antibody (designated M1.4) that recognized a 240 kDa polypeptide was characterized. This antibody stained the intermediate filaments in several cell lines, and biochemical characteristics of the 240 kDa polypeptide led us to conclude that it was a novel intermediate filament-associated protein, which we termed gyronemin. Here we report that gyronemin is expressed in adult rat organs that contain a substantial smooth muscle component. Taking advantage of this observation, this protein was purified from bovine uterine tissue and, by biochemical, immunological and amino acid sequence analysis, found to be homologous to the actin-associated protein filamin. Three novel monoclonal antibodies raised using purified bovine gyronemin as the immunogen show this protein to be associated with actin-containing stress fibers, although our original M1.4 antibody continued to be localized along vimentin filaments. Since two-dimensional electrophoretic analysis did not demonstrate a difference in either relative molecular mass or isoelectric point of this polypeptide when associated with either filamentous system, we conclude that filamin is a bifunctional protein capable of associating with both the intermediate filament and actin cytoskeletal systems.


Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments. Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain ('coil 1a' and 'coil 2b', respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.


Vertebrate p34cdc2 phosphorylation site mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe. We have used the fission yeast Schizosaccharomyces pombe to analyse the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34(cdc2) protein, Thr 14, Tyr 15, Thr 161 and Ser 277, upon cell cycle progression. We have studied both the effect of overexpression of mutant proteins in a cdc2+ background and assayed their ability to rescue null and temperature-sensitive alleles of cdc2. Mutations of Thr 14 and Tyr 15 within the ATP binding domain of p34(cdc2) that mimic constitutive phosphorylation cause dominant negative cell cycle arrest when overexpressed. In contrast, some substitutions that simulate permanent dephosphorylation of the corresponding sites advance dephosphorylation of the corresponding sites advance mitosis. These data confirm the model that p34(cdc2) function is negatively regulated by phosphorylation of residues in the ATP binding site. Mutagenesis of the conserved residue Thr 161 functionally inactivates p34(cdc2), and our data suggest that both phosphorylation and dephosphorylation events at Thr 161 are required for progression through the cell cycle. Mutations at the fourth site of phosphorylation. Ser 277, lead to cold-sensitive cell cycle arrest, in minimal but not rich growth medium, suggesting that this site is involved in monitoring the nutritional status of the cell.


Extrajunctional distribution of N-cadherin in cultured human endothelial cells. Human endothelial cells contain prominent Ca(2+)-dependent intercellular adherens-type junctions (AJ), which are associated, at their cytoplasmic surfaces, with actin, vinculin and plakoglobin. The transmembrane adhesion molecules present in these sites are members of the cadherin family, which are recognized by a pancadherin serum, directed against the conserved C terminus of these molecules. Immunoblotting analysis of cultured human endothelial cells using these antibodies revealed three immunoreactive bands with apparent molecular masses of 135, 130 and 120 kDa. Cloning and sequencing of the 135 kDa cadherin from an endothelial cDNA expression library indicated that this molecule is a typical cadherin, essentially identical to N-cadherin. Transfection of cDNA encoding this molecule into CHO cells resulted in the induction of AJ formation and an apparent epithelialization of the cells. Immunofluorescent labeling with antibodies to chicken N-cadherin indicated that the molecule is associated with intercellular junctions in the transfectants. In contrast, cultured human umbilical cord endothelial cells exhibited a largely diffuse N-cadherin labeling over the entire cell surface with only occasional enrichment in cell-cell junctions. Comparison of this pattern with the discrete junctional labeling obtained with the pan-cadherin antibody suggests that different cadherins, co-expressed in the same endothelial cells, may undergo differential surface distribution.


Preparative chromatographic separation of enantiomers. The potential of the chromatographic separation of enantiomers on a preparative scale as a tool for the isolation of optically pure compounds is gaining increasing recognition. This review surveys the different chiral stationary phases (CSPs) used for preparative chromatography, emphasizing the advantages and drawbacks of each. The strategy to be followed for preparative separations is discussed and tables summarizing separations reported in the literature give an overview of practical applications. Cellulose triacetate has been used most frequently, probably because of its broad application range and its low production costs in comparison with more recently introduced CSPs. Nevertheless, the high efficiency of some of the novel CSPs is likely to contribute to the further development and expansion of the method.


Separation and identification of the 4-hydroxyantipyrine sulphoconjugate. In a previous study we observed, during separation of total antipyrine metabolites by high-performance liquid chromatography and after enzymatic hydrolysis, an unidentified peak corresponding to an ionic compound with pyrazolinone features. In the present study, this compound was identified as the 4-hydroxyantipyrine sulphoconjugate, and its structure was definitively confirmed by gas chromatographic-mass spectrometric analysis and by the use of pure synthetic substance. We also demonstrated the inhibitory effect of sodium metabisulphite, a necessary preservative of urinary samples, on hydrolysis of this conjugate in the presence of sulphatases from Helix pomatia or Aerobacter aerogenes. This inhibitory effect makes it impossible to perform a global assay of antipyrine metabolites after enzymatic or chemical hydrolysis and confirms the value of direct assay of the 4-hydroxyantipyrine sulphoconjugate.


Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. III. Determination of the arotinoid sumarotene and its Z-isomer in human and animal plasma. A fully automated and sensitive high-performance liquid chromatographic method, using on-line solid-phase extraction, automated column switching and ultraviolet detection, was developed for the third-generation retinoid (arotinoid) sumarotene (methyl p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propenyl]phe nyl sulphone; Ro 14-9706) and its Z-isomer. Nearly quantitative recoveries for human, rat and dog plasma were obtained by addition of acetonitrile (final content ca. 17%) to the plasma sample prior to injection. No isomerization was observed when the samples were stored in the autosampler for more than 20 h. The injection volume was 0.5 ml, resulting in quantification limits of 1 ng/ml for sumarotene and 2 ng/ml for the Z-isomer. More than 40 injections could be made on to one precolumn, allowing routine overnight injections. Using a 1-ml injection volume, the limit of quantification for sumarotene could be improved to 0.5 ng/ml. The method was applied to toxicokinetic studies in rats and dogs, and was used to monitor human plasma samples after repeated topical application. The method could also be adapted to etarotene (Ro 15-1570), which was used as an internal standard, and which is at present in clinical development.


Determination of piroxicam and its major metabolites in the plasma, urine and bile of humans by high-performance liquid chromatography. A simple and sensitive liquid chromatographic method with ultraviolet detection is described for the determination of the nonsteroidal anti-inflammatory drug piroxicam and its major metabolites in human plasma, urine and bile. Separation of these components occurs on a reversed phase C10CN column with a mobile phase consisting of acetonitrile-water-sodium dihydrogenphosphate solution. The detection limit of the assay was 50 ng/ml with intra- and inter-assay coefficients of variation for piroxicam of the order of 2 and 5%, respectively. The assay linearity was good (typically r = 0.9999). This method can be readily utilised for clinical pharmacokinetic and mass-balance studies.


Reversed-phase ion-pair high-performance liquid chromatographic determination of triclabendazole metabolites in serum and urine. An ion-pair high-performance liquid chromatographic method was developed for measuring the concentrations of triclabendazole metabolites (sulphoxide and sulphone) in plasma and urine samples. The diluted biological fluids are ultrafiltered before chromatography through a 30,000 relative molecular mass cut-off filter and then injected into a C18 column. They are then isocratically eluted with a mobile phase consisting of 0.05 M phosphate buffer (pH 7.0)-acetonitrile (55:45, v/v) with addition of 1.0 mmol/l sodium decanesulphonate and monitored by ultraviolet-visible spectrophotometry at 312 nm. Recoveries over the range 0.01-9.0 micrograms/ml for triclabendazole sulphoxide and sulphone are, respectively, 91.7% and 91.6% in serum and 90.3% and 90.2% in urine. For both metabolites, the limit of detection is 10 ng/ml in both urine and serum.


Determination of the coumarin derivative cloricromene acid in rabbit plasma and platelets. Two methods for the determination of cloricromene acid in biological samples are described. Cloricromene acid is a catabolite of cloricromene, a coumarin derivative which is active in the cardiovascular system. After oral administration of cloricromene to a rabbit, plasma and platelets were taken at different times and cloricromene acid was then isolated by solid-phase extraction with Sep-Pak C18 cartridges using acetonitrile-tetrahydrofuran-20% aqueous acetic acid (15:11:74, v/v/v) as eluent. The analyses were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with fluorescence detection with excitation at 310 nm and emission at 390 nm. The limit of quantification by RP-HPLC was about 50 pg. The catabolite in the plasma was identified by continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS), also used as a complementary means of RP-HPLC determination. The results obtained by RP-HPLC and CF-FAB-MS showed good agreement.


Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high-performance liquid chromatography in plasma and cardiac tissue. A rapid ion-pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous detection of creatine, phosphocreatine, hypoxanthine, inosine, adenosine, AMP, ADP, ATP, 8-azaguanine, 2-chloroadenosine, and 2'-O-methyladenosine. This method has proven useful for measuring changes in nucleotide concentrations in both heart tissue and plasma samples. Separation of the compounds of interest is achieved in less than 8 min with re-equilibration in 7 min, making the total run time 15 min. Separation is performed on a 3-microns Ultrasphere ODS column employing tetrabutylammonium phosphate as the ion-pair agent and dipotassium hydrogenphosphate as the counter ion. The accuracy, rapid separation, and re-equilibration time make this method particularly useful for the routine analysis of a large number of samples.


Simple, rapid and sensitive determination of plasma taurine by high-performance liquid chromatography using pre-column derivative formation with fluorescamine. A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The plasma taurine contents of yellowtail fish, Seriola quinqueradiata, beef cattle, dairy cows and chickens were determined to be 125 +/- 54, 5.6 +/- 1.4, 2.2 +/- 0.7 and 20.0 +/- 9.6 micrograms/ml, respectively.


Bioanalysis of the investigational anti-tumour drug 5,10-dideaza-5,6,7,8-tetrahydrofolic acid by high-performance liquid chromatography with ultraviolet detection. A high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 278 nm is presented for the determination of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid in plasma. Sample pretreatment was achieved by using cation-exchange solid-phase extraction columns with methotrexate as internal standard. Chromatographic separation was based on ion-pair HPLC with 1-octanesulphonic acid as the ion-pairing compound. The detection limit was 10 ng/ml using an 500-microliters sample volume. The assay was linear from the detection limit up to 5000 ng/ml with good reproducibility. The applicability of the assay was demonstrated in a study in the rat.


Determination of valproic acid by high-performance liquid chromatography with photodiode-array and fluorescence detection. The derivatization of valproic acid and undecylenic acid with 4-bromomethyl-7-methoxycoumarin is described. The derivatives were detected by photodiode-array and fluorescence detectors. The optimum monitoring conditions and the stability of the suspension solution and derivatives were investigated. A high-performance liquid chromatographic (HPLC) method with isocratic or gradient elution has been established for the analysis. This method has been used for the determination of total and free valproic acid in serum. There is a satisfactory correlation between the results obtained by this HPLC method and those measured by the enzyme immunoassay. Some other common anti-epileptic drugs did not interfere with the analysis. The method is simple and fast and has better sensitivity and linearity than enzyme immunoassay. It is suited for routine therapeutic drug monitoring.


Determination of glycyrrhizin in rabbit plasma by high-performance liquid chromatography with photodiode-array ultraviolet detection and its pharmacokinetics application. A simple and sensitive high-performance liquid chromatographic method for the determination of glycyrrhizin in rabbit plasma has been developed. Up to 0.1 ml of plasma containing glycyrrhizin was deproteinized by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected onto a LiChrospher RP-18 column using a methanol-water-ammonia solution (80:20:0.1, v/v, pH 3.0-3.2, adjusted with perchloric acid) as the mobile phase and ultraviolet detection at 254 nm, followed by ultraviolet spectrum identification (between 200 and 380 nm) with a photodiode-array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of glycyrrhizin in rabbit, after a 2 mg/kg intravenous administration. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. Compartmental analysis yielded a two-compartment model.


Simultaneous determination of a new gastrointestinal prokinetic agent (HSR-803) and its metabolites in human serum and urine by high-performance liquid chromatography using automated column-switching. A method based on high-performance liquid chromatography using column-switching is described for the simultaneous determination of HSR-803 and its metabolites in human serum and urine. The system uses a six-port valve with a Nucleosil CN pre-column for on-line sample clean-up, and direct injection of samples. The limits of quantitation in serum and urine were 5 and 20 ng/ml for HSR-803 and 50 and 200 ng/ml for the metabolites, respectively. The coefficients of variation for the intra- and inter-day accuracies were between 0.8 and 7.1% for each compound. This method was applied to the pharmacokinetic studies in humans after oral administration of HSR-803.


Simultaneous determination of cortisol and cortisone in human plasma by stable-isotope dilution mass spectrometry. A method for the simultaneous determination of cortisol and cortisone in human plasma was developed using capillary gas chromatography-mass spectrometry-selected ion monitoring. [2H5]Cortisol and [2H5]cortisone were used as internal standards. Cortisol and cortisone in plasma were determined from the peak-height ratios of the [M-31] fragment ions of the methoxime-trimethylsilyl derivatives of cortisol and [2H5]cortisol (m/z 605 and 610) and of cortisone and [2H5]cortisone (m/z 531 and 536). Sensitivity, specificity, precision, accuracy and reproducibility of the method were demonstrated to be satisfactory for measuring the circulating concentrations of cortisol and cortisone.


Simple and rapid method for determination of short-chain fatty acids in biological materials by high-performance liquid chromatography with ultraviolet detection. A new and versatile method for the identification and quantification of short-chain fatty acids, such as formic, acetic, propionic, butyric, isobutyric, valeric, isovaleric and mercaptoacetic acids, in biological specimens by high-performance liquid chromatography is described. After sample purification by vacuum transfer and concentration by alkaline freeze-drying, the acids were measured without any further preparative step, using a sulphonated polystyrene-divinylbenzene column as stationary phase. Ultraviolet detection of the native acids was done at 214 nm. Peak resolution and reproducibility were as good as with gas chromatography. Many examples of the application of this method to a variety of biological specimens and fluids both from the rats and humans are described.


Determination of free N-acetylamino acids in biological samples and N-terminal acetylamino acids of proteins. N-Acetylamino acids were derivatized with 9-anthryldiazomethane to the corresponding esters. The anthryl esters were separated by high-performance liquid chromatography and detected fluorimetrically (excitation at 365 nm; fluorescence emission measured at 412 nm). N-Acetyl derivatives of Asn, Gln, Ser, Thr, Gly, Ala, Tyr, Pro, Met, Val, Ile and Leu as well as N-formyl-Met could be separated and identified in the same chromatographic run. The detection limit was from 0.10 pmol for AcGln to 5.5 pmol for AcIle and AcLeu. When the acetylamino acids listed above were added to the 700 g supernatant of a rat liver homogenate, the mean recovery was 72%. AcAla and AcTyr were found in free form in baker's yeast. Proteins with an acetylated N-terminus were digested by a protease, and the peptides formed were treated with an N-acylamino acid-releasing enzyme. This method was applied to end-group determination of four proteins (each 0.5 nmol).


Single-step method for purification of human transferrin from a by-product of chromatographic fractionation of plasma. A rapid, simple and convenient method is described for the isolation, on a pilot scale, of pure and functional human transferrin from an unexploited by-product of chromatographic fractionation of plasma. In a single chromatographic step on DEAE-Spherodex, 97% pure transferrin was obtained in 75% yield. A virus inactivation treatment was included in the preparative process in order to guarantee the safety of the final product, which could be used in culture media.


Determination of alkaline phosphatase isoenzymes in serum by high-performance liquid chromatography with post-column reaction detection. A weak anion-exchange high-performance liquid chromatographic procedure with post-column reaction detection for simultaneous determination of alkaline phosphatase (EC 3.1.3.1, ALP) isoenzymes is described. We identified six peaks with ALP isoenzyme activity in normal serum. The peaks were, in order of elution, one intestinal/bone, two bone and three liver ALP isoenzymes. This new assay with automatic injection, on-line post-column reaction detection and powerful integration data system could be of significant value in the routine clinical biochemistry laboratory. The advantages include improved sensitivity and selectivity over previous methods for the determination of ALP isoenzymes.


Dye-affinity purification of transthyretin from an unexploited by-product of human plasma chromatographic fractionation. Dye-affinity chromatography of human plasma transthyretin on Remazol Yellow GGL-Sepharose from an unexploited by-product of chromatographic fractionation of plasma was optimized for large-scale preparation of a therapeutic product. With this system, transthyretin is only weakly bound to the gel. The residence time on gel and the transthyretin level in the by-product were observed to have no influence on the binding capacity of gel, and the optimum amount of transthyretin to be applied to the gel was found to be 1 g/l of gel. The adsorbent can be used more than ten times. The procedure resulted in the isolation, with a 30% yield with respect to plasma, of an 80% pure protein, which retained its thyroxine-binding capacity. Although the purity is acceptable for substitutive therapy, it can be improved further with a second chromatography on Cibacron Blue-Sepharose.


Quantification of erythrocyte S-adenosyl-L-methionine levels and its application in enzyme studies. A highly selective high-performance liquid chromatographic method for the quantification of human erythrocyte S-adenosyl-L-methionine levels is described. A strong cation-exchange sorbent with propylsulphonic acid functional groups was used to extract S-adenosyl-L-methionine and S-adenosylethionine (internal standard) from erythrocytes. Quantification of erythrocyte S-adenosyl-L-methionine levels was achieved by using reversed-phase high-performance liquid chromatography and ultraviolet detection at 254 nm. This method was adapted to measure methionine-adenosyltransferase activity in erythrocytes, which enables us to study the possible role of altered methylation in different diseases.


Specific gas chromatography-mass spectrometry analytical method for the determination of cyhexatin in animal feed. The acaricide tricyclohexyltin hydroxide (cyhexatin) was determined in animal feed samples, using gas chromatographic--mass spectrometry in the electron impact mode. Sample extraction and derivatization (converting the analyte to an alkylated derivative) were performed using a tricyclopentyl analogue of this acaricide as internal standard to obtain a better analytical precision.


Simplified derivatization for determining sphingolipid fatty acyl composition by gas chromatography-mass spectrometry. A simple procedure for simultaneously derivatizing non-hydroxy and hydroxy fatty acids prior to GC analysis [I. Ciucanu and F. Kerek, J. Chromatogr., 284 (1984) 179] has been evaluated for its usefulness in determining sphingolipid acyl composition. The method uses methyl iodide in polar aprotic solvents to generate methyl esters of carboxyl groups and methyl ethers of hydroxyl groups. Methylation efficiency is examined as a function of hydroxyl group presence and location in free fatty acids as well as a function of 2-hydroxy fatty acid chain length. Conditions are also reported for efficient saponification and derivatization of sphingolipid fatty acyl chains as is illustrated using bovine brain galactosylceramide.


Determination of diclazuril in animal feed by liquid chromatography. A method is described for the determination of diclazuril (Janssen Research Compound R64433; trademark Clinacox) in chicken feed at the mg kg(-1) level. Compound R062646, a structure analogous to diclazuril, was used as the interna standard. The drug was extracted from food with acidified methanol. Diclazuril was then isolated by means of solid-phase extraction with a cartridge containing a C18 phase. The eluate was evaporated and the residue redissolved in dimethylformamide. An aliquot was injected onto a reversed-phase high-performance liquid chromatographic column and the drug substance quantified at 280 nm by an ultraviolet detector. Extraction (absolute) recoveries of 85% for both internal standard and diclazuril were obtained. The method is suitable for diclazuril concentrations ranging from 0.1 to 1.5 mg kg(-1). Method validation data are presented.


Determination of the unusual amino acid hypusine at the lower picomole level by derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride and reversed-phase high-performance or medium-pressure liquid chromatography. Hypusine, an unusual amino acid formed by post-translational modification of lysine, is normally determined by specific metabolic labelling followed by measurement of released radioactivity after protein hydrolysis. This paper describes a sensitive non-radioactive method for the determination of hypusine, involving complete protein hydrolysis and precolumn derivatization of the released amino acids with 4-dimethylaminoazobenzene-4'-sulphonyl chloride, followed by reversed-phase high-performance or medium-pressure liquid chromatography of the dabsylated derivatives. The detection limit of hypusine was about 500 fmol. Additionally, the hypusine-containing protein from the archaebacterium Sulfolobus acidocaldarius was purified. By applying the dabsylation method to the analysis of tryptic peptides derived from this protein, it was possible to determine the correct positioning of the hypusine residue in the amino acid sequence, which was not possible by the amino acid sequencing procedure alone.


Resumption of pituitary and ovarian activity post-partum: endocrine and ultrasonic observations in bromocriptine-treated women. The resumption of pituitary and ovarian activity was investigated by hormonal measurements and ultrasound scanning in 45 healthy post-partum women who were treated with bromocriptine. Bromocriptine, 50 mg (Parlodel LAR) was administered within 24 h post-partum. Plasma luteinizing hormone, follicle stimulating hormone, oestradiol, progesterone and prolactin concentrations were measured repeatedly and correlated with ultrasound measurements. Forty-one of the 45 women completed the study. A prompt fall in prolactin levels to normal (non-pregnant) values was observed within 24 h post-partum. Hereafter, a rapid return of the normal menstrual cycle was observed. Endocrine evidence of ovulation was obtained in 19/41 women within 1 month post-partum. Ultrasound measurements started at day 10 and were repeated regularly in 40 women. Ultrasound evidence of ovulation was found in 25/40 women. Ultrasound findings appeared not to correspond with the hormonal measurements. It is concluded that the resumption of pituitary and ovarian secretory functions post-partum is not always paralleled by a concomitant recovery of normal follicular growth and ovulation. Application of a progesterone threshold to judge resumption of ovulation post-partum should be reconsidered.


Reliability of home urinary LH tests for timing of insemination: a consumer's study. In this study, intrauterine insemination (IUI) was timed either after the detection of a urinary luteinizing hormone (LH) surge at home by the patient (group A), or following a positive LH test as interpreted by the gynaecologist (group B). Afterwards, samples tested by the patient were retested by the gynaecologist and vice versa. The gynaecologist also rechecked his own findings and the results were correlated with ultrasound data and charts of basal body temperature. Forty-seven cycles were evaluated. The patient's and the gynaecologist's readings agreed (+/- 12 h) in 42 of the cases (89%), and in five cycles (11%) a difference greater than 24 h was found. The intra-observer variation in the gynaecologist's results was +/- 12 h in four cycles (8.5%). These findings suggest that the LH test can be used as a reliable home device for the prediction of pending ovulation and timing of IUI.


Induction of multiple follicular growth by pulsatile gonadotrophin-releasing hormone (GnRH) treatment in women with normal cycles: the role of the route of administration. In a prospective study, seven patients with normal menstrual cycles were treated with pulsatile gonadotrophin-releasing hormone. They received one cycle of intravenous (i.v.) therapy and one cycle of subcutaneous (s.c.) therapy. Another four volunteers with normal cycles were treated for one s.c. cycle only. Cycles were compared to each other and to the normal unstimulated cycles of 14 other volunteers. Multiple follicular development could be achieved using both the i.v. and the s.c. routes. However, the i.v. route showed significantly higher luteinizing hormone (LH) and luteal steroid levels. Comparing s.c. cycles to controls, significantly lower LH levels were observed, resulting in significantly lower steroid production if calculated per large follicle. We conclude that the i.v. route is superior to the s.c. route in inducing multiple follicular growth in women with normal cycles.


Luteal phase support and severe ovarian hyperstimulation syndrome. The incidence and statistical associations of the ovarian hyperstimulation syndrome (OHSS) were studied in 304 egg retrievals with gonadotrophin-releasing hormone agonist suppression, gonadotrophin administration and follicular aspiration. In addition to preserving corpus luteum function, the luteal phase administration of human chorionic gonadotrophin (HCG) was associated with a higher incidence of severe OHSS than was supplementation with progesterone alone (12 versus 0%, P less than 0.001). Severe OHSS occurred in 3.7% and 12% of retrievals without and with pregnancy respectively (P less than 0.01). Stepwise logistic regression showed that the occurrence of moderate or severe OHSS was statistically predicted by the log of the serum oestradiol on the day the initial HCG was given (P less than 0.0001), treatment with luteal phase HCG (P less than 0.0003), and fetal number (P less than 0.0079). In the late luteal phase of cycles without luteal HCG, the serum oestradiol concentration was one-tenth and the serum progesterone concentration was one-fifth of the luteal phase value with HCG support (P less than 0.001). Without luteal phase HCG, oestradiol was two-fold higher (P less than 0.001) and progesterone was 1.4-fold higher (P less than 0.005) in pregnant than in non-pregnant women. With luteal phase HCG, oestradiol was 1.4-fold higher in pregnant than in non-pregnant women (P less than 0.05), and progesterone was 1.7-fold higher (P less than 0.001). Oestradiol upper limits of 4400 and 14,700 pmol/l (1200 and 4000 pg/ml) for cycles with and without luteal phase HCG respectively correspond to approximately 5% risk of moderate or severe OHSS with a singleton pregnancy under these conditions.


Human preovulatory follicular fluid: inhibin and free steroids related to optimal follicular maturation in ovarian stimulation regimes and possible function in ovulation. Concentrations of inhibin, total and free oestradiol and progesterone were determined in preovulatory follicular fluid from 15 women undergoing in-vitro fertilization and embryo transfer treatment. The women underwent ovarian stimulation using clomiphene citrate and human menopausal gonadotrophin (HMG) (69 follicular fluid samples) in one cycle, and gonadotrophin-releasing hormone agonist (GnRHa) and HMG stimulation in the next treatment cycle (64 follicular fluid samples). The women thereby served as their own control. Inhibin, total oestradiol and progesterone were measured by radioimmunoassay. Concentrations of free steroid were calculated after quantitation of the steroid binding proteins, i.e. sex-hormone binding globulin (SHBG), cortisol binding protein (CBP) and albumin. Levels of inhibin and free and total progesterone were significantly higher in follicular fluids collected after stimulation with the GnRHa compared to the clomiphene regime (P less than 0.05, P less than 0.001, P less than 0.001, respectively). In contrast, levels of total and free oestradiol in follicular fluid were significantly lower after stimulation with GnRHa than after clomiphene stimulation (P less than 0.001). These results indicate that the follicles have achieved a more optimal maturation during the GnRHa regimen than during the clomiphene regime. It is suggested that the concentration of free biologically active steroids in follicular fluid, released into the peritoneal cavity during ovulation, may be physiologically important in stimulating the oviduct and the uterus in connection with ovulation, pre-embryo development and implantation.


Clinical and endocrinological changes in women following ovulation induction using buserelin acetate/human menopausal gonadotrophin augmented with biosynthetic human growth hormone. Biosynthetic human growth hormone added to an ovarian stimulation regime of human menopausal gonadotrophin (HMG) for IVF treatment improves the response of women who were previously resistant. This study investigated the efficacy of growth hormone (GH)/buserelin/HMG treatment in women with a previous normal response to buserelin/HMG stimulation. Ten patients (28-36 years, mean 32.5 years) were treated with GH (6 IU/day) plus buserelin/HMG. A control group of 10 women (28-37 years mean 31.0 years) received buserelin/HMG alone. All were given buserelin 500 micrograms and 2 ampoules (150 IU) HMG daily once pituitary suppression had been confirmed. There was no improvement in the GH group as assessed by follicular growth rate or number, oocyte number per woman and pregnancy rate. There was no effect of GH upon the serum oestradiol level and the follicular fluid levels of oestradiol, GH and inhibin. Serum IGF-1 increased significantly during GH administration, returning to pre-treatment levels 2 days after the last dose of GH. Follicular IGF-1 was much higher in the GH-treated group than the controls. Significant correlations were found in the GH-treated group between follicular fluid GH and follicular fluid oestradiol concentrations and between follicular GH and follicular size. Follicular IGF-1 was correlated with the serum IGF-1 concentration on day 8 of the GH/HMG treatment. In conclusion GH/buserelin/HMG treatment in women with a previous normal response to buserelin/HMG stimulation increased their serum and follicular IGF-1 concentrations. However, it does not improve the clinical ovarian response or the follicular secretion of oestradiol or inhibin.


The detrimental influence of functional ovarian cysts during in-vitro fertilization cycles. Reviewing 780 in-vitro fertilization (IVF) cycles, where buserelin was commenced in the preceding luteal phase and human menopausal gonadotrophin on day 4 of the ensuing menses, 53 cycles were identified with sonolucent cysts (30-50 mm diameter). Of the latter 53 cycles, the serum oestradiol was significantly greater on day 4 in 22 cycles abandoned for poor follicular development than in 31 cycles which proceeded to oocyte retrieval (P less than 0.05). Of the 31 cycles proceeding to oocyte retrieval, nine had a day 4 serum oestradiol greater than 200 pmol/l (95th centile for day 4 oestradiol in patients without apparent cysts), and these cycles produced significantly fewer grade 1 embryos than the cycles with day 4 oestradiol levels less than or equal to 200 pmol/l (P less than 0.05). Six of the 53 cycles with cysts resulted in conception, and all of these cycles had a day 4 serum oestradiol less than 200 pmol/l. Among the 53 cycles with ovarian cysts, the serum progesterone on the day of abandonment in four cycles and on the day of human chorionic gonadotrophin administration in one non-abandoned cycle, was above the range established for 104 cycles without cysts. No significant difference was seen in day 4 serum androstenedione levels, and the day 4 serum progesterone was less than 5 nmol/l in all but one patient. Functional activity of ovarian cysts is associated with an adverse influence on IVF cycles.


The significance of antisperm antibodies for sperm-cervical mucus interaction. An overview is presented of the effects of antisperm antibodies on the sperm-cervical mucus interaction. Antisperm IgA on spermatozoa or in cervical mucus can severely inhibit sperm penetration of cervical mucus and migration through it. Disturbance of the sperm-cervical mucus interaction is the only firmly established effect of these antisperm antibodies and leads to reduced fertility, as shown by a poor or negative result of the post-coital test. The presence of antisperm IgA in the male or female partner can be investigated more specifically with the sperm-cervical mucus contact test and can be confirmed by the sperm agglutination test on bromelinliquefied cervical mucus, by the mixed antiglobulin reaction test or by the immunobead test for IgA.


Infertility treatment: relative effectiveness of conventional and assisted conception methods. The effectiveness of infertility treatments is still questioned, particularly the assisted conception methods because of their complexity and cost. Furthermore, pregnancies often occur independent of treatment but many treatments have not been properly evaluated. The most basic audit of outcome with or without treatment requires pregnancy and preferably birth rates to be calculated in a cycle-specific and/or time-specific way; cumulative rates are the preferable method of calculation, in order to account for the usual tendency for fecundity to fall progressively. The choice of treatment usually depends on a balance of the chances of conceiving with or without treatment, and with more or less complicated treatments, and on other factors such as duration of infertility and the woman's age. This review aims to address those choices by assessing the actual and comparative effectiveness of treatments insofar as there are well defined and strictly comparable time-specific or cycle-specific published data available. Cumulative rates are described wherever possible and presented graphically for easy reference.


Influence of number of pre-embryos transferred, progesterone level and oestradiol/progesterone ratio at thaw on pregnancy results during replacement of cryopreserved pre-embryos in natural cycles. When frozen-thawed pre-embryos were transferred in a natural menstrual cycle, a clinical pregnancy rate of 22% per transfer cycle was obtained. The pregnancy rates were higher when serum progesterone levels at thaw were less than or equal to 3.4 ng/ml than when they were greater than or equal to 3.5 ng/ml. This was reflected in a significantly higher oestradiol/progesterone ratio at thaw in the conception cycles than in the non-conception cycles.


A randomized prospective study comparing pregnancy rates following clomiphene citrate and human menopausal gonadotrophin therapy. The anti-oestrogenic effect of clomiphene citrate causes an increased rate of associated cervical factor problems. Theoretically, it may also cause an adverse effect on the endometrium. The hostile mucus may be completely or partially corrected by the use of additional oestrogen after stopping clomiphene, or by intrauterine insemination (IUI). The prospective randomized study presented here was designed to determine whether a higher pregnancy rate might be achieved using human menopausal gonadotrophin (HMG) compared to clomiphene citrate. Thirty-seven women treated with clomiphene citrate for 165 cycles were compared to 33 treated with HMG for 179 cycles. A pregnancy was achieved in 81.1% of clomiphene-treated women (18.2%/cycle) versus 72.7% (13.3% per cycle) of HMG-treated women. The abortion rates of 13.3 and 12.5% respectively, were similar. The data suggest no adverse effect of clomiphene compared to HMG as far as pregnancy rates are concerned. IUI seems to be an effective means of treating cervical factor problems induced by clomiphene.


Fallopian tube sperm perfusion used in a donor insemination programme. Ninety-six couples were enrolled in a programme of Fallopian tube sperm perfusion (FSP). FSP is a method combining ovarian stimulation, ovulation induction and intra-uterine insemination with a 4 ml volume of sperm suspension. Frozen/thawed donor semen was used in all cycles. A total of 196 treatment cycles were started and of these 172 were completed. Twenty cycles were cancelled because of maturation of too many follicles, a low ovarian response, or formation of ovarian cysts. Five of the women enrolled in the programme did not have any inseminations. Forty-eight pregnancies occurred (27.9% per cycle) among 45 women (49.5%). There were five spontaneous abortions and one ectopic pregnancy. Sixteen pregnancies are on-going (greater than 12 weeks) and 26 women have delivered (including two sets of triplets and one set of twins). The pregnancy rate declined from the first attempt (34.1%) to the fourth attempt (14.3%). The pregnancy rate was significantly higher in women with three to five mature follicles compared to women with two or fewer mature follicles (P less than 0.05). Cycles in which human chorionic gonadotrophin (HCG) was administered on day 11 or later had a higher pregnancy rate than cycles where HCG was administered on day 10 (P less than 0.01). There was no difference in pregnancy rates between insemination with 8-10 x 10(6) motile spermatozoa and insemination with greater than 30 x 10(6) motile spermatozoa. FSP used in a programme of artificial insemination by donor using frozen/thawed donor semen is relatively simple to perform and gives a high pregnancy rate per cycle.


Hormone and ultrasound parameters in ovarian stimulation cycles for direct intraperitoneal insemination. To determine hormonal and ultrasound parameters associated with pregnancies, 115 women with unexplained infertility (n = 82), endometriosis (n = 22) or cervical factor (n = 11) were treated with direct intraperitoneal insemination (DIPI) after ovarian stimulation with clomiphene citrate and human menopausal gonadotrophins (HMG). Twenty women conceived and were compared with the remaining 95 non-pregnant women during one treatment cycle. Women with basal FSH levels less than or equal to 1.25 micrograms/l responded with higher oestradiol levels (P less than 0.0001), with the development of more follicles (P less than 0.05) and higher progesterone levels (P less than 0.05) than women with basal FSH levels greater than 1.25 micrograms/l, but the conception rates were similar. Women with miscarriages or biochemical pregnancies had a higher basal FSH value than both the women with term pregnancies and the non-pregnant women. Women with at least 3 preovulatory follicles greater than or equal to 15 mm had a higher pregnancy rate than those with fewer follicles, but a further increase was not observed above that number. The endometrium was thicker on the day of ovulation induction in cycles leading to a term pregnancy than in cycles without conception or with a biochemical pregnancy. No term pregnancy was observed when the endometrium was thinner than 8 mm. Women with a short luteal phase (less than 12 days) had a higher ratio of oestradiol/progesterone in the midluteal phase than women with a luteal phase of greater than or equal to 12 days and pregnant women.(ABSTRACT TRUNCATED AT 250 WORDS)


Endometriosis: a new approach is needed. Endometriosis is generally regarded and treated as an endocrine and/or a surgical disease. Endocrine and surgical approaches to treatment are effective only in treating the symptoms of the disease and not its pathophysiology. Consequently, the success of such therapy is at best short-lived, and probably non-existent in mild disease. This thesis therefore argues for a new therapeutic approach towards endometriosis. In view of increasing evidence in favour of an immunological aetiology for this condition, it is suggested that such an approach may involve immunological rather than endocrine manipulation. It is also proposed that so-called unexplained infertility may in many cases represent a precursor stage of endometriosis, since immunological abnormalities in both of these conditions are so similar. Large-scale, prospective, multicentre clinical trials of immunotherapy in unexplained infertility and endometriosis appear to be indicated at this time if endometriosis is to lose its importance as one of the major contributors to female infertility.


Meiotic segregation of a 21;22 Robertsonian translocation. In-vitro fertilization of hamster oocytes with human spermatozoa has been used to study heterozygotes for structural chromosome abnormalities. To date only four men heterozygous for Robertsonian translocations have been examined. In this study, 150 sperm chromosome complements from a 21;22 translocation were investigated. There was no evidence of an interchromosomal effect since the frequency of abnormalities unrelated to the translocation was within the range of normal donors. The frequency of unbalanced complements was 3.4%, which is similar to other Robertsonian translocations. As expected, an equivalent number of normal (n = 74) and balanced (n = 70) karyotypes was observed.


A new sperm collection method for in-vitro fertilization: collection at tail of epididymis. We have developed a method for the collection of spermatozoa from the tail of the epididymis in patients with severe asthenozoospermia. The treatment was applied to 25 cycles, of which 16 cycles provided spermatozoa in good condition with concentration and motility greater than 50 x 10(4)/ml and greater than 50% respectively. Spermatozoa thus obtained were subjected to IVF in 11 cycles and to ZIFT in five cycles; five IVF cases reached cleavage and two of those cases resulted in pregnancy. Two of the five ZIFT cases also achieved a pregnancy. Despite limits on frequency, the sperm collection process is thought to be very useful for the treatment of severe asthenozoospermia.


Perforated appendicitis following transvaginal oocyte retrieval for in-vitro fertilization and embryo transfer. A case of appendicitis following transvaginal oocyte retrieval is presented. The time sequence and the finding of puncture holes in the appendix indicate a causal relationship between the two events. Although minor infectious complications have been reported previously, this is the first report of appendicitis caused by transvaginal oocyte retrieval.


The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development. A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.


The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte. We have investigated the effect of ultra-rapid versus slow freezing on the meiotic spindle of the mouse oocyte. A slow freezing protocol [1.5 M dimethyl sulphoxide (DMSO)] and an ultra-rapid protocol (3.5 M DMSO/0.5 M sucrose) have been compared. Oocytes were fixed at different time points as follows: after prefreeze equilibration, immediately after thawing and after 60 or 180 min of post-thaw recuperation. The spindle was visualized with a monoclonal anti-alpha-tubulin antibody followed by an immunogold-silver staining technique. The chromosomes were stained with 4',6-diamidino-2-phenylindole (DAPI). Spindle morphology was classified as follows: normal barrel-shaped, abnormal shaped, partial/multipolar or absent. After slow freezing with 1.5 M DMSO, these spindle morphologies were found respectively in 67%, 17%, 11% and 6% of the oocytes after 60 min of post-thaw recuperation and in 72%, 11%, 11% and 6% after 180 min. After ultra-rapid freezing with 3.5 M DMSO/0.5 M sucrose, we observed the same categories of spindle morphology in 84%, 13%, 3% and 0% of oocytes after 60 min of post-thaw recuperation and in 86%, 7%, 7% and 0% after 180 min. The present study demonstrates that the majority of spindles exhibit a normal morphology after both slow and ultra-rapid freezing. The ultra-rapid freezing protocol preserved spindle integrity to the highest extent. Nevertheless, the occurrence of abnormal spindles and chromosome dislocation indicate that the genetic risk of oocyte freezing has to be evaluated in further detail.


Structural heterogeneity of platelet-activating factor produced by murine preimplantation embryos. Platelet-activating factor (PAF) is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine with the alkyl moiety predominantly a mixture of saturated hexadecyl and octadecyl chains (C16:0 and C18:0 PAF, respectively). Previously, a PAF bioassay was compared with a radioimmunoassay for PAF (NEN Du Pont). Both assays were sensitive and quantitative, but the correlation between PAF measured by the bioassay compared to the radioimmunoassay was poor for murine embryo-derived PAF (r = 0.773, n = 88), while being completely adequate (r = 0.961) for a PAF standard which was an equimolar mixture of C16:0 and C18:0 PAF (C16:0/C18:0 PAF). This study compared a larger sample size of murine embryo-derived PAF (n = 154) and found that the poor correlation between the two assays persisted (r = 0.791). When dose-response curves were generated with C16:0, C16:0/C18:0 and C18:0 PAF (over a concentration range of 0.3-30 ng/ml), the concentrations which gave a 50% response were equivalent in the bioassay (i.e. 6 ng/ml), but differed in the radioimmunoassay (i.e. 1.5, 3 and 6 ng/ml, respectively). Following separation of murine embryo-derived PAF (from medium in which 30 two-cell embryos had been cultured for 24 h) into C16:0 and C18:0 PAF by reverse phase high performance liquid chromatography, 9/20 cultures produced 100% C16:0 PAF, 2/20 cultures produced 100% C18:0 PAF and the remaining 9/20 cultures produced varying proportions of both.(ABSTRACT TRUNCATED AT 250 WORDS)


Pretreatment of hamster oocytes with Ca2+ ionophore to facilitate fertilization by ooplasmic micro-injection. The fertilization rate after ooplasmic micro-injection in mammals is low. In hamsters, micro-injection of sperm nuclei into oocytes resulted in the release of the second polar body and the formation of female and male pronuclei, but disappearance of the cortical granules was minimal. These unfavourable results seem to be associated with insufficient activation of oocytes by the current techniques of micro-injection. Therefore, we attempted to increase the fertilization rate by pretreating the oocytes with a Ca2+ ionophore, A23187 (10 microM, 3 min). Sperm nuclei were then micro-injected into the treated oocytes and 5 h later the percentage of oocytes at the pronuclear stage was examined: 53.1% for untreated controls and 85.7% for the treated group (P less than 0.05). However, when the number of pronucleate oocytes was divided by the number of A23187-activated oocytes, the percentage was 81.0% for the controls and 85.7% for the treated group. These findings suggest that pretreatment of oocytes with ionophore does not facilitate fertilization of activated oocytes, but that it indirectly enhances their development to the pronuclear stage by increasing the efficiency of egg activation.


Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. A heterogeneous group of cells interact with each other and with the surrounding matrix to form the complex structure of human endometrium. Since the integrin superfamily of molecules is involved in the cell-cell and cell-matrix interactions, this study was designed to screen, in situ, the cellular distribution of CDW49a-f molecules in human endometrium throughout the menstrual cycle. The integrin molecules were localized by immunohistochemistry using monoclonal antibodies. Glandular epithelium expressed all integrin molecules. With the exception of CDW49d (alpha 4 beta 1), surface epithelium also expressed all these molecules. Endothelial cells were positive for all integrin molecules except CDW49a (alpha 1 beta 1). Endometrial lymphoid cells were positively immunostained for CDW49a, d and e (alpha 5 beta 1) and were negative for CDW49b (alpha 2 beta 1), CDW49c (alpha 3 beta 1) and CDW49f (alpha 6 beta 1). Regional differences in the expression of integrin molecules were observed. As compared to the functionalis epithelium, basalis epithelium characteristically exhibited higher expression of CDW49a, d and e. Two integrins in endometrium, CDW49a and d exhibited changes related to the menstrual cycle. CDW49a, which was not expressed in glandular epithelial cells in the proliferative phase, was strongly expressed in these cells after ovulation and its expression was diminished in the late secretory phase. This molecule was not expressed in the stromal cells, however, predecidual cells characteristically expressed this molecule in the late secretory phase. CDW49d was only expressed in the glandular epithelial cells in the mid-proliferative to mid-secretory phases.(ABSTRACT TRUNCATED AT 250 WORDS)


Turner's syndrome patients lack tight junctions between uterine epithelial cells. Eleven endometrial biopsies, taken from six Turner's syndrome patients receiving hormone replacement therapy prior to treatment by oocyte donation and embryo transfer, were assessed by freeze fracture followed by electron microscopy for epithelial tight junctions. Nine of the eleven biopsies had no discernible tight junctions; the other two biopsies had reduced and disorganized junctional structures. Two patients subsequently became pregnant following embryo transfer. It is concluded that compromised epithelial integrity does not prevent embryo implantation in the human, an observation that is consistent with a barrier role for the epithelium except at times when appropriately conditioned with oestrogen and progesterone to induce receptivity for implantation.


Serological responses to various Coccidioides antigen preparations in a new enzyme immunoassay. A new enzyme immunoassay (EIA) was used to investigate immunoglobulin M (IgM) and IgG responses to various coccidioidal antigen preparations. Direct binding and inhibition assays both revealed that the IgG responses of many serum specimens were directed largely to the tube precipitin (TP) antigen even when the microwells were coated with a combination of coccidioidal antigens (CCAs). From a panel of 28 serum specimens containing antibodies to CCAs, 4 serum specimens yielded high IgG signals (absorbance, greater than 1.0) and were negative for IgM (absorbance, less than 0.2), yet all four IgG responses were inhibited by at least 90% with partially purified TP antigens (2.4 micrograms/ml). The IgM and IgG fractions of several serum specimens were separated by ion-exchange chromatography and assayed by using different antigen preparations adsorbed to the microwells. The binding of both IgM and IgG peaks to the microwells coated with the CCA preparation was inhibited significantly by preincubation with TP antigens. One serum specimen (specimen 26) yielded a large IgG response (absorbance, greater than 2.0) with the CCA preparation and also bound directly to microwells coated only with TP antigens. The IgG signal (absorbance) of serum specimen 26 was reduced by 98% when it was preincubated with TP antigens prior to the assay. Significant IgG signals from several other serum specimens were observed when microwells were coated with TP antigen preparations, but they were absent when periodate-treated preparations were used. Two cerebrospinal fluid specimens yielded IgG signals with CCA-coated microwells, which were not inhibited by TP antigens, and yielded no signal with microwells coated only with TP antigens. The results are consistent with the concept that the typical serologic response to TP antigens occurs early in disease progression, but they suggest that TP antigens stimulate both IgM and IgG responses.


Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification. The presence of hepatitis B virus DNA in the sera of individuals is the most definitive marker of an active viral infection. We have used polymerase chain reaction detection of hepatitis B virus DNA directly on whole blood dried as a spot on filter paper. The method is rapid, specific, and sensitive and has the ability to detect as little as 10 virus particles by ethidium bromide staining of the polymerase chain reaction-amplified products. The method is cost-effective, and the stability of the spots makes the collection and transportation of potentially infectious blood safe.


Laboratory and clinical evaluations of media for the primary isolation of Haemophilus species. There has not previously been an objective comparison of medium formulations for the primary isolation of Haemophilus species. This study was undertaken to evaluate the components required for the optimal growth of large, easily identifiable colonies of these bacteria. We compared six medium bases and seven supplements for their ability to support the growth of 86 strains of Haemophilus influenzae and 17 strains of other species of Haemophilus. By using a growth index that combines colony size and the dilution factor, a formulation of GC agar base with 1% yeast autolysate and 5% sheep blood (chocolated) promoted the growth of large, easily recognizable colonies of H. influenzae and other Haemophilus species. This medium was designated GCYSB. The addition of hematin to supplements that supplied NAD (or factor V) to the medium was inhibitory to the growth of all of the Haemophilus species tested. In a clinical comparison of GCYSB with routinely used chocolate agar medium in two laboratories for the primary isolation of Haemophilus species, overall GCYSB promoted better growth of 124 strains of H. influenzae and H. parainfluenzae. GCYSB is easy to prepare and inexpensive compared with the ease of preparation and expense of other Haemophilus isolation media.


Development of polymerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples. In order to improve the diagnosis of Listeria meningitis or meningoencephalitis, especially in patients who have received antibiotics before their cerebrospinal fluid (CSF) has been examined, two assays for the detection of Listeria monocytogenes based on the polymerase chain reaction (PCR) were evaluated. After a standard PCR, the amplified DNA was detected either by a second round of PCR with internal primers followed by gel electrophoresis and ethidium bromide staining (nested PCR) or by dot blot hybridization to an internal digoxigenin-labeled probe (PCR-dot blot). For PCR, two sets of primers within the invasion-associated protein gene (iap gene) were chosen. They allowed for the highly specific detection of all L. monocytogenes reference strains tested (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, and 7). These primers did not detect amplification products from various other gram-positive or gram-negative bacterial DNAs or human DNA. The sensitivities of both assays were assessed on sterile CSF samples that were artificially seeded with serial dilutions of L. monocytogenes serotype 4b cells. By both methods the limit of detection was less than 10 cells in the initial reaction. Since the nested PCR is more prone to contamination because of manipulation of the amplified products, a standard PCR assay followed by dot blot hybridization was applied to 52 CSF samples in a retrospective study. Of 28 CSF samples which were sterile or positive for bacteria other than Listeria species, 24 were PCR negative. In contrast, from 17 patients with culture-proven Listeria meningitis, 14 of 17 initial CSF samples were PCR positive, as were 3 of 7 culture-negative followup CSF samples taken after patients received antibiotics. These results support the usefulness of this approach in the diagnosis of Listeria meningitis, in particular, when antibiotic administration precedes culture of CSF.


Polymerase chain reaction-gene probe detection system specific for pathogenic strains of Yersinia enterocolitica. The polymerase chain reaction technique was used to develop a rapid diagnostic assay for detection of pathogenic Yersinia enterocolitica strains. The assay targeted a stretch of 163 bp of the yst gene and could be applied to both pure cultures and crude DNA extracted from feces. The defined primer pair amplified the targeted sequence from only pathogenic strains and fecal samples seeded with the serotype O:3 strain of Y. enterocolitica, whereas neither nonpathogenic strains nor normal stools yielded any amplified fragments. Of the other Yersinia species and non-Yersinia species tested, only two strains of Y. kristensenii yielded the same amplified product. A 20-mer oligonucleotide probe specifically hybridized within the amplified yst fragment of Y. enterocolitica but did not hybridize with the amplified yst fragment of Y. kristensenii by Southern and dot blot hybridizations. This confirms the reliability of this diagnostic assay in both clinical and epidemiological studies. The availability of the extracted DNA for the polymerase chain reaction was checked by simultaneous amplification of a part of the 16S rDNA and the yst gene. The entire diagnostic assay, including a simplified technique for DNA extraction, the amplification process, and gel electrophoresis, could be completed within 1 working day, which is better than the time required for the time-consuming traditional techniques used in clinical laboratories.


Staphylococcus lugdunensis endocarditis. Staphylococcus lugdunensis is a recently described coagulase-negative species which has been associated with human infections, including infective endocarditis. A case of native valve endocarditis caused by this organism is described. The initial laboratory detection of S. lugdunensis is facilitated by a positive test for ornithine decarboxylase. The identification of such isolates should not cause difficulty unless undue reliance is placed upon a small number of tests.


Epidemiology of Enterococcus faecalis urinary tract infection in a teaching hospital in London, United Kingdom. Enterococcus faecalis is a frequent cause of urinary tract infection in hospitalized patients. Recent reports have suggested that the organism may frequently be acquired by cross-infection from other patients. In this study, we used total DNA restriction patterns to type 135 urine isolates of E. faecalis from four sets of patients. Isolates were placed into types (all bands identical) and into groups (most bands identical). Most isolates were discriminated by the typing method, and the results suggested that direct cross-infection occurred rarely if at all. However, two groups of clonally related isolates occurred frequently in the urine specimens and also in feces from hospital-associated patients and were often associated with antibiotic resistance. Isolates from these two groups were found less frequently in feces from people not associated with the hospital.


Primary humoral antibody response to Coxiella burnetii, the causative agent of Q fever. Of 147 patients with acute Q fever diagnosed during a major outbreak in Birmingham, England, in early summer 1989, 41 provided sets of sera which allowed us to make a detailed analysis of the primary humoral immune response. Antibody titers specific for Coxiella burnetii were measured by the complement fixation test and by an immunoglobulin M (IgM)- and IgG-specific indirect immunofluorescence test. The relative avidity of specific IgGs was determined by the indirect immunofluorescence test with and without treatment of antigen-antibody complexes with 8 M urea. The IgG subclass responses after primary infection and their avidities were also determined for a limited number of paired serum specimens. Specific IgM titers persisted for more than 6 months in the majority of cases and were therefore not a sufficient criterion for the diagnosis of recent infection. However, for serial samples the antibody titer ratios (IgG/IgM) and the ratios (IgG titer with treatment/IgG titer without treatment) that indicated relative avidity changed significantly, depending on the time postinfection. Within the IgG class, the C. burnetii-specific antibody response over time was almost exclusively represented by subclass 1 molecules, which thus showed affinity maturation.


Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. Chlamydia pneumoniae has proved to be difficult to isolate and propagate in cell culture. We compared the growth of three strains of C. pneumoniae, TW-183 and two clinical isolates from Brooklyn, N.Y., in five cell lines, including HeLa 229, McCoy, HL, HEp-2, and HTED, an immortalized human tracheal cell line. HEp-2 was the most sensitive cell line tested. When 10-fold dilutions of three C. pneumoniae strains at known titers were inoculated into the different cell lines, the mean number of inclusion-forming units per milliliter was 1 to 2 log units higher in the HEp-2 than in the other cell lines. This difference was statistically significant. Omission of pretreatment with DEAE-dextran resulted in larger inclusions than those seen in pretreated cells, with the exception of McCoy and HTED cells. Retrieval of clinical specimens previously cultured on HeLa 229 cells and comparison of mean inclusion counts in fresh clinical specimens simultaneously inoculated on HeLa 229 and HEp-2 cells suggested that culture in HEp-2 cells may require only the initial inoculation and one passage, compared with three to four passages, as required by culture in HeLa 229 cells.


Characterization of a distinct group of slowly growing mycobacteria by biochemical tests and lipid analyses. A group of slowly growing mycobacterial strains (n = 14) isolated from respiratory tract specimens was collected from 1971 to 1990 on the basis of growth characteristics and uncommon biochemical and glycolipid profiles. Growth at 25 to 45 degrees C, a negative Tween 80 hydrolysis test, a strong positive reaction in a 14-day arylsulfatase test, and susceptibility to ethambutol in combination with resistance to cycloserine were important for the initial separation. The strains had a distinctive glycolipid pattern which was unlike those of other mycobacterial species. Analyses of cellular fatty acids by gas-liquid chromatography and mycolic acids by thin-layer chromatography further characterized this homogeneous group of mycobacteria. The presence of 2-eicosanol (2-OH-20:0alc) and hexacosanoic acid (26:0) combined with the lack of 2-docosanol (2-OH-22:0alc) differentiated this group from other slowly growing mycobacteria.


Collaborative investigation of broth microdilution and semisolid agar dilution for in vitro susceptibility testing of Candida albicans. A study was performed in two laboratories to evaluate the effect of growth medium and test methodology on inter- and intralaboratory variations in the MICs of amphotericin B (AMB), flucytosine (5FC), fluconazole (FLU), itraconazole (ITRA), and the triazole Sch 39304 (SCH) against 14 isolates of Candida albicans. Testing was performed by broth microdilution and semisolid agar dilution with the following media, buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS): buffered yeast nitrogen base (BYNB), Eagle's minimal essential medium (EMEM), RPMI 1640 medium (RPMI), and synthetic amino acid medium for fungi (SAAMF). Inocula were standardized spectrophotometrically, and endpoints were defined by the complete absence of growth for AMB and by no more than 25% of the growth in the drug-free control for all other agents. Comparative analyses of median MICs, as determined by each test method, were made for all drug-medium combinations. Both methods yielded similar (+/- 1 twofold dilution) median MICs for AMB in EMEM and RPMI, 5FC in all media, and FLU in EMEM, RPMI, and SAAMF. In contrast, substantial between-method variations in median MICs were seen for AMB in BYNB and SAAMF, FLU In BYNB, and ITRA and SCH in all media. Interlaboratory concordance of median MICs was good for AMB, 5FC, and FLU but poor for ITRA and SCH in all media. Endpoint determinations were analyzed by use of kappa statistical analyses for evaluating the strength of observer agreement. Moderate to almost perfect interlaboratory agreement occurred with AMB and 5FC in all media and with FLU in EMEM, RPMI, and SAAMF, irrespective of the test method. Slight to almost perfect interlaboratory agreement occurred with ITRA and SCH in EMEM, RPMI, and SAAMF when tested by semisolid agar dilution but not broth microdilution. Kappa values assessing intralaboratory agreement between methods were high for 5FC in all media, for AMB in BYNB, ENEM, and RPMI, and for FLU in EMEM, RPMI, and SAAMF. One laboratory, but not the other, reported substantial to almost perfect agreement between methods for ITRA, and SCH in EMEM, RPMI, and SAAMF. Both laboratories reported poor agreement between methods for the azoles in BYNB. Discrepancies noted in azole-BYNB combinations were largely due to the greater inhibitory effect of these agents in BYNB than in other media. These results indicate that the semisolid agar dilution and broth microdilution methods with EMEM or RPMI yield equivalent and reproducible MICs for AMB, 5FC, and FLU but not ITRA and SCH.


Pulsed-field gel electrophoresis for epidemiologic studies of Campylobacter hyointestinalis isolates. Campylobacter hyointestinalis was isolated from five members of the same family who had previously consumed raw milk. Pulsed-field gel electrophoresis of genomic DNAs from the five strains, after digestion with restriction endonuclease SalI, revealed that three strains had identical genome patterns and therefore appeared to be related, whereas the other two had completely different genome patterns and appeared to be unrelated. We report here for the first time the isolation of C. hyointestinalis from family members who had consumed raw milk. Our study also demonstrates the usefulness of pulsed-field gel electrophoresis for epidemiologic studies of this unusual campylobacter.


Evaluation of commercial and standard methodology for determination of oxacillin susceptibility in Staphylococcus aureus. Agar dilution with and without 4% NaCl, broth microdilution with 2% NaCl, the dried MicroScan Rapid Positive MIC 1 panel (Baxter Health Care Corp., West Sacramento, Calif.), the Vitek GPS-SA card (Vitek Systems, Hazelwood, Mo.), and the oxacillin agar screen plate were compared with a DNA probe encoding the mec gene for their abilities to detect oxacillin resistance in 506 clinical isolates of Staphylococcus aureus. The results of testing for the mec gene showed that there were 254 oxacillin-resistant and 252 oxacillin-susceptible isolates of S. aureus. There were 14.2% very major errors with Vitek (a resistant isolate was interpreted as susceptible) and 6.7% very major errors with MicroScan. Fewer major errors were seen: 0.8% with MicroScan (a susceptible isolate was interpreted as resistant) and 0.4% with Vitek. No very major errors but 2.4% major errors occurred by agar dilution with 4% NaCl supplementation, whereas there were 0.8% very major and 0.4% major errors without 4% NaCl supplementation. By broth microdilution there were 2.0% very major and 0.8% major errors. The results of the oxacillin agar screen plate method were 100% concordant with those of the mec gene probe method.


BioArgos: a fully automated blood culture system. BioArgos (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) is a fully automated blood culture system that detects carbon dioxide production by infrared spectroscopy through a glass bottle. This hands-off system was compared with the BACTEC NR-660 system (Becton Dickinson Diagnostic Instrument Systems, Towson, Md.). A total of 336 microorganisms belonging to 74 taxa were tested in simulated blood cultures by both systems. Experimental data showed no significant differences between the two systems. The inclusive detection times (+/- the standard deviations) were 33.2 +/- 28.7 and 35.0 +/- 30.6 h with BioArgos and BACTEC, respectively. Anaerobes were detected earlier with BioArgos, whereas detection of some organisms that need oxygen to grow was slightly delayed. In conclusion, BioArgos is as reliable and accurate as BACTEC NR-660 and shows better practicability owing to noninvasive detection, reduction of vial manipulation, and absence of daily maintenance.


Application of ubiquinone systems and electrophoretic comparison of enzymes to identification of clinical isolates of Aspergillus fumigatus and several other species of Aspergillus. The ubiquinone systems and electrophoretic comparison of enzymes were used to determine the relatedness among 64 isolates of seven Aspergillus spp. These were 31 clinical and 3 nonclinical isolates of Aspergillus fumigatus Fres., 2 isolates of A. nidulellus Samson & W. Gams, 8 isolates of A. terreus Thom, 4 isolates of A. flavus Link, 1 isolate of A. oryzae (Ahlburg) Cohn, 14 isolates of A. niger van Tieghem, and 1 isolate of A. japonicus Saito. The enzymes glucose 6-phosphate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, fumarase, and malate dehydrogenase were examined. The relative mobilities were analyzed numerically. The results were presented as a dendrogram. Isolates from clinical and nonclinical sources within the same species had identical ubiquinone systems and identical or very similar enzyme patterns. In the dendrogram, 64 of the tested isolates were separated into seven major clusters at a 60% similarity level. Each major cluster corresponds to a single species. On the dendrogram, A. fumigatus isolates showed homogeneity, whereas A. niger isolates showed relative heterogeneity; in particular, A. niger MF-24 and the other A. niger isolates were distantly linked to each other. All A. fumigatus isolates had the Q-10 ubiquinone system and formed a single major cluster at a similarity level of 73% or greater. Glucose 6-phosphate dehydrogenase and glutamate dehydrogenase were key enzymes for differentiating all clinical and nonclinical isolates of A. fumigatus from the other Aspergillus spp. Ubiquinone systems and enzyme patterns appear to be objective and useful indicators for use in the precise identification of clinical isolates of Aspergillus spp.


Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives. Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies to Mycobacterium tuberculosis, M. avium, and M. gordonae. The patients had significantly higher IgA responses to mycobacterial antigens than did either their relatives or the controls. On the other hand, both the patients and their relatives had significantly higher IgG responses against these antigens than did the controls. The elevated IgA response was more pronounced against isopentanol-extracted whole bacterial cells than it was against soluble protein extracts, and it appeared to be directed against fixed surface antigens that lie under the loosely bound peptidoglycolipid or glycolipid antigens of mycobacteria.


Standardization of an enzyme immunoassay for human antibody to Haemophilus ducreyi. We standardized a serologic enzyme immunoassay (EIA) for human immunoglobulin G and M antibodies against Haemophilus ducreyi. We evaluated the performance of this test with respect to the time from acute chancroid and coinfection with human immunodeficiency virus (HIV). Antibody to a crude, soluble bacterial antigen of one H. ducreyi strain was detected in a panel of serum samples from clinically and microbiologically confirmed cases of chancroid and from controls. Test interpretation was standardized for optimal sensitivity and specificity. Performance of the EIA was enhanced in the period of early convalescence from acute primary chancroid and was not diminished in the presence of HIV coinfection. The EIA performed adequately as a serologic screening test for field evaluation and epidemiologic application in conjunction with sexually transmitted disease and HIV detection and control efforts.


Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein. A polymerase chain reaction (PCR) assay for the rapid detection of mycobacterial DNA is described. Oligonucleotide primers, derived from the sequence of a gene coding for the 32-kDa antigen of Mycobacterium tuberculosis, amplified DNA from all 28 species of mycobacteria tested. All nonmycobacterial species tested were negative. An oligonucleotide probe hybridized to the PCR products of the strains belonging to the M. tuberculosis complex. This method could detect as little as 50 fg, as tested with purified M. tuberculosis DNA. By this amplification method, 127 sputum specimens were tested, with 7.9% of the specimens proving to be inhibitory in PCR. The sensitivity of detection by PCR compared with that by culture was 55.9%; when the inhibitory specimens were excluded, the sensitivity was 70.4%. The specificity of PCR combined with hybridization was 100%.


Development of revised quality control limits for disk diffusion susceptibility tests of selected cephem antibiotics with Haemophilus influenzae and description of a new control strain. Inconsistent quality control results in disk diffusion testing of cefaclor, cefamandole, cefonicid, and cefuroxime with Haemophilus influenzae ATCC 49247 and Haemophilus test medium (HTM) prompted a search for an alternative control strain that would provide more reliable results. A five-laboratory study was conducted to evaluate two candidate H. influenzae strains as possible alternatives to the aforementioned strain. Repetitive testing of the candidate strains and H. influenzae ATCC 49247 over several days with a total of six different lots of HTM documented consistent performance of the two candidate strains and confirmed inconsistent results for some of the antibiotics with H. influenzae ATCC 49247. In particular, certain lots of HTM failed to yield cefaclor and cefamandole zone sizes within the quality control range advocated by the National Committee for Clinical Laboratory Standards. Because of the greater consistency offered by the new strains, one was selected (now designated H. influenzae ATCC 49766) to be recommended for routine quality control testing of cefaclor, cefamandole, cefonicid, cefuroxime, and the related carbacephem loracarbef. The new control strain and zone size ranges proposed here have been approved by the National Committee for Clinical Laboratory Standards in place of the previously recommended strain and zone size limits for testing of these five cephem antibiotics.


Haemophilus influenzae ATCC 49766, an alternative quality control strain for monitoring broth microdilution susceptibility tests with selected beta-lactams. A beta-lactamase-negative, ampicillin-resistant strain of Haemophilus influenzae is currently used for quality control of broth microdilution tests performed with Haemophilus test medium (HTM). Studies with eight lots of HTM broth documented the fact that MIC limits for some antimicrobial agents are unrealistically stringent; i.e., only three of eight lots of HTM broth were satisfactory for testing cefaclor. An alternative, ampicillin-susceptible strain of H. influenzae (ATCC 49766) was found to provide much more reproducible results with five problematic drugs (cefaclor, cefuroxime, cefamandole, loracarbef, and cefonicid). Multilaboratory studies defined MIC control limits for both control strains tested against 12 antimicrobial agents.


Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays. A total of 320 stool specimens obtained from 262 patients suspected of having Clostridium difficile-associated gastrointestinal disease were examined with two cytotoxicity assays (CTAs) and two commercially available enzyme immunoassays (EIAs). The CTAs were an in-house-developed procedure (University of Massachusetts Medical Center [UMMC], Worcester, Mass.) and a commercial test (Bartels CTA; Baxter Healthcare Corp., West Sacramento, Calif.). One EIA was a monoclonal antibody-based assay for C. difficile toxins A and B (Cambridge Biotech Corp. [CBC], Worcester, Mass.). The other EIA employed monoclonal antibodies directed against only toxin A (Meridian Diagnostics, Cincinnati, Ohio). True-positive and true-negative results were defined on the basis of the results of the four assays, clinical assessments of patients, and the results of other laboratory tests. The sensitivities of the four assays were as follows: Bartels CTA, 100%; UMMC CTA, 97.2%; CBC EIA, 84.5%; and Meridian EIA, 69.0%. The Bartels CTA demonstrated a specificity of 99.2%. The other three assays had a specificity of 100%.


Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination. Genetic relationships among 254 isolates of Staphylococcus aureus resistant to methicillin recovered between 1961 and 1992 from nine countries on four continents were determined by analyzing electrophoretically demonstrable allelic variation at 15 chromosomal enzyme loci. Fifteen distinctive electrophoretic types, marking clones, were identified. The mec gene is harbored by many divergent phylogenetic lineages representing a large portion of the breadth of chromosomal diversity in the species, a result that is interpreted as evidence that multiple episodes of horizontal transfer and recombination have contributed to the spread of this resistance determinant in natural populations. Isolates recovered in the United Kingdom, Denmark, Switzerland, Egypt, and Uganda in the 1960s are of a single multilocus enzyme genotype and probably are progeny of an ancestral methicillin-resistant clone. There is geographic variation in the frequency of recovery of the common methicillin-resistant clones, an observation that may in part explain reported regional differences in natural history correlates of resistant organisms.